(en)Compositions and methods for the therapy and diagnosis of cancer, particularly ovarian cancer, are disclosed. Illustrative compositions comprise one or more ovarian tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly ovarian cancer.

1.ApplicationNumber: US-71355000-A
1.PublishNumber: US-6617109-B1
2.Date Publish: 20030909
3.Inventor: XU JIANGCHUN
STOLK JOHN A.

4.Inventor Harmonized: XU JIANGCHUN(US)
STOLK JOHN A(US)

5.Country: US
6.Claims:
(en)Compositions and methods for the therapy and diagnosis of cancer, particularly ovarian cancer, are disclosed. Illustrative compositions comprise one or more ovarian tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly ovarian cancer.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 09/656,668, filed Sep. 7, 2000, which is a CIP of U.S. application Ser. No. 09/640,173, filed Aug. 15, 2000, which is a is a CIP of U.S. application Ser. No. 09/561,778, filed May 1, 2000, now abandoned which is a CIP of U.S. application Ser. No. 09/394,374, filed Sep. 10, 1999, now abandoned all pending and incorporated by reference in their entirety herein.


TECHNICAL FIELD
The present invention relates generally to ovarian cancer therapy. The invention is more specifically related to polypeptides comprising at least a portion of an ovarian carcinoma protein, and to polynucleotides encoding such polypeptides, as well as antibodies and immune system cells that specifically recognize such polypeptides. Such polypeptides, polynucleotides, antibodies and cells may be used in vaccines and pharmaceutical compositions for treatment of ovarian cancer.
BACKGROUND OF THE INVENTION
Ovarian cancer is a significant health problem for women in the United States and throughout the world. Although advances have been made in detection and therapy of this cancer, no vaccine or other universally successful method for prevention or treatment is currently available. Management of the disease currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. However, the use of established markers often leads to a result that is difficult to interpret, and high mortality continues to be observed in many cancer patients.
Immunotherapies have the potential to substantially improve cancer treatment and survival. Such therapies may involve the generation or enhancement of an immune response to an ovarian carcinoma antigen. However, to date, relatively few ovarian carcinoma antigens are known and the generation of an immune response against such antigens has not been shown to be therapeutically beneficial.
Accordingly, there is a need in the art for improved methods for identifying ovarian tumor antigens and for using such antigens in the therapy of ovarian cancer. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, this invention provides compositions and methods for the therapy of cancer, such as ovarian cancer.
In one aspect, the present invention provides polynucleotide compositions comprising a sequence selected from the group consisting of:
(a) sequences provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205;
(b) complements of the sequences provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205;
(c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205;
(d) sequences that hybridize to a sequence provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205 under moderately stringent conditions;
(e) sequences having at least 75% identity to a sequence provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205;
(f) sequences having at least 90% identity to a sequence provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205; and
(g) degenerate variants of a sequence provided in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205.
In one preferred embodiment, the polynucleotide compositions of the invention are expressed in at least about 20%, more preferably in at least about 30%, and most preferably in at least about 50% of ovarian tumors samples tested, at a level that is at least about 2-fold, preferably at least about 5-fold, and most preferably at least about 10-fold higher than that for normal tissues.
In one aspect, the present invention provides polypeptides comprising an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished. Within certain embodiments, the ovarian carcinoma protein comprises a sequence that is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 4-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, and 203-205, and complements of such polynucleotides.
The present invention further provides polynucleotides that encode a polypeptide as described above or a portion thereof, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
The present invention further provides polypeptide compositions comprising an amino acid sequence selected from the group consisting of sequences recited in SEQ ID NOs: 200-202.
In certain preferred embodiments, the polypeptides of the present invention are immunogenic, i.e., they are capable of eliciting an immune response, particularly a humoral and/or cellular immune response, as further described herein.
The present invention further provides fragments, variants and/or derivatives of the disclosed polypeptide sequences, wherein the fragments, variants and/or derivatives preferably have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% of the level of immunogenic activity of a the ovarian carcinoma protein comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs: 1-185, 187-199, and 203-205.
Within other aspects, the present invention provides pharmaceutical compositions comprising a polypeptide and/or polynucleotide as described above and a physiologically acceptable carrier.
Within a related aspect of the present invention, the pharmaceutical compositions, e.g., vaccine compositions, are provided for prophylactic or therapeutic applications. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the invention and an immunostimulant, such as an adjuvant.
The present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of the present invention, or a fragment thereof, and (b) a physiologically acceptable carrier.
Within further aspects, the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient. Illustrative antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.
Within related aspects, pharmaceutical compositions are provided that comprise: (a) an antigen presenting cell that expresses a polypeptide as described above and (b) an immunostimulant.
The present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins, typically in the form of pharmaceutical compositions, e.g., vaccine compositions, comprising a physiologically acceptable carrier and/or an immunostimulant. The fusions proteins may comprise multiple immunogenic polypeptides or portions/variants thereof, as described herein, and may further comprise one or more polypeptide segments for facilitating the expression, purification and/or immunogenicity of the polypeptide(s).
Within further aspects, the present invention provides methods for stimulating an immune response in a patient, preferably a T cell response in a human patient, comprising administering a pharmaceutical composition described herein. The patient may be afflicted with ovarian cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.
Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition as recited above. The patient may be afflicted with ovarian cancer, in which case the methods provide treatment for the disease, or patient considered at risk for such a disease may be treated prophylactically.
The present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a polypeptide of the present invention, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the protein from the sample.
Within related aspects, methods are provided for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated as described above.
Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a polypeptide of the present invention, comprising contacting T cells with one or more of: (i) an ovarian carcinoma polypeptide as described above; (ii) a polynucleotide encoding such a polypeptide; and/or (iii) an antigen presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Isolated T cell populations comprising T cells prepared as described above are also provided.
Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
The present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4 + and/or CD8 + T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of polypeptide disclosed herein; (ii) a polynucleotide encoding such a polypeptide; and (iii) an antigen-presenting cell that expressed such a polypeptide; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient. Proliferated cells may, but need not, be cloned prior to administration to the patient.
Within further aspects, the present invention provides methods for determining the presence or absence of a cancer, preferably an ovarian cancer, in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within preferred embodiments, the binding agent is an antibody, more preferably a monoclonal antibody.
The present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient. Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.
The present invention further provides, within other aspects, methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide of the present invention; (b) detecting in the sample a level of a polynucleotide, preferably mRNA, that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within certain embodiments, the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as recited above, or a complement of such a polynucleotide. Within other embodiments, the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.
In related aspects, methods are provided for monitoring the progression of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a polypeptide of the present invention; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.
Within further aspects, the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, and 193-199 are described in Tables III-VII below.
SEQ ID NO: 200 is the amino acid sequence of a polypeptide encoded by the polynucleotide recited in SEQ ID NO: 182;
SEQ ID NO: 201 is the amino acid sequence of a polypeptide encoded by the polynucleotide recited in SEQ ID NO: 182;
SEQ ID NO: 202 is the amino acid sequence of a polypeptide encoded by the polynucleotide recited in SEQ ID NO: 182.
SEQ ID NO: 203 is the determined extended cDNA sequence for SEQ ID NO: 197.
SEQ ID NO: 204 is the determined extended cDNA sequence for SEQ ID NO: 198.
SEQ ID NO: 205 is the determined extended cDNA sequence for SEQ ID NO: 199.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed generally to compositions and their use in the therapy and diagnosis of cancer, particularly ovarian cancer. As described further below, illustrative compositions of the present invention include, but are not restricted to, polypeptides, particularly immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (e.g., T cells).
The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
Polypeptide Compositions
As used herein, the term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.
Particularly illustrative polypeptides of the present invention comprise those encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199 or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions, to a polynucleotide sequence identified above. Certain other illustrative polypeptides of the invention comprise amino acid sequences as set forth in any one of SEQ ID NOs: 200-202.
The polypeptides of the present invention are sometimes herein referred to as ovarian tumor proteins or ovarian tumor polypeptides, as an indication that their identification has been based at least in part upon their increased levels of expression in ovarian tumor samples. Thus, a “ovarian tumor polypeptide” or “ovarian tumor protein,” refers generally to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide, that is expressed in a substantial proportion of ovarian tumor samples, for example preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50% or more of ovarian tumor samples tested, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in normal tissues, as determined using a representative assay provided herein. An ovarian tumor polypeptide sequence of the invention, based upon its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.
In certain preferred embodiments, the polypeptides of the invention are immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with ovarian cancer. Screening for immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.
As would be recognized by the skilled artisan, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention. An “immunogenic portion,” as used herein, is a fragment of an immunogenic polypeptide of the invention that itself is immunologically reactive (i.e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide. Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology , 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are “antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well-known techniques.
In one preferred embodiment, an immunogenic portion of a polypeptide of the present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Preferably, the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70% and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide. In some instances, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.
In certain other embodiments, illustrative immunogenic portions may include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted. Other illustrative immunogenic portions will contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.
In another embodiment, a polypeptide composition of the invention may also comprise one or more polypeptides that are immunologically reactive with T cells and/or antibodies generated against a polypeptide of the invention, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof.
In another embodiment of the invention, polypeptides are provided that comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.
The present invention, in another aspect, provides polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide compositions set forth herein, such as those set forth in SEQ ID NOs: 200-202, or those encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199.
In another aspect, the present invention provides variants of the polypeptide compositions described herein. Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein.
In one preferred embodiment, the polypeptide fragments and variants provide by the present invention are immunologically reactive with an antibody and/or T-cell that reacts with a full-length polypeptide specifically set for the herein.
In another preferred embodiment, the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90% or more of that exhibited by a full-length polypeptide sequence specifically set forth herein.
A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity as described herein and/or using any of a number of techniques well known in the art.
For example, certain illustrative variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other illustrative variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.
In many instances, a variant will contain conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, immunogenic variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table 1.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.






TABLE 1



Amino Acids
Codons















Alanine
Ala
A
GCA
GCC
GCG
GCU



Cysteine
Cys
C
UGC
UGU

Aspartic acid
Asp
D
GAC
GAU

Glutamic acid
Glu
E
GAA
GAG

Phenylalanine
Phe
F
UUC
UUU

Glycine
Gly
G
GGA
GGC
GGG
GGU

Histidine
His
H
CAC
CAU

Isoleucine
Ile
I
AUA
AUC
AUU

Lysine
Lys
K
AAA
AAG

Leucine
Leu
L
UUA
UUG
CUA
CUC
CUG
CUU

Methionine
Met
M
AUG

Asparagine
Asn
N
AAC
AAU

Proline
Pro
P
CCA
CCC
CCG
CCU

Glutamine
Gln
Q
CAA
CAG

Arginine
Arg
R
AGA
AGG
CGA
CGC
CGG
CGU

Serine
Ser
S
AGC
AGU
UCA
UCC
UCG
UCU

Threonine
Thr
T
ACA
ACC
ACG
ACU

Valine
Val
V
GUA
GUC
GUG
GUU

Tryptophan
Trp
W
UGG

Tyrosine
Tyr
Y
UAC
UAU








































In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
When comparing polypeptide sequences, two sequences are said to be “identical” if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol . 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy , Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol . 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res . 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol . 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one preferred approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.
Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide.
The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med ., 336:86-91, 1997).
In one preferred embodiment, the immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. patent application Ser. No. 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis . The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. patent application Ser. No. 60/158,585; see also, Skeiky et al., Infection and Immun . (1999) 67:3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as a soluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ra12 polypeptide or a portion thereof.
Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the invention, when fused with this targeting signal, will associate more efficiently with MHC class II molecules and thereby provide enhanced in vivo stimulation of CD4 + T-cells specific for the polypeptide.
Polypeptides of the invention are prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further described below. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc . 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.
In general, polypeptide compositions (including fusion polypeptides) of the invention are isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
Polynucleotide Compositions
The present invention, in other aspects, provides polynucleotide compositions. The terms “DNA” and “polynucleotide” are used essentially interchangeably herein to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. “Isolated,” as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
As will be understood by those skilled in the art, the polynucleotide compositions of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
As will be also recognized by the skilled artisan, polynucleotides of the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide/protein of the invention or a portion thereof) or may comprise a sequence that encodes a variant or derivative, preferably and immunogenic variant or derivative, of such a sequence.
Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that comprise some or all of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199, complements of a polynucleotide sequence set forth as described above, and degenerate variants of a polynucleotide sequence set forth as described above. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above.
In other related embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199, for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein). The term “variants” should also be understood to encompasses homologous genes of xenogenic origin.
In additional embodiments, the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that “intermediate lengths”, in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65° C. or 65-70° C.
In certain preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
When comparing polynucleotide sequences, two sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol . 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy , Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol . 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res . 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol . 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.
Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of immunogenic variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.
Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nuclcotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.
As used herein, the term “oligonucleotide directed mutagenesis procedure” refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term “oligonucleotide directed mutagenesis procedure” is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224, specifically incorporated herein by reference in its entirety.
In another approach for the production of polypeptide variants of the present invention, recursive sequence recombination, as described in U.S. Pat. No. 5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to “evolve” individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.
In other embodiments of the present invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.
Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.
Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Pat. No. 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
The nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50° C. to about 70° C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
According to another embodiment of the present invention, polynucleotide compositions comprising antisense oligonucleotides are provided. Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A receptor and human EGF (Jaskulski et al., Science. Jun. 10, 1988;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. Jun. 15, 1998;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683).
Therefore, in certain embodiments, the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein. Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, T m , binding energy, and relative stability. Antisense compositions may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA, are those which are at or near the AUG translation initiation codon, and those sequences which are substantially complementary to 5′ regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software and/or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).
The use of an antisense delivery method employing a short peptide vector, termed MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp4l and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., Nucleic Acids Res. Jul. 15, 1997;25(14):2730-6). It has been demonstrated that several molecules of the MPG peptide coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.
According to another embodiment of the invention, the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of the tumor polypeptides and proteins of the present invention in tumor cells. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. December 1987;84(24):8788-92; Forster and Symons, Cell. Apr. 24, 1987;49(2):211-20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. December 1981;27(3 Pt 2):487-96; Michel and Westhof, J. Mol Biol. Dec. 5, 1990;216(3):585-610; Reinhold-Hurek and Shub, Nature. May 14, 1992;357(6374):173-6). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., Proc Natl Acad Sci USA. Aug. 15, 1992;89(16):7305-9). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif Examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. Sep. 11, 1992;20(17):4559-65. Examples of hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz, Biochemistry Jun. 13, 1989;28(12):4929-33; Hampel et al., Nucleic Acids Res. Jan. 25, 1990;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitis δ virus motif is described by Perrotta and Been, Biochemistry. Dec. 1, 1992;31(47):11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al., Cell. December 1983;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. May 18, 1990;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. Oct. 1, 1991;88(19):8826-30; Collins and Olive, Biochemistry. Mar. 23, 1993;32(11):2795-9); and an example of the Group I intron is described in (U.S. Pat. No. 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.
Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.
Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.
Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells Ribozymes expressed from such promoters have been shown to function in mammalian cells. Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).
In another embodiment of the invention, peptide nucleic acids (PNAs) compositions are provided. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey ( Trends Biotechnol June 1997;15(6):224-9). As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell to which such PNA compositions have been administered.
PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., Science Dec. 6, 1991;254(5037):1497-500; Hanvey et al., Science . Nov. 27, 1992;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. January 1996;4(1):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.
PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., Bioorg Med Chem. April 1995;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.
Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem. April 1995;3(4):437-45; Petersen et al., J Pept Sci. 1995 May-June;1(3):175-83; Orum et al., Biotechniques. September 1995;19(3):472-80; Footer et al., Biochemistry. Aug. 20, 1996;35(33):10673-9; Griffith et a Nucleic Acids Res. Aug. 11, 1995;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci U.S. A. Jun. 6, 1995;92(12):5592-6; Boffa et al., Proc Natl Acad Sci U.S.A. Mar. 14, 1995;92(6):1901-5; Gambacorti-Passerini et al., Blood. Aug. 15, 1996;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U.S.A. Nov. 11, 1997;94(23):12320-5; Seeger et al, Biotechniques. September 1997;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.
Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. Dec. 15, 1997;65(24):3545-9) and Jensen et al. (Biochemistry. Apr. 22, 1997;36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcore™ technology.
Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.
Polynucleotide Identification, Characterization and Expression
Polynucleotides compositions of the present invention may be identified, prepared and/or manipulated using any of a variety of well established techniques (see generally, Sambrook et al., Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, and other like references). For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using the microarray technology of Affymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.
Many template dependent processes are available to amplify a target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
Any of a number of other template dependent processes, many of which are variations of the PCR Tm amplification technique, are readily known and available in the art. Illustratively, some such methods include the ligase chain reaction (referred to as LCR), described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in Great Britain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822 describes a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Other amplification methods such as “RACE” (Frohman, 1990), and “one-sided PCR” (Ohara, 1989) are also well-known to those of skill in the art.
An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a tumor cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5′ and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5′ sequences.
For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
Alternatively, amplification techniques, such as those described above, can be useful for obtaining a full length coding sequence from a partial cDNA sequence. One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res . 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as “rapid amplification of cDNA ends” or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a poly A region or vector sequence, to identify sequences that are 5′ and 3′ of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic . 1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res . 19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.
In other embodiments of the invention, polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser . 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser . 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).
A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTI plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
In bacterial systems, any of a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem . 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol . 153:516-544.
In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J . 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J . 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ . 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci . 91 :3224-3227).
In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci . 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ . 20:125-162).
In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci . 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol . 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci . 85:8047-51). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol . 55:121-131).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells that contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983 ; J. Exp. Med . 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992 , Prot. Exp. Purif . 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993 ; DNA Cell Biol . 12:441-453).
In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc . 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
Antibody Compositions, Fragments Thereof and Other Binding Agents
According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that exhibit immunological binding to a tumor polypeptide disclosed herein, or to a portion, variant or derivative thereof. An antibody, or antigen-binding fragment thereof, is said to “specifically bind,” “immunogically bind,” and/or is “immunologically reactive” to a polypeptide of the invention if it reacts at a detectable level (within, for example, an ELISA assay) with the polypeptide, and does not react detectably with unrelated polypeptides under similar conditions.
Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K off /K on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K d . See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.
An “antigen-binding site,” or “binding portion” of an antibody refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.”
Binding agents may be further capable of differentiating between patients with and without a cancer, such as ovarian cancer, using the representative assays provided herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. Preferably, a statistically significant number of samples with and without the disease will be assayed. Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.
Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol . 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
A number of therapeutically useful molecules are known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the “F(ab)” fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the “F(ab′) 2 ” fragment which comprises both antigen-binding sites. An “Fv” fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V H ::V L heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
A single chain Fv (“sFv”) polypeptide is a covalently linked V H ::V L heterodimer which is expressed from a gene fusion including V H - and V L -encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.
Each of the above-described molecules includes a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain FR set which provide support to the CDRS and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.
As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRS. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures—regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.
A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No. 519,596, published Dec. 23, 1992). These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
As used herein, the terms “veneered FRs” and “recombinantly veneered FRs” refer to the selective replacement of FR residues from, e.g., a rodent heavy or light chain V region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen-binding site which retains substantially all of the native FR polypeptide folding structure. Veneering techniques are based on the understanding that the ligand binding characteristics of an antigen-binding site are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other, and their interaction with the rest of the V region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.
The process of veneering makes use of the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1987), updates to the Kabat database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities of V region amino acids can be deduced from the known three-dimensional structure for human and murine antibody fragments. There are two general steps in veneering a murine antigen-binding site. Initially, the FRs of the variable domains of an antibody molecule of interest are compared with corresponding FR sequences of human variable domains obtained from the above-identified sources. The most homologous human V regions are then compared residue by residue to corresponding murine amino acids. The residues in the murine FR which differ from the human counterpart are replaced by the residues present in the human moiety using recombinant techniques well known in the art. Residue switching is only carried out with moieties which are at least partially exposed (solvent accessible), and care is exercised in the replacement of amino acid residues which may have a significant effect on the tertiary structure of V region domains, such as proline, glycine and charged amino acids.
In this manner, the resultant “veneered” murine antigen-binding sites are thus designed to retain the murine CDR residues, the residues substantially adjacent to the CDRs, the residues identified as buried or mostly buried (solvent inaccessible), the residues believed to participate in non-covalent (e.g., electrostatic and hydrophobic) contacts between heavy and light chain domains, and the residues from conserved structural regions of the FRs which are believed to influence the “canonical” tertiary structures of the CDR loops. These design criteria are then used to prepare recombinant nucleotide sequences which combine the CDRs of both the heavy and light chain of a murine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies which exhibit the antigen specificity of the murine antibody molecule.
In another embodiment of the invention, monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 90 Y, 123 I, 125 I, 131 I, 186 Re, 188Re, 211 At, and 212 Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.
T Cell Compositions
The present invention, in another aspect, provides T cells specific for a tumor polypeptide disclosed herein, or for a variant or derivative thereof. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the Isolex™ System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
T cells may be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide of interest. Preferably, a tumor polypeptide or polynucleotide of the invention is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
T cells are considered to be specific for a polypeptide of the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res . 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to a tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4 + and/or CD8 + . Tumor polypeptide-specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
For therapeutic purposes, CD4 + or CD8 + T cells that proliferate in response to a tumor polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a tumor polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a tumor polypeptide. Alternatively, one or more T cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
Pharmaceutical Compositions
In additional embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody compositions disclos ed herein in pharmaceutically-acceptable carriers f or admini stration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a signficant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions.
Therefore, in another aspect of the present invention, pharmaceutical compositions are provided comprising one or more of the polynucleotide, polypeptide, antibody, and/or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise immunogenic polynucleotide and/or polypeptide compositions of the invention for use in prophylactic and theraputic vaccine applications. Vaccine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),” Plenum Press (N.Y., 1995). Generally, such compositions will comprise one or more polynucleotide and/or polypeptide compositions of the present invention in combination with one or more immunostimulants.
It will be apparent that any of the pharmaceutical compositions described herein can contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts can be prepared, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
In another embodiment, illustrative immunogenic compositions, e.g., vaccine compositions, of the present invention comprise DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered within any of a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate polynucleotide expression systems will, of course, contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and terminating signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as Bacillus - Calmette - Guerrin ) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems. In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).
Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
Additional viral vectors useful for delivering the polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup.(−) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Pat. Nos. 5,505,947 and 5,643,576.
Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.
Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci . 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res . 73:1202-1207, 1993.
In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.
In another embodiment of the invention, a polynucleotide is administered/delivered as “naked” DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
In still another embodiment, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, Wis.), some examples of which are described in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Pat. No. 0500 799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.
In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, Oreg.), some examples of which are described in U.S. Pat. Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.
According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
Within certain embodiments of the invention, the adjuvant composition is preferably one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol . 7:145-173, 1989.
Certain preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL® adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such as Carbopol R to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (Corixa, Hamilton, Mont.), RC.-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general formula


HO(CH 2 CH 2 O) n —A—R, (I):


wherein, n is 1-50, A is a bond or —C(O)—, R is C 1-50 alkyl or Phenyl C, 50 alkyl.
One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C 1-50 , preferably C 4 -C 20 alkyl and most preferably C 12 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.
The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.
According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med . 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Mecl . 4:594-600, 1998).
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will typically vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.
Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems. such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.
The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate.
The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature Mar. 27, 1997;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
Typically, these formulations will contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J. Controlled Release Mar 2, 1998;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol July 1998;16(7):307-21; Takakura, Nippon Rinsho March 1998;56(3):691-5; Chandran et al., Indian J. Exp Biol. August 1997;35(8):801-9; Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety).
Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., J. Biol Chem. Sep. 25, 1990;265(27):16337-42; Muller et al., DNA Cell Biol. April 1990;9(3):221-9). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.
In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm. December 1998;24(12):1113-28). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J. Pharm Biopharm. March 1998;45(2):149-55; Zambaux et al. J. Controlled Release. Jan. 2, 1998;50(1-3):31-40; U.S. Pat. No. 5,145,684.
Cancer Therapeutic Methods
In further aspects of the present invention, the pharmaceutical compositions described herein may be used for the treatment of cancer, particularly for the immunotherapy of ovarian cancer. Within such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. As discussed above, administration of the pharmaceutical compositions may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.
Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).
Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8 + cytotoxic T lymphocytes and CD4 + T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Pat. No. 4,918,164) for passive immunotherapy.
Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157: 177, 1997).
Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.
Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 μg to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
Cancer Detection and Diagnostic Compositions, Methods and Kits
In general, a cancer may be detected in a patient based on the presence of one or more ovarian tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as ovarian cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding an ovarian tumor protein, which is also indicative of the presence or absence of a cancer. In general, a ovarian tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue
There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length ovarian tumor proteins and polypeptide portions thereof to which the binding agent binds, as described above.
The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with ovarian cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited above.
The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of a cancer, such as ovarian cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine , Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such tumor protein specific antibodies may correlate with the presence of a cancer.
A cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample. Within certain methods, a biological sample comprising CD4 + and/or CD8 + T cells isolated from a patient is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37° C. with polypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of tumor polypeptide to serve as a control. For CD4 + T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8 + T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer in the patient.
As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.
To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a tumor protein of the invention that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence as disclosed herein. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol ., 51:263, 1987; Erlich ed., PCR Technology , Stockton Press, N.Y., 1989).
One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.
In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.
Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.
As noted above, to improve sensitivity, multiple tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.
The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA encoding a tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a tumor protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a tumor protein.


The following Examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Identification of Representative Ovarian Carcinoma cDNA Sequences
Primary ovarian tumor and metastatic ovarian tumor cDNA libraries were each constructed in kanamycin resistant pZErO™-2 vector (Invitrogen) from pools of three different ovarian tumor RNA samples. For the primary ovarian tumor library, the following RNA samples were used: (1) a moderately differentiated papillary serous carcinoma of a 41 year old, (2) a stage IIIC ovarian tumor and (3) a papillary serous adenocarcinoma for a 50 year old caucasian. For the metastatic ovarian tumor library, the RNA samples used were omentum tissue from: (1) a metastatic poorly differentiated papillary adenocarcinoma with psammoma bodies in a 73 year old, (2) a metastatic poorly differentiated adenocarcinoma in a 74 year old and (3) a metastatic poorly differentiated papillary adenocarcinoma in a 68 year old.
The number of clones in each library was estimated by plating serial dilutions of unamplified libraries. Insert data were determined from 32 primary ovarian tumor clones and 32 metastatic ovarian tumor clones. The library characterization results are shown in Table I.





TABLE I



Characterization of cDNA Libraries








# Clones
Clones with
Insert Size
Ave Insert

Library
in Library
Insert (%)
Range (bp)
Size (bp)



Primary Ovarian
1,258,000
97
175-8000
2356

Tumor

Metastatic Ovarian
1,788,000
100
150-4300
1755

Tumor

























Four subtraction libraries were constructed in ampicillin resistant pcDNA3.1 vector (Invitrogen). Two of the libraries were from primary ovarian tumors and two were from metastatic ovarian tumors. In each case, the number of restriction enzyme cuts within inserts was minimized to generate full length subtraction libraries. The subtractions were each done with slightly different protocols, as described in more detail below.







A. POTS 2 Library: Primary Ovarian Tumor Subtraction Library








Tracer:
10 μg primary ovarian tumor library, digested with Not I

Driver:
35 μg normal pancreas in pcDNA3.1(+)


20 μg normal PBMC in pcDNA3.1(+)


10 μg normal skin in pcDNA3.1(+)


35 μg normal bone marrow in pZErO ™ -2


Digested with Bam HI/Xho I/Sca I

























Two hybridizations were perfonned, and Not 1-cut pcDNA3.1(+) was the cloning vector for the subtracted library. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table II.





TABLE II



Ovarian Carcinoma Sequences






Sequence
SEQ ID NO









21909
2


21920
9


21921
10


25099
143


25101
144


25103
145


25107
146


25111
148


25113
149


25115
150


25116
151


25752
156


25757
158


25769
161


21907
1


21911
5


25763
160


25770
162

















































B. POTS 7 Library: Primary Ovarian Tumor Subtraction Library








Tracer:
10 μg primary ovarian tumor library, digested with Not I

Driver:
35 μg normal pancreas in pcDNA3.1(+)


20 μg normal PBMC in pcDNA3.1(+)


10 μg normal skin in pcDNA3.1(+)


35 μg normal bone marrow in pZErO ™ -2


Digested with Bam HI/Xho I/Sca I


˜25 μg pZErO ™ -2, digested with Bam HI and Xho I


























Two hybridizations were performed, and Not I-cut pcDNA3.1(+) was the cloning vector for the subtracted library. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table III.





TABLE III



Ovarian Carcinoma Sequences






Sequence
SEQ ID NO





24937
125


24940
128


24946
132


24950
133


24951
134


24956
137


25791
166


25796
167


25797
168


25804
171


24955
136







































C. OS1D Library: Metastatic Ovarian Tumor Subtraction Library








Tracer:
10 μg metastatic ovarian library in pZErO ™ -2,


digested with Not I

Driver:
24.5 μg normal pancreas in pcDNA3.1


14 μg normal PBMC in pcDNA3.1


14 μg normal skin in pcDNA3.1


24.5 μg normal bone marrow in pZErO ™ -2


50 μg pZErO ™ 2, digested with Bam HI/Xho I/Sfu I


























Three hybridizations were performed, and the last two hybridizations were done with an additional 15 μg of biotinylated pZErO™-2 to remove contaminating pZErO™-2 vectors. The cloning vector for the subtracted library was pcDNA3.1/Not I cut. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table IV.





TABLE IV



Ovarian Carcinoma Sequences






Sequence
SEQ ID NO









24635
57


24647
63


24661
69


24663
70


24664
71


24670
72


24675
75


23645.1
13


23660.1
16


23666.1
19


23679.1
23


24651
65


24683
78












































D. OS1F Library: Metastatic Ovarian Tumor Subtraction Library








Tracer:
10 μg metastatic ovarian tumor library, digested with Not I

Driver:
12.8 μg normal pancreas in pcDNA3.1


7.3 μg normal PBMC in pcDNA3.1


7.3 μg normal skin in pcDNA3.1


12.8 μg normal bone marrow in pZErO ™ -2


25 μg pZErO ™ -2, digested with Bam HI/Xho I/Sfu I

























One hybridization was performed. The cloning vector for the subtracted library was pcDNA3.1/Not I cut. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table V.





TABLE V



Ovarian Carcinoma Sequences




Sequence
SEQ ID NO






24344
33

24356
42

24368
53

24696
86

24699
89

24701
90

24703
91

24707
95

24709
97

24732
111

24745
120

24746
121

24337
28

24348
35

24351
38

24358
44

24360
46

24361
47

24690
81

24692
82

24694
84

24705
93

24711
98

24713
99

24727
107

24741
17

24359 (78% Human mRNA for KIAA0111 gene,
45

complete cds)

24336 (79% with H. sapiens mitochondrial genome
27

(consensus sequence))

24737 (84% Human ADP/ATP translocase mRNA)
114

24363 (87% Homo sapiens eukarvotic translation
49

elongation factor 1 alpha 1 (EEF1A1)

24357 (87% S. scrofa mRNA for UDP glucose
43

pyrophosphorylase)

24362 (88% Homo sapiens Chromosome 16 BAC clone
48

CIT987SK-A-233A7)

24704 (88% Homo sapiens chromosome 9, clone
92

hRPK.401_G_18)

24367 (89% Homo sapiens 12p13.3 BAC RCPI11-
52

935C2)

24717 (89% Homo sapiens proliferation-associated gene
103

A (natural killer-enhancing factor A) (PAGA)

24364 (89% Human DNA sequence from PAC 27K14 on
50

chromosome Xp11.3-Xp11.4)

24355 (91% Homo sapiens chromosome 17, clone
41

hCIT.91_J_4)

24341 (91% Homo sapiens chromosome 5, BAC clone
32

249h5 (LBNL H149)

24714 (91% Human DNA sequence from clone 125N5
100

on chromosome 6q26-27)










































































The sequences in Table VI, which correspond to known sequences, were also identified in the above libraries.





TABLE IV



Ovarian Carcinoma Sequences






Identity
SEQ ID NO
Sequence
Library








Genomic sequence from Human 9q34
56
24634
OS1D

Homo sapiens 12p13.3 PAC RPCI1-96H9 (Roswell
66
24653
OS1D

Park Cancer Institute Human PACLibrary)

Homo sapiens annexin II (lipocortin II) (ANX2)
60
24640
OS1D

mRNA

Homo sapiens eukaryotic translation elongation
55
24627
OS1D

factor 1 alpha 1 (EEF1A1)

Homo sapiens ferritin, heavy polypeptide 1 (FTH1)
64
24648
OS1D

Homo sapiens FK506-binding protein 1A (12kD)
22
23677.1
OS1D

(FKBP1A) mRNA

Homo sapiens growth arrest specific transcript 5 gene
73
24671
OS1D

Homo sapiens keratin 18 (KRT18) mRNA
68
24657
OS1D

Homo sapiens mRNA; cDNA DKFZp564H182
76
24677
OS1D

Homo sapiens ribosomal protein S7 (RPS7)
74
24673
OS1D

Homo sapiens ribosomal protein, large, P0 (RPLP0)
14
23647.1
OS1D

mRNA

Homo sapiens T cell-specific tyrosine kinase mRNA
67
24655
OS1D

Homo sapiens tubulin, alpha, ubiquitous (K-ALPHA-
61
24642
OS1D

1)

HSU78095 Homo sapiens placental bikunin mRNA
18
23662.1
OS1D

Human BAC clone GS055K18 from 7p15-p21
11
23636.1
OS1D

Human insulin-like growth factor-binding protein-3
58
24636
OS1D

gene

Human mRNA for ribosomal protein
79
24687
OS1D

Human non-histone chromosomal protein HMG-14
62
24645
OS1D

mRNA

Human ribosomal protein L3 mRNA, 3′ end
59
24638
OS1D

Human TSC-22 protein mRNA
77
24679
OS1D

HUMGFIBPA Human growth hormone-dependent
12
23637.1
OS1D

insulin-like growth factor-binding protein

HUMMTA Homo sapiens mitochondrial DNA
17
23661.1
OS1D

HUMMTCG Human mitochondrion
21
23673.1
OS1D

HUMT1227HC Human mRNA for TI-227H
20
23669.1
OS1D

HUMTRPM2A Human TRPM-2 mRNA
15
23657.1
OS1D

Genomic sequence from Human 13
80
24689
OS1F

H. sapiens CpG island DNA genomic Mse1 fragment,
104
24719
OS1F

clone 84a5

H. sapiens RNA for snRNP protein B
110
24730
OS1F

Homo sapiens (clone L6) E-cadherin (CDH1) gene
108
24728
OS1F

Homo sapiens atrophin-1 interacting protein 4 (AIP4)
37
24350
OS1F

mRNA

Homo sapiens CGI-08 protein mRNA
102
24716
OS1F

Homo sapiens clone 24452 mRNA sequence
54
24374
OS1F

Homo sapiens clone IMAGE 286356
83
24693
OS1F

Homo sapiens cornichon protein mRNA
113
24735
OS1F

Homo sapiens hypothetical 43.2 Kd protein mRNA
87
24697
OS1F

Homo sapiens interleukin 1 receptor accessory
29
24338
OS1F

protein (IL1RAP) mRNA.

Homo sapiens K-Cl cotransporter KCC4 mRNA,
31
24340
OS1F

complete cds

Homo sapiens keratin 8 (KRT8) mRNA
115
24739
OS1F

Homo sapiens mRNA for DEPP (decidual protein
36
24349
OS1F

induced by progesterone)

Homo sapiens mRNA for KIAA0287 gene
101
24715
OS1F

Homo sapiens mRNA for KIAA0762 protein
118
24742
OS1F

Homo sapiens mRNA for zinc-finger DNA-binding
24
24333
OS1F

protein, complete cds

Homo sapiens mRNA; cDNA DKFZp434K114
112
24734
OS1F

Homo sapiens mRNA; cDNA DKFZp564E1962
25
24334
OS1F

(from clone DKFZp564E1962)

Homo sapiens nuclear chloride ion channel protein
34
24345
OS1F

(NCC27) mRNA

Homo sapiens ribosomal protein L13 (RPL13)
109
24729
OS1F

Homo sapiens senescence-associated epithelial
94
24706
OS1F

membrane protein (SEMP1)

Homo sapiens tumor protein, translationally-
26
24335
OS1F

controlled 1 (TPT1) mRNA.

Homo sapiens tumor suppressing subtransferable
51
24366
OS1F

candidate 1 (TSSC1)

Homo sapiens v-fos FBJ murine osteosarcoma viral
85
24695
OS1F

oncogene homolog(FOS) mRNA

Homo sapiens zinc finger protein slug (SLUG) gene
106
24722
OS1F

Human clone 23722 mRNA
105
24721
OS1F

Human clones 23667 and 23775 zinc finger protein
119
24744
OS1F

mRNA

Human collagenase type IV mRNA, 3′ end.
39
24352
OS1F

Human DNA sequence from PAC 29K1 on
116
24740
OS1F

chromosome 6p21.3-22.2.

Human ferritin H chain mRNA
96
24708
OS1F

Human heat shock protein 27 (HSPB1) gene exons
88
24698
OS1F

1-3

Human mRNA for KIAA0026 gene
30
24339
OS1F

Human mRNA for T-cell cyclophilin
40
24354
OS1F

Genomic sequence from Human 9q34, complete
140
25092
POTS2

sequence [Homo sapiens]

H. sapiens DNA for muscle nicotinic acetylcholine
3
21910
POTS2

receptor gene promotor, clone ICRFc105F02104

Homo sapiens breast cancer suppressor candidate 1
142
25098
POTS2

(bcsc-1) mRNA, complete cds

Homo sapiens CGI-151 protein mRNA, complete cds
8
21916
POTS2

Homo sapiens complement component 3 (C3) gene,
4
21913
POTS2

exons 1-30.

Homo sapiens mRNA for hepatocyte growth factor
159
25758
POTS2

activator inhibitor type 2, complete cds

Homo sapiens preferentially expressed antigen of
153
25745
POTS2

melanoma (PRAME) mRNA

Homo sapiens prepro dipeptidyl peptidase I (DPP-I)
152
25117
POTS2

gene, complete cds

Homo sapiens SKB1 ( S. cerevisiae ) homolog (SKB1)
147
25110
POTS2

mRNA.

Homo sapiens SWI/SNF related, matrix associated,
6
25914
POTS2

actin dependent regulator of chromatin, subfamily a,

member 4 (SMARCA4)

Human 12S RNA induced by poly(rI), poly(rC) and
155
25749
POTS2

Newcastle disease virus

Human ferritin Heavy subunit mRNA, complete cds.
7
21915
POTS2

Human glyceraldehyde-3-phosphate dehydrogenase
141
25093
POTS2

(GAPDH) mRNA, complete cds.

Human mRNA for fibronectin (FN precursor)
157
25755
POTS2

Human translocated t(8;14) c-myc (MYC) oncogene,
154
25746
POTS2

exon 3 and complete cds

H. sapiens vegf gene, 3′UTR
169
25799
POTS7

Homo sapiens 30S ribosomal protein S7 homolog
170
25802
POTS7

mRNA, complete cds

Homo sapiens acetyl-Coenzyme A acetyltransferase 2
172
25808
POTS7

(acetoacetyl Coenzyme A thiolase) (ACAT2) mRNA

Homo sapiens amyloid beta precursor protein-binding
138
24959
POTS7

protein I, 59kD (APPBP1) mRNA.

Homo sapiens arylacetamide deacetylase (esterase)
129
24942
POTS7

(AADAC) mRNA.

Homo sapiens clone 23942 alpha enolase mRNA,
165
25787
POTS7

partial cds

Homo sapiens echinoderm microtubule-associated
130
24943
POTS7

protein-like EMAP2 mRNA, complete cds

Homo sapiens IMP (inosine monophosphate)
164
25775
POTS7

dehydrogenase 2 (IMPDH2) mRNA

Homo sapiens megakaryocyte potentiating factor
126
24938
POTS7

(MPF) mRNA.

Homo sapiens mRNA for KIAA0552 protein,
163
25771
POTS7

complete cds

Homo sapiens Norrie disease protein (NDP) mRNA
173
25809
POTS7

Homo sapiens podocalyxin-like (PODXL) mRNA.
131
24944
POTS7

Homo sapiens synaptogyrin 2 (SYNGR2) mRNA.
135
24952
POTS7

Human aldose reductase mRNA, complete cds.
139
24969
POTS7

Human cyclooxygenase-1(PTSG1) mRNA, partial
124
24935
POTS7

cds

Human H19 RNA gene, complete cds.
122
24933
POTS7

Human mRNA for Apo1_Human (MER5(Aop1-
127
24939
POTS7

Mouse)-like protein), complete cds

Human triosephosphate isomerase mRNA, complete
123
24934
POTS7

cds.




































































































































































Still further ovarian carcinoma polynucleotide and/or polypeptide sequences identified from the above libaries are provided below in Table VII. Sequences O574S (SEQ ID NOs: 183&185), O584S (SEQ ID NO: 193) and O585S (SEQ ID NO: 194) represent novel sequences. The remaining sequences exhibited at least some homology with known genomic and/or EST sequences.







TABLE VII



SEQ ID:
Sequence
Library



174:
O565S_CRABP
OS1D

175:
O566S_Ceruloplasmin
POTS2

176:
O567S_41191.SEQ(1 > 487)
POTS2

177:
O568S_KIAA0762.seq(1 > 3999)
POTS7

178:
O569S_41220.seq(1 > 1069)
POTS7

179:
O570S_41215.seq(1 > 1817)
POTS2

180:
O571S_41213.seq(1 > 2382)
POTS2

181:
O572S_41208.seq(1 > 2377)
POTS2

182:
O573S_41177.seq(1 > 1370)
OS1F

183:
O574S_47807.seq(1 > 2060)
n/a

184:
O568S/VSGF DNA seq
n/a

185:
O5745_47807.seq(1 > 3000)
n/a

186:
O5685/VSGF protein seq
n/a

187:
449H1(57581)
OS1D

188:
451E12(57582)
OS1D

189:
453C7_3′(57583.1)Osteonectin
OS1D

190:
453C7_5′(57583.2)
OS1D

191:
456G1_3′(57584.1)Neurotensin
OS1F

192:
456G1_5′(57584.2)
OS1F

193:
O5845_465G5(57585)
OS1F

194:
O5855_469B12(57586)
POTS2

195:
O5695_474C3(57587)
POTS7

196:
483B1_3′(24934.1)Triosephosphate
POTS7

197:
57885 Human preferentially
POTS2


expressed antigen of melanoma

198:
57886 Chromosome 22q12.1 clone
POTS2


CTA-723E4

199:
57887 Homologous to mouse brain
POTS2


cDNA clone MNCb-0671













































Further studies on the clone of SEQ ID NO: 182 (also referred to as O573S) led to the identification of multiple open reading frames that encode the amino acid sequences of SEQ ID NO: 200-202.
Example 2
Analysis of cDNA Expression Using Microarray Technology
In additional studies, sequences disclosed herein were found to be overexpressed in specific tumor tissues as determined by microarray analysis. Using this approach, cDNA sequences are PCR amplified and their mRNA expression profiles in tumor and normal tissues are examined using cDNA microarray technology essentially as described (Shena et al., 1995). In brief, the clones are arrayed onto glass slides as multiple replicas, with each location corresponding to a unique cDNA clone (as many as 5500 clones can be arrayed on a single slide or chip). Each chip is hybridized with a pair of cDNA probes that are fluorescence-labeled with Cy3 and Cy5 respectively. Typically, 1 μg of polyA + RNA is used to generate each cDNA probe. After hybridization, the chips are scanned and the fluorescence intensity recorded for both Cy3 and Cy5 channels. There are multiple built-in quality control steps. First, the probe quality is monitored using a panel of ubiquitously expressed genes. Secondly, the control plate also can include yeast DNA fragments of which complementary RNA may be spiked into the probe synthesis for measuring the quality of the probe and the sensitivity of the analysis. Currently, the technology offers a sensitivity of 1 in 100,000 copies of mRNA. Finally, the reproducitility of this technology can be ensured by including duplicated control cDNA elements at different locations.
The microarray results for clones 57885 (SEQ ID NO:197), 57886 (SEQ ID NO:198) and 57887 (SEQ ID NO:199) are as follows.
Clone 57885: 16/38 (42%) of ovarian tumors showed an expression signal value of >0.4. The mean value for all ovary tumors was 0.662 with a mean value of 0.187 for all normal tissues, which yields a 3.64 fold overexpression level in ovary tumor relative to essential normal tissues. Normal tissue expression was elevated (>0.4) in peritoneum, skin and thymus.
Clone 57886: 16/38 (420/o) of ovarian tumors showed an expression signal value of >0.4. The mean value for all ovary tumors was 0.574 with a mean value of 0.166 for all normal tissues which yields a 3.46 fold overexpression level in ovary tumor relative to essential normal tissues. Normal tissue expression was elevated (>0.4) in heart, pancreas and small intestive.
Clone 57887: 17/38 (44%) of ovarian tumors showed an expression signal value of >0.4. The mean value for all ovary tumors is 0.744 with a mean value of 0.184 for all normal tissues which yields a 4.04 fold overexpression level in ovary tumor relative to essential normal tissues. Normal tissue expression was elevated (>0.4) in esophagus.
Example 3
Synthesis of Polypeptides
Polypeptides are synthesized on a Perkin Elmer/Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence is attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide. Cleavage of the peptides from the solid support is carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides are precipitated in cold methyl-t-butyl-ether. The peptide pellets are then dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) is used to elute the peptides. Following lyophilization of the pure fractions, the peptides are characterized using electrospray or other types of mass spectrometry and by amino acid analysis.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.




205


1
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


1
caacctcact agtaaatgaa agaaatattg taatttgtat ttgatctgct gggtctttgg 60
agtcagaact ggttttatca gcagtttgat cttctgaggt ctggtatgta gtttgctggc 120
ccacagaacc ttcacgtgta ttcacagcct caatgccata aggaaactct tttagaagtt 180
ctgacagctg gtcatgtagg tataagacag gtgccttatc actgtggatt tcatttcttg 240
caggatcttg gggagtatag ttgctggatg catctatttc ctgagggtaa atatcctcct 300
ggncgacgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 360
tgccttctan ttgccancca tntgttgttt gcccct 396


2
396
DNA
Homo sapien

2
cgaccaaaaa gtaaactcca agtgaacatc aaatcaaatc taatcctttt ggccacatga 60
ctggttgttc tttatctcat agttacaatg aatcatataa actgtagact gccactacca 120
cgatacttct gtgacacaga aggaatgtcc tatttgccta tctatctgag gaatgttaaa 180
tagagaaaaa tagattataa aacaacctgg aggtcacagg attctgagat aatccctctg 240
ttaaaaaaca tctgaacagc aaatgtccaa tctgtaataa aatagttaaa ggtccaagtc 300
aagtccactt ctacttggct ggcccagcac aagaaatcta acagcacttt gtaatcattt 360
tgcttttcta attttcccgg aggacatggg ccattg 396


3
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


3
cgcccttttt tttttttttt tnattggnnn aantcncttt nantnnaaaa acntgnangg 60
naancccann cccnnggnac cannnccagg agttgggtgg anactgagtg gggtttgtgt 120
gggtgagggg gcatctactc ctnttgcaac aagccaaaag tagaacagcc taaggaaaag 180
tgacctgcct tggagcctta gtccctccct tagggccccc tcagcctacc ctatccaagt 240
ctgaggctat ggaagtctcc ctcctagttc actagcaggt tccccatctt ttccaggctg 300
cccctagcac tccacgtttt tctgaaaaaa tctanacagg ccctttttgg gtacctaaaa 360
cccagctgag gttgtgagct tgtaaggtaa agcaag 396


4
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


4
gaccaatcct tgncncacta ncaaaangac cccnctnacc nccaggaact gaacctnnnt 60
gtnnacctcc nnctgcnnag ccntatntcc aanatcaccc accgtatcca ctgggaatct 120
gccagcctcc tgcgatcaga agagaccaat cgaaaatgag ggtttcacan tcacagctga 180
aggaaaaggc caaggcacct tgtcggnggn gacaatgtac catgctaagg ccaaagatca 240
actcacctgt aataaattcg acctcaaggt caccataaaa ccagcaccgg aacagaaaaa 300
gaggcctnag gatgcccaag aaacactttt gatcctttga aaactgtacc aaggtaccgg 360
ggggagaccc aggaaaggnc cnttatgtnt nnntnt 396


5
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


5
gacgccggag ctgccgcgcc agtcgcctag caggtcctct accggcttat tcctgtgccg 60
gatcttcatc ggcacagggg ccactgagac gtttctgcct ccctctttct tcctccgctc 120
tttctcttcc ctctngttta gtttgcctgg gagcttgaaa ggagaaagca cnggggtcgc 180
cccaaaccct ttctgcttct gcccatcaca agtgccacta ccgccatggg cctcactatc 240
tcctccctct tctcccgact atttggcaag aagcagatgc gcattttgat ggttggattg 300
gatgctgctg gcaagacaac cattcttgat aaactgaaag tanggganat aagnaccacc 360
atttctacca ttgggtttaa tgggggaaac agtana 396


6
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


6
acgggaggcg ccgggaagtc gacggcgccg gcggctcctg caggaggcca ctgtctgcag 60
ctcccgtgaa gatgtccact ccagacccac ccctgggcgg aactcctcgg ccaggtcctt 120
ccccgggccc tgcccttccc ctggagccat gctgggccct agcccgggtc cctcgccggg 180
ctccgcccac agcatgatgg ggcccagccc angggccgcc ctcagcagga caccccatcc 240
ccacccaggg gcctggaggg taccctcagg acaacatgca ccagatgcac aagcccatgg 300
agtccatgca tgagaagggc atgtcggacg acccgcgcta caaccagatg aaaggaatgg 360
ggatgcggtc agggggccat gctgggatgg ggcccc 396


7
396
DNA
Homo sapien

7
acccgagagt cgtcggggtt tcctgcttca acagtgcttg gacggaaccc ggcgctcgtt 60
ccccaccccg gccggccgcc catagccagc cctccgtcac ctcttcaccg caccctcgga 120
ctgccccaag gcccccgccg ccgctccagc gccgcgcagc caccgccgcc gccgccgcct 180
ctccttagtc gccgccatga cgaccgcgtc cacctcgcag gtgcgccaga actaccacca 240
ggactcagag gccgccatca accgccagat caacctggag ctctacgcct cctacgttta 300
cctgtccatg tcttactact ttgaccgcga tgatgtggct ttgaagaact ttgccaaata 360
ctttcttcac caatctcatg aggagaggga acatgc 396


8
396
DNA
Homo sapien

8
cgacaacaag gttaatacct tagttcttaa catttttttt ctttatgtgt agtgttttca 60
tgctaccttg gtaggaaact tatttacaaa ccatattaaa aggctaattt aaatataaat 120
aatataaagt gctctgaata aagcagaaat atattacagt tcattccaca gaaagcatcc 180
aaaccaccca aatgaccaag gcatatatag tatttggagg aatcaggggt ttggaaggag 240
tagggaggag aatgaaggaa aatgcaacca gcatgattat agtgtgttca tttagataaa 300
agtagaaggc acaggagagg tagcaaaggc caggcttttc tttggttttc ttcaaacata 360
ggtgaaaaaa acactgccat tcacaagtca aggaac 396


9
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


9
tcgacatcgc ggcaactttt tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc 60
agtgctacca gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg 120
tgaattgcac ggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg 180
ggatcatgta ccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt 240
accagtcctt ctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc 300
ctctttgtaa cgggccaagg nccaaaaaaa ggggaaagtt ctgncctcgg ccctcaggcc 360
agggctccgc accaccatcc tgttcctcaa attagc 396


10
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


10
cctttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt tttttttttt tttttttttt tttttttttt ttttaaaaaa aaaannnttt 120
tttttttttn aaaaaaangg gnnnnntttt ttncccnnnn gggngggggg ggggnnnnnt 180
ttnaaanaaa aaaaccnnaa annnnngggg nnnannnaan nncccncccc naancnntaa 240
aaaannnggn aaaanagggg gggnannnnn nnggggggna aaantttttt ttttttnaag 300
ggnnnggnaa aaaantnnnn nnnttttttt ttnnaanngg gnnaaaaaaa aaaaaaaaaa 360
attttttngg gntnaggggn ngggggaaaa ncccna 396


11
396
DNA
Homo sapien

11
agaacacagg tgtcgtgaaa actaccccta aaagccaaaa tgggaaagga aaagactcat 60
atcaacattg tcgtcattgg acacgtagat tcgggcaagt ccaccactac tggccatctg 120
atctataaat gcggtggcat cgacaaaaga accattgaaa aatttgagaa ggaggctgct 180
gagatgggaa agggctcctt caagtatgcc tgggtcttgg ataaactgaa agctgagcgt 240
gaacgtggta tcaccattga tatctccttg tggaaatttg agaccagcaa gtactatgtg 300
actatcattg atgccccagg acacagagac tttatcaaaa acatgattac agggacatct 360
caggctgact gtgctgtcct gattgttgct gctggt 396


12
396
DNA
Homo sapien

12
cgaaaacctt taaaccccgg tcatccggac atcccaacgc atgctcctgg agctcacagc 60
cttctgtggt gtcatttctg aaacaagggc gtggatccct caaccaagaa gaatgtttat 120
gtcttcaagt gacctgtact gcttggggac tattggagaa aataaggtgg agtcctactt 180
gtttaaaaaa tatgtatcta agaatgttct agggcactct gggaacctat aaaggcaggt 240
atttcgggcc ctcctcttca ggaatcttcc tgaagacatg gcccagtcga aggcccagga 300
tggcttttgc tgcggccccg tggggtagga gggacagaga gacagggaga gtcagcctcc 360
acattcagag gcatcacaag taatggcaca attctt 396


13
396
DNA
Homo sapien

13
accacaggct ggccacaaga agcgctggag tgtgctggcg gctgcaggcc tacggggcct 60
ggtccggctg ctgcacgtgc gtgccggctt ctgctgcggg gtcatccgag cccacaagaa 120
ggccatcgcc accctgtgct tcagccccgc ccacgagacc catctcttca cggcctccta 180
tgacaagcgg atcatcctct gggacatcgg ggtgcccaac caggactacg aattccaggc 240
cagccagctg ctcacactgg acaccacctc tatccccctg cgcctctgcc ctgtcgcctc 300
ctgcccggac gcccgcctgc tggccggctg cgagggcggc tgctgctgct gggacgtgcg 360
gctggaccag ccccaaaaga ggagggtgtg tgaagt 396


14
396
DNA
Homo sapien

14
acggcgtcct cgtggaagtg acatcgtctt taaaccctgc gtggcaatcc ctgacgcacc 60
gccgtgatgc ccagggaaga cagggcgacc tggaagtcca actacttcct taagatcatc 120
caactattgg atgattatcc gaaatgtttc attgtgggag cagacaatgt gggctccaag 180
cagatgcagc agatccgcat gtcccttcgc gggaaggctg tggtgctgat gggcaagaac 240
accatgatgc gcaaggccat ccgagggcac ctggaaaaca acccagctct ggagaaactg 300
ctgcctcata tccgggggaa tgtgggcttt gtgttcacca aggaggacct cactgagatc 360
agggacatgt tgctggccaa taaggtgcca gctgct 396


15
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


15
accgcgcggg cacagggtgc cgctgaccga ggcgtgcaaa gactccagaa ttggaggcat 60
gatgaagact ctgctgctgt ttgtggggct gctgctgacc tgggagagtg ggcaggtcct 120
gggggaccag acggtctcag acaatgagct ccaggaaatg tccaatcagg gaagtaagta 180
cgtcaataag gaaattcaaa atgcttgtca acggggtgaa acagataaag actctcatag 240
aaaaaacaaa cgaagagcgc aagacactgc tcagcaacct agaagaagcc aagaagaaga 300
aagaggatgc cctaaatgag accagggaat canagacaaa gctgaaggag ctcccaggag 360
tgtgcaatga gaccatgatg gccctctggg aagagt 396


16
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


16
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttngggggg 120
nnnaaanttt tttntnanan nnnngggnaa aaaaaaaaaa aanaangggg gnnntnnggc 180
ccnnnanaaa aaaanngnna annaancccc ccnnnnnnnc ccncnnntnn ggaaananna 240
aaaccccccc cngggnnggg nnaaaaannc ccnggggnan tttttatnnn annccccccc 300
ccnggggggg gnggaaaaaa aaaantnccc ccnannaaaa nnggggnccc cccnttttnc 360
aaaanggggg nccgggcccc ccnnantntt nggggg 396


17
396
DNA
Homo sapien

17
accacactaa ccatatacca atgatggcgc gatgtaacac gagaaagcac ataccaaggc 60
caccacacac cacctgtcca aaaaggcctt cgatacggga taatcctatt tattacctca 120
gaagtttttt tcttcgcagg atttttctga gccttttacc actccagcct agcccctacc 180
ccccaactag gagggcactg gcccccaaca ggcatcaccc cgctaaatcc cctagaagtc 240
ccactcctaa acacatccgt attactcgca tcaggagtat caatcacctg agctcaccat 300
agtctaatag aaaacaaccg aaaccaaata attcaagcac tgcttattac aattttactg 360
ggtctctatt ttaccctcct acaagcctca gagtac 396


18
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


18
tttttttttt tttttttttt tttttttttt tttttttttt ttttttttta ntcnaaaggg 60
gaaggnccct ttttattaaa nttggncatt ttactttnct tttttnaaaa ngctaanaaa 120
aaanttttnt ttntncttaa aaaaaccctn natntcacna ncaaaaaaaa cnattcccnc 180
ntncnttttg tgataaaaaa aaaggcaatg gaattcaacn tancctaana aaactttncc 240
tgggaggaaa aaaaattnnt ccgngggaaa cacttggggc tntccaaant gnanccatnc 300
tangaggacc ntctntaaga tttccaaang aaaccccttc ctnccaaang nantaccccg 360
ntgcctacnn cccataaaaa aaacctcanc cntaan 396


19
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


19
tttttttttt tttttttttt tttttttttt tttttttttt ttttttntgg tctgggcttt 60
tattttacna aaaanctaan ggnaaanntn cnttaaacta antngaanac aaagtnttaa 120
ngaaaaaggn ctgggggnnt cntttacaaa aanggncngg gncanntttg ggcttaaaan 180
ttcaaaaagg gnncntcaaa ngggtttgca tttgcatgtt tcancnctaa ancgnangaa 240
naaacccngg ngnccnctgg gaaaagttnt tnanctncca aaanatnaan tntttgnanc 300
agggnntttt tgggnaaaaa aannanttcc anaaactttc catcccctgg ntttgggttc 360
ggccttgngt tttcggnatn atntccntta angggg 396


20
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


20
tttttttttt tttttttttt ttttttctna acaaaccctg ttnttgggng ggngngggta 60
taatactaag ttganatgat ntcatttacg ggggaaggcn ctttgtgaan naggccttat 120
ttctnttgnc ctttcgtaca gggaggaatt tgaagtaaan anaaaccnac ctggattact 180
ccggtctgaa ctcaaatcac gtaggacttt aatcgttgaa caaacaaacc tttaatagcg 240
gctgcnccat tgggatgtcc tgatccaaca tcgaggncgt aaaccctatt gttgatatgg 300
actctaaaaa taggattgcg ctgttatccc tagggtaact tgttcccgtg gtcaaagtta 360
ttggatcaat tgagtataag tagttcgctt tgactg 396


21
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


21
acatanatnt tatactanca ttnaccatct cacttgnagg aanactanta tatcnctcac 60
acctnatatc ctncntacta tgcctagaag gaataatact atngctgttn attatancta 120
ctntnataac cctnaacacc cactccctct tanccaatat tgtgcctatt gccatactag 180
tntttgccgc ctgcnaagca gnggngggcc tanccntact agnctcaatc tccaacacnt 240
atggcctana ctacgtacat aacctaaacc tactcnaatg ctaaaactaa tcnncccaac 300
anttatntta ctaccactga catgactttc caaaaaacac atantttgaa tcaacncanc 360
cacccacanc ctanttatta ncatcatccc cntact 396


22
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


22
tttttttttt ttttganaaa agccggcata aagcactttt attgcaataa taaaacttga 60
gactcataaa tggtgctggg ggaagggtgc agcaacgatt tctcaccaaa tcactacaca 120
ggacagcaaa ggggtgagaa ggggctgagg gaggaaaagc caggaaactg agatcagcag 180
agggagccaa gcatcaaaaa acaggagatg ctgaagctgc gatgaccagc atcattttct 240
taanagaaca ttcaaggatt tgtcatgatg gctgggcttt cactgggtgt taagtctaca 300
aacagcacct tcaattgaaa ctgtcaatta aagttcttaa gatttaggaa gtggtggagc 360
ttggaaagtt atgagattac aaaattcctg aaagtc 396


23
396
DNA
Homo sapien

23
acaaaggcgg ttccaagcta aggaattcca tcagtgcttt tttcgcagcc accaaattta 60
gcaggcctgt gaggttttca tatcctgaag agatgtattt taaagctttt tttttttaat 120
gaaaaaatgt cagacacaca caaaagtaga atagtaccat ggagtcccca cgtacccagc 180
ctgcagcttc aacagttacc acatttgcca accggagaga ctgccaaggc aggaaaaagc 240
cctggaaagc ccacggcccc tttttccctt gggtcagagg ccttagagct ggctgccaaa 300
gcagccaacc aaaggggcag ctcagctcct tcgtggcacc agcagtgttc ctgatgcagt 360
tgaagagttg atgtctttga caacatacgg acactg 396


24
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


24
cgactatcct ctcagattct tatctggcac taatttataa ctattatatt atcagagact 60
atgtagcaat atatcagtgc acaggcgcat cccaggcctg tacagatgta tgtctacacg 120
taagtataaa tgaatttgca taccaggttt tacacttgca tctctaatag agattaaaaa 180
caacaaattg gcctcttcct aagtatatta atatcattta tccttacatt ttatgcctcc 240
ccctaaatta atgactgagt tggtggaaag cggctaggtt ttattcatac tgttttttgt 300
tctcaacttc aanagtaatc tacctctgaa aaatttntan tttaatattn nnnnnnagga 360
atttgngcca ctttannnct tncnntntnn tnnccn 396


25
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


25
tttttttttt tttttttttt gtcttttaaa aaatataaaa gtgttattat tttaaaacat 60
caagcattac agactgtaaa atcaattaan aactttctgt atatgaggac aaaaatacat 120
ttaanacata tacaanaaga tgctttttcc tgagtagaat gcaaactttt atattaagct 180
tctttgaatt ttcaaaatgt aaaataccaa ggctttttca catcagacaa aaatcaggaa 240
tgttcacctt cacatccaaa aagaaaaaaa aaaaaaancc aattttcaag ttgaagttna 300
ncaanaatga tgtaaaatct gaaaaaagtg gccaaaattt taanttncaa canannngnn 360
ncagntttna tggatctntn nnnnnncttc nnntnn 396


26
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


26
gacgctcccc cctccccccg agcgccgctc cggctgcacc gcgctcgctc cgagtttcag 60
gctcgtgcta agctagcgcc gtcgtcgtct cccttcagtc gccatcatga ttatctaccg 120
ggacctcatc agccacgatg agatgttctc cgacatctac aagatccggg agatcgcgga 180
cgggttgtgc ctggaggtgg aggggaagat ggtcagtagg acagaaggta acattgatga 240
ctcgctcatt ggtggaaatg cctccgctga aggccccgag ggcgaaggta cccgaaagca 300
cagtaatcac tgnngncnat nttgtcatga accatcacct gcnngaaaca annttnacaa 360
aanaancctn cnnnnannnc ctnnnnnatt ncnnnn 396


27
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


27
tttttttttt tttttttttt tttttttttt tttttttttt tggctaaant ttatgtatac 60
nggttnttca aangnggggg aggggggggg gcatccatnt anncncncca ggtttatggn 120
gggntnttnt actattanna nttttcnctt caaancnaag gnttntcaaa tcatnaaaat 180
tattaanatt ncngctgnta aaaaaangaa tgaaccnncn nanganagga nntttcatgg 240
ggggnatgca tcggggnann ccnaanaacc ncggggccat tcccganagg cccaaaaaat 300
gtttnnnnaa aaagggtaaa nttacccccn tnaantttat annnnaaann nnannnnagc 360
ccaannnttn nnnnnnnnnn nnnccnnnna nnnnnn 396


28
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


28
cgaccttttt tttttttttt atagatgaaa gagggtttat ttattaatat atgatagcct 60
tggctcaaaa aagacaaatg agggctcaaa aaggaattac agtaacttta aaaaatatat 120
taaacatatc caagatccta aatatattat tctccccaaa agctagctgc ttccaaactt 180
gatttgatat tttgcatgtt ttccctacgt tgcttggtaa atatatttgc ttctcctttc 240
tgcaatcgac gtctgacagc tgatttttgc tgttttgnca acntgacgtt tcaccttntg 300
tttcaccant tctggaggaa ttgttnaaca ncttacanca ctgccttgaa naaannnnan 360
gcctcaaaag ntcttgnnct atnctnnttc ntnnnt 396


29
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


29
gacttgctca tttagagttt gcaggaggct ccatactagg ttcagtctga aagaaatctc 60
ctaatggtgc tatagagagg gaggtaacag aaagactctt ttagggcatt tttctgactc 120
atgaaaagag cacagaaaag gatgtttggc aatttgtctt ttaagtctta accttgctaa 180
tgtgaatact gggaaagtga tttttttctc actcgttttt gttgctccat tgtaaagggc 240
ggaggtcagt cttagtggcc ttgagagttg cttttggcat ttaaatattc taagagaatt 300
aactgtattt cctgtcacct attcactant gcangaaata tacttgctcc aaataagtca 360
ntatgagaag tcactgtcaa tgaaanttgn tttgtt 396


30
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


30
tttttttttt tttttttttg aaatttanaa acaaatttta tttaagatct gaaatacaat 60
tcctaaaata tcaacttttc canaaaaccg tggctacaca ataatgcatt gcctctatca 120
tgttanaacg tgcattanac tcaaatacaa aaaccatgaa acaaatcacc atccttcaac 180
aatttgagca aagatagaat gcctaagaac aacatagatg gacttgcaga ggatgggctg 240
ttttacttca agcnccataa aaaaaaaaaa gagcncaaat gcattgggtt ttcaggtnta 300
tacattaagn ngaacctttg gcactaggaa tcagggcgtt ttgtcacata gcnttaacac 360
atnttaaaaa attntgtant gtcaaaggga tangaa 396


31
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


31
gacgggccag ggccatctgg aaagggaact cggcttttcc agaacgtggt ggatcatctg 60
tcgggtgtgt ggtgaacacg ttcagttcat cagggcctac gctccgggaa ggggccccca 120
gctgtggctc tgccatgccg ggctgtgttt gcagctgtcc gagtctccat ccgcctttag 180
aaaaccagcc acttcttttc ataagcactg acagggccca gcccacagcc acaggtgcga 240
tcagtgcctc acgcaggcaa atgcactgaa acccaggggc acacncncgc agagtgaaca 300
gtgagttccc ccgacagccc acgacagcca ggactgccct ccccaccccn ccccgacccc 360
angancacgg cacacanntc ancctctnan ctngct 396


32
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


32
cgactggcct cataccttgt ctacacagtc cctgcacagg gttcctaacc tgtggttagt 60
aaagaatgtc actttctaac aggtctggaa gctccgagtt tatcttggga actcaagagg 120
agaggatcac ccagttcaca ggtatttgag gatacaaacc cattgctggg ctcggcttta 180
aaagtcttat ctgaaattcc ttgtgaaaca gagtttcatc aaagccaatc caaaaggcct 240
atgtaaaaat aaccattctt gctgcacttt atgcaaataa tcaggccaaa tataagacta 300
cagtttattt acaatttgtt tttaccaaaa atgaggacta nagagaaaaa tggtgctcca 360
aagcttatca tacatttgtc attaagtcct agtctc 396


33
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


33
cctttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120
nngnnntntn nnnnannaaa aaaaaaaaaa aannnnnnna aaaaaaannn nnnnnnnnnt 180
tttnnggggg gnttttnann gnannttnnn nttnnnnnaa anccccnnng ggnngggggg 240
nntnnnnnng gnaaaaaaan nnnnnggggn cnnnngggnc cncncccnan nnnnaaaann 300
nnnggntttt ttnnttttna aaaaaanngn nnnnnaacaa aantttttnn nnaanttttn 360
gggggaaann ncccntttnt ttttttnnan nnnnnn 396


34
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


34
acggaccnag ctggaggagc tgggtgtggg gtgcgttggg ctggtgggga ggcctagttn 60
gggtgcaagt angtctgatt gagcttgtgt tgtgctgaag ggacagccct gggtctaggg 120
ganagagncc ctgagtgtga gacccacctt ccccngtccc agcccctccc anttccccca 180
gggacggcca cttcctgntc cccgacncaa ccatggctga agaacaaccg caggtcgaat 240
tgttcntgaa ggctggcagt gatggggcca agattgggaa ctgcccattc tcccacagac 300
tgttnatggt actgtggctc aaggnagtca ccttcaatgt taccaccnnt gacaccaaaa 360
ggcggaccna nacagtgcan aagctgtgcc canngg 396


35
396
DNA
Homo sapien

35
tcgaccaaaa tcaaatctgg cactcacaag ccctggccga cccccaatgg gttttaccac 60
tccccctcta gaccctgtct tgcaaaatcc tctccctagc cagctagtat tttctgggct 120
aaagactgta caaccagttc ctccatttta tagaagttta ctcactccag gggaaatggt 180
gagtcctcca acctcccttt caaccagtcc catcattcca accagtggta ccatagagca 240
gcaccccccg ccaccctctg agccagtagt gccagcagtg atgatggcca cccatgagcc 300
cagtgctgac ctggcaccca agaaaaagcc caggaagtca agcatgcctg tgaagattga 360
gaaggaaatt attgataccg ccgatgagtt tgatga 396


36
396
DNA
Homo sapien

36
tcgacgggaa gagcctgcta cggtggactg tgagactcag tgcactgtcc tcctcccagc 60
gaccccacgc tggaccccct gccggaccct ccacccttcg gcccccaagc ttcccagggg 120
cttcctttgg actggactgt ccctgctcat ccattctcct gccaccccca gacctcctca 180
gctccaggtt gccacctcct ctcgccagag tgatgaggtc ccggcttctg ctctccgtgg 240
cccatctgcc cacaattcgg gagaccacgg aggagatgct gcttgggggt cctggacagg 300
agcccccacc ctctcctagc ctggatgact acgtgaggtc tatatctcga ctggcacagc 360
ccacctctgt gctggacaag gccacggccc agggcc 396


37
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


37
cgacggtgtc agcaactggc catgccacag cacataaaga ttacagtgac aagaaaaaca 60
ttgtttgagg attcctttca acagataatg agcttcagtc cccaagatct gcgaagacgt 120
ttgtgggtga tttttccagg agaagaaggt ttagattatg gaggtgtagc aagagaatgg 180
ttctttcttt tgtcacatga agtgttgaac ccaatgtatt gcctgtttga atatgcaggg 240
aaggataact actgcttgca gataaacccc gcttcttaca tcaatccaga tcacctgaaa 300
tattttcgtt ttattggcag atttattgcc atggctctgt tccatgggaa aattcataga 360
cacgggtttt tctttnccat tctataagcg tatctt 396


38
396
DNA
Homo sapien

38
cgaccaaaat gataaatagc tttaagaatg tgctaatgat aaatgattac atgtcaattt 60
aatgtactta atgtttaata ccttatttga ataattacct gaagaatata ttttttagta 120
ctgcatttca ttgattctaa gttgcacttt ttacccccat actgttaaca tatctgaaat 180
cagaatgtgt cttacaatca gtgatcgttt aacattgtga caaagtttaa tggacagttt 240
tttcccatat gtatatataa aataatgtgt tttacaatca gtggcttaga ttcagtgaaa 300
tacagtaatt cattcaatta tgatagtatc tttacagaca ttttaaaaat aagttatttt 360
tatatgctaa tattctatgt tcaagtggaa tttgga 396


39
396
DNA
Homo sapien

39
tcgaccaaga atagatgctg actgtactcc tcccaggcgc cccttccccc tccaatccca 60
ccaaccctca gagccacccc taaagagata ctttgatatt ttcaacgcag ccctgctttg 120
ggctgccctg gtgctgccac acttcaggct cttctccttt cacaaccttc tgtggctcac 180
agaacccttg gagccaatgg agactgtctc aagagggcac tggtggcccg acagcctggc 240
acagggcaag tgggacaggg catggccagg tggccactcc agacccctgg cttttcactg 300
ctggctgcct tagaaccttt cttacattag cagtttgctt tgtatgcact ttgttttttt 360
ctttgggtct tgtttttttt ttccacttag aaattg 396


40
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


40
tttttttttt ttttgttatt tagtttttat ttcataatca taaacttaac tctgcaatcc 60
agctaggcat gggagggaac aaggaaaaca tggaacccaa agggaactgc agcgagagca 120
caaagattct aggatactgc gagcaaatgg ggtggagggg tgctctcctg agctacagaa 180
ggaatgatct ggtggttaan ataaaacaca agtcaaactt attcgagttg tccacagtca 240
gcaatggtga tcttcttgct ggtcttgcca ttcctggacc caaagcgctc catggcctcc 300
acaatattca tgccttcttt cactttgcca aacaccacat gcttgccatc caaccactca 360
gtcttggcag tgcanatgaa aaactgggaa ccattt 396


41
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


41
tcgacctctt gtgtagtcac ttctgattct gacaatcaat caatcaatgg cctagagcac 60
tgactgttaa cacaaacgtc actagcaaag tagcaacagc tttaagtcta aatacaaagc 120
tgttctgtgt gagaattttt taaaaggcta cttgtataat aacccttgtc atttttaatg 180
tacaaaacgc tattaagtgg cttagaattt gaacatttgt ggtctttatt tactttgctt 240
cgtgtgtggg caaagcaaca tcttccctaa atatatatta cccaaagnaa aagcaagaag 300
ccagattagg tttttgacaa aacaaacagg ccaaaagggg gctgacctgg agcagagcat 360
ggtgagaggc aaggcatgag agggcaagtt tgttgt 396


42
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


42
cttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
aaaanccnna nnaananang gnaannnann aaaaaannca aaccncntnt anaaaangcc 120
nntntnaggg ggggggttca aaaccaaang gnngntngga ngnaaannna aaanttnnnn 180
gggggnanaa anaaaaaggg nngaaanntg acccnanaan gaccngaaan cccgggaaac 240
cnngggntan aaaaaaagnt ganccctaaa nncccccgna aaanggggga agggnaannc 300
caaatccnnt gngggttggg ggnggggaaa aaaaaaaccc cnaaaaantg naaaaaaccg 360
ggnttnaaan atttgggttc gggggntttn tnttaa 396


43
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


43
tttttttttt ttttgcttca ctgctttatt tttgaaatca caagcaattc aaagtgatca 60
tcattgaggc ttctgttaaa agttcttcca aagttgccca gttttaanat taaacaatat 120
tgcactttaa gatgaactaa cttttgggat tctcttcaaa gaaggaaagt attgctccat 180
ctgtgctttt cttanactaa aagcatactg canaaaactc tattttaaaa atcaacactg 240
cagggtacag taacatagta aagtacctgc ctattttana atcctanaga acatttcatt 300
gtaagaaact agcccattat ttaagtgtcc acagtatttt tcatttcant ggtccaagat 360
gccaaggttt ccaaacacaa tcttgttctc taatac 396


44
396
DNA
Homo sapien

44
gacctagttt tacctcttaa atatctctgt tcccttctaa gttgtttgct gtgttttctt 60
cagagcaaga aggttatatt ttttaaaatt tacttagtaa tgcacattca aaacacacat 120
caagtcttca ggataaagtt caaaaccgct gtcatggccc catgtgatct ctccctcccc 180
tacccctcta tcatttagtt tcttctgcgc aagccactct ggcttccttt cagttttgtg 240
gttcccgttt ttagctagtt cagtggtttt caatgggcat ttcttgcctt tttttttcta 300
aacgacaaat agaaatacat cttctttatt atcctccaaa tccaattcag aggtaatatg 360
ctccacctac acacaatttt agaaataaat taaaaa 396


45
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


45
tttttttttt ttttaaannt tntaaatttt taatgaaann ganttagaac aatgtattat 60
tnacatgtaa ataaaaaaag agancataan ccccatatnc tcnnnaaagg aaggganacn 120
gcnggccntt tatnagaana nnnnncatat aagaccccat taagaagaat ctggatctaa 180
anacttncaa acaggagttc acagtangtg aacagcannc cctaatccca ctgatgtgat 240
gnttcanata aaatcancan cgntgatcgg gnatcnnanc aatntgancg gaanannact 300
gctcnatatn tttnaggann cngatgtggt cattttttac aaagataatg gccacaccct 360
tccngnccga atcgancnga nctcccnntt ctgtgn 396


46
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


46
tttttttttt tttttttttc tganacagag tctcattctg ttgcctaggc tggattgcag 60
tggtgccatc tcggctcact gcaacctccg cctcctgggt tccanaaatt ctcctgcctc 120
agcctcccgg gtagctggga ctanaggcac acgccaccac gccaggctaa tttttatatt 180
tttagtanan atggcgtttc accatgttga ccanactgat ctcgaactcc cgacctcgtg 240
atccacccac ctcggcctcc caaagtgctg ggattacagg cgtgaaacca ccaggcccgg 300
cctgaaatat ctatttnttt tcagattatt tttaaaattc catttgatga atcttttaaa 360
gtgagctana naaagtgngt gtgtacatgc acacac 396


47
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


47
tttttttttt tttttttgct gttgccaact gtttattcag ggccctgaac gggtggtgcg 60
tggacatgca acacactcgg gcccacagca gcgtgaccgg ccgctcccaa gccccgggcg 120
cacaaccaca gccaggagca gcccctgcca ccactgggcc accgtccagg gccccacagg 180
accagccgaa ggtgccccgg gccgaggcca gctgggtcag gtgtacccct agcctggggt 240
tgagtgagga gcggcacccc cagtatcctg tgtaccccaa gttgcccagn aggccgaggg 300
ggccttgggc tccatctgca ctggccaccc cgtgccaagc atcacagctg cgtgagcagg 360
tttgtgtgtg agcgtgtggc ggggcctggt tgtccc 396


48
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


48
ctgggcctgt gccgaagggt ctgggcagat cttccaaaga tgtacaaaat gtagaaattg 60
ccctcaagca aatgcaaaga tgctcaacac ccttagtcat caagaaaatg caaatggaat 120
ccacagagag atactgcaca ctgacaaaga tggtcgtatt actaaaggtg aataaccagc 180
gcggggggca cgtggagtca ctggaacatt tgtgcaatgc tggtgggaat gtcaacccgt 240
gcggccctct ggaataagcc tggcagctcc tccaagagtt acccgtgtga cccagcaatt 300
ccactcctag ctccacccac aggaattgaa agcaaagacg caaacagatg cctgtgcacc 360
aaagttcacg gcagcatcct tcgccatagt ggnaan 396


49
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


49
accccaaaat gggaaaggaa aagactcata tnaacattgn cgtnattgga cacgtacatt 60
cggncaagtn caccactact ggncatntga tntataaatg cggnggcatc gacanaanaa 120
ccatngnaan atttganaag gaggctgctg atatnggaaa gggctccntc nantntgcct 180
gggtcttgga tnaactgaaa nctgancntg aacgtggnnt caccattgat atctncttgt 240
ggaaatntna gaccancann tactatgtna ctatcattga tgccccagga cacaganact 300
ttatcnaaan catgattacn nggacatnta nagctgactg tgctngcctg attgtngctg 360
ctggtgttgg tgaatttgaa nctggtatnt ccaana 396


50
396
DNA
Homo sapien

50
cgacttcttg ctggtgggtg gggcagtttg gtttagtgtt atactttggt ctaagtattt 60
gagttaaact gcttttttgc taatgagtgg gctggttgtt agcaggtttg tttttcctgc 120
tgttgattgt tactagtggc attaactttt agaatttggg ctggtgagat taattttttt 180
taatatccca gctagagata tggcctttaa ctgacctaaa gaggtgtgtt gtgatttaat 240
tttttcccgt tcctttttct tcagtaaacc caacaatagt ctaaccttaa aaattgagtt 300
gatgtcctta taggtcacta cccctaaata aacctgaagc aggtgttttc tcttggacat 360
actaaaaaat acctaaaagg aagcttagat gggctg 396


51
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


51
tttttttttt ttcagcgngg atttatttta tttcattttt tactctcaag anaaagaana 60
gttactattg caggaacaga cattttttta aaaagcgaaa ctcctgacac ccttaaaaca 120
gaaaacattg ttattcacat aataatgngg ggctctgtct ctgccgacag gggctgggtt 180
cgggcattag ctgtgccgtc gacaatagcc ccattcaccc cattcataaa tgctgctgct 240
acaggaaggg aacagcggct ctcccanaga gggatccacc ctggaacacg agtcacctcc 300
aaagagctgc gactgtttga naatctgcca anaggaaaac cactcaatgg gacctggata 360
acccaggccc gggagtcata gcaggatgtg gtactt 396


52
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


52
acctcgctaa gtgttcgcta cgcggggcta ccggatcggt cggaaatggc agaggtggag 60
gagacactga agcgactgca nagccagaag ggagtgcagg gaatcatcgt cgtgaacaca 120
gaaggcattc ccatcaagag caccatggac aaccccacca ccacccagta tgccagcctc 180
atgcacagnt tcatcctgaa ggcacggagc accgtgcgtg acatcgaccc ccagaacgat 240
ctcaccttcc ttcgaattcg ctccaagaaa aatgaaatta tggttgcacc agataaagac 300
tatttcctga ttgtgattca gaatccaacc gaataagcca ctctcttggc tccctgtgtc 360
attccttaat ttaatgcccc ccaagaatgt taatgt 396


53
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


53
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt ttannttntt ttttnttttn 240
cctttntttt aattcanaaa aagaanaaga aaanataana nnnancnnan nnnnnnnatn 300
ntncttnata ntnnttnnnn nanngggnnn gcgagnnnnn nnnnnnnnnn nntctnnnnt 360
tnnnnnnctt gcnccccttn nnttngnnnn angcaa 396


54
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


54
ctcttggggc tgctgggact cgcgtcggtt ggcgactccc ggacgtaggt agtttgttgg 60
gccgggttct gaggccttgc ttctctttac ttttccactc taggccacga tgccgcagta 120
ccagacctgg gaggagttca gccgcgctgc cgagaagctt tacctcgctg accctatgaa 180
ggcacgtgtg gttctcaaat ataggcattc tgatgggaac ttgtgtgtta aagtaacaga 240
tgatttagtt tgtttggtgt ataaaacaga ccaagctcaa gatgtaaaga agattgagaa 300
attccacagt caactaatgc gacttatggt agccaaggaa gcccgcaatg ttaccatgga 360
aactgantga atggtttgaa atgaagactt tgtcgt 396


55
396
DNA
Homo sapien

55
cgacggtttg ccgccagaac acaggtgtcg tgaaaactac ccctaaaagc caaaatggga 60
aaggaaaaga ctcatatcaa cattgtcgtc attggacacg tagattcggg caagtccacc 120
actactggcc atctgatcta taaatgcggt ggcatcgaca aaagaaccat tgaaaaattt 180
gagaaggagg ctgctgagat gggaaagggc tccttcaagt atgcctgggt cttggataaa 240
ctgaaagctg agcgtgaacg tggtatcacc attgatatct ccttgtggaa atttgagacc 300
agcaagtact atgtgactat cattgatgcc ccaggacaca gagactttat caaaaacatg 360
attacaggga catctcaggc tgactgtgct gtcctg 396


56
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


56
tttttttttt ttttttctca tttaactttt ttaatgggtc tcaaaattct gtgacaaatt 60
tttggtcaag ttgtttccat taaaaagtac tgattttaaa aactaataac ttaaaactgc 120
cacacgcaaa aaanaaaacc aaagnggtcc acaaaacatt ctcctttcct tctgaaggtt 180
ttacgatgca ttgttatcat taaccagtct tttactacta aacttaaatg gccaattgaa 240
acaaacagtt ctganaccgt tcttccacca ctgattaana gtggggtggc aggtattagg 300
gataatattc atttagcctt ctgagctttc tgggcanact tggngacctt gccagctcca 360
gcagccttnt tgtccactgc tttgatgaca cccacc 396


57
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


57
cctttttttt tttttttttt tttttttttt tttttttttt tttttttttt tnaaaanntt 60
ntttttgcaa anccnancaa aaanggnngg aangaaaaan nggaaaaatt ntttttncnt 120
ntttgggaac nnnnagccct tnntttgaaa aaangnggnc ttaaaanngn tgaannaaag 180
gnnanncccn gntncttnnn tttaaaaana anggggnngn ttttttttaa anaanatttt 240
ttttttccct aanancnncn anntgaaacn ngncccnacn nctnncttna aagggnnnaa 300
atnanangnn aaaaaanccc tnancccccc cccttanntt tncnannana naaagncntt 360
ttgggncntg naaaaanaan cctttttnnt gcnttn 396


58
396
DNA
Homo sapien

58
cgacctcaaa tatgccttat tttgcacaaa agactgccaa ggacatgacc agcagctggc 60
tacagcctcg atttatattt ctgtttgtgg tgaactgatt ttttttaaac caaagtttag 120
aaagaggttt ttgaaatgcc tatggtttct ttgaatggta aacttgagca tcttttcact 180
ttccagtagt cagcaaagag cagtttgaat tttcttgtcg cttcctatca aaatattcag 240
agactcgagc acagcaccca gacttcatgc gcccgtggaa tgctcaccac atgttggtcg 300
aagcggccga ccactgactt tgtgacttag gcggctgtgt tgcctatgta gagaacacgc 360
ttcaccccca ctccccgtac agtgcgcaca ggcttt 396


59
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


59
cttttttttt tttttttttt tcagnggaaa ataactttta ttganacccc accaactgca 60
aaatctgttc ctggcattaa gctccttctt cctttgcaat tcggtctttc ttcagnggtc 120
ccatgaatgc tttcttctcc tccatggtct ggaagcggcc atggccaaac ttggaggngg 180
tgtcaatgaa cttaaggnca atcttctcca nagcccgccg cttcntctgc accancaagg 240
acttgcggag ggngagcacc cgcttnttgg ttcccaccac ncagcctttc agcatgacaa 300
agtcattggt cacttcacca tagnggacaa agccacccaa agggttgatg ctccttggca 360
aataggncat agtcacngga ggcattgtnc ttgatc 396


60
396
DNA
Homo sapien

60
acctcagctc tcggcgcacg gcccagcttc cttcaaaatg tctactgttc acgaaatcct 60
gtgcaagctc agcttggagg gtgatcactc tacaccccca agtgcatatg ggtctgtcaa 120
agcctatact aactttgatg ctgagcggga tgctttgaac attgaaacag ccatcaagac 180
caaaggtgtg gatgaggtca ccattgtcaa cattttgacc aaccgcagca atgcacagag 240
acaggatatt gccttcgcct accagagaag gaccaaaaag gaacttgcat cagcactgaa 300
gtcagcctta tctggccacc tggagacggt gattttgggc ctattgaaga cacctgctca 360
gtatgacgct tctgagctaa aagcttccat gaaggg 396


61
396
DNA
Homo sapien

61
tagcttgtcg gggacggtaa ccgggacccg gtgtctgctc ctgtcgcctt cgcctcctaa 60
tccctagcca ctatgcgtga gtgcatctcc atccacgttg gccaggctgg tgtccagatt 120
ggcaatgcct gctgggagct ctactgcctg gaacacggca tccagcccga tggccagatg 180
ccaagtgaca agaccattgg gggaggagat gactccttca acaccttctt cagtgagacg 240
ggcgctggca agcacgtgcc ccgggctgtg tttgtagact tggaacccac agtcattgat 300
gaagttcgca ctggcaccta ccgccagctc ttccaccctg agcagctcat cacaggcaag 360
gaagatgctg ccaataacta tgcccgaggg cactac 396


62
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


62
tcgacgtttc ctaaagaaaa ccactctttg atcatggctc tctctgccag aattgtgtgc 60
actctgtaac atctttgtgg tagtcctgtt ttcctaataa ctttgttact gtgctgtgaa 120
agattacaga tttgaacatg tagtgtacgt gctgttgagt tgtgaactgg tgggccgtat 180
gtaacagctg accaacgtga agatactggt acttgatagc ctcttaagga aaatttgctt 240
ccaaatttta agctggaaag ncactggant aactttaaaa aagaattaca atacatggct 300
ttttagaatt tcnttacgta tgttaagatt tgngtacaaa ttgaantgtc tgtnctganc 360
ctcaaccaat aaaatctcag tttatgaaan aaannn 396


63
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


63
ttnttttttt nttttntntt ttntcnttgn ttgnacngaa cccggcgctn nttccccacn 60
nnnnacggcc gcccntattc annnntncnt canntannna ccgcaccctc ggactgcnnn 120
tngggccccg ccgncnannc nccnncnccc anttcnccgc cgccgccgcc gccttttttt 180
attggcnncc atnanaaccg gggncacctc ncangngcgc cnaaantngg ggcangactc 240
anagggggcc atcaaccncc aagnncaanc tgganctcta caaacggcct acgntttntg 300
nccatgnggg tagggnttta cccgcnatga tgannatgnn aanaactttn ncaanccctt 360
tattaaccaa tgnggtgngg agacggaacn tggtta 396


64
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


64
tcgacgtcgg ggtttcctgc ttcaacagtg cttggacgga acccggcgct cgttccccac 60
cccggccggc cgcccatagc cagccctccg tcacctcttc accgcaccct cggactgccc 120
caaggccccc gccgccgctc cagcgccgcg cagccaccgc cgccgccgcc gcctntnctt 180
agtcgccgcc atgacgaccg cgtccacctc gcaggtgcgc cagaactacc accaggactc 240
agaggccgcc atcaaccgcc agatcaacct ggagctctac gcctcctacg tttacctgtc 300
catgtcttac tactttgacc gcgatgatgt ggctttgaan aactttgcca aatactttct 360
tcccaatctc atgaggagaa ggaacatgct ganaaa 396


65
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


65
tttttttttt tttttttttt tttttnacca ataatgcttt tattttccac atcaanatta 60
atttatatgt tagttttagt acaagtacta aaatgtatac ttnttgccct aatagctaag 120
gnatacataa gcttcaccat acatnttgca nccncctgtc tgtcctatgt cattgttata 180
aatgtanana ttttaggaaa ctnttttatt caacctggga catntatact gtaggagtta 240
gcactgacct gatgtnttat ttaaaagtaa tgnatattac ctttacatat attccttata 300
tattnaaacg tatttccatg ttatccagct taaaatcaca tggnggttaa aagcatgagt 360
tctgagtcaa atctggactg aaatcctgat gctccc 396


66
396
DNA
Homo sapien

66
tcgacttttt tttttccagg acattgtcat aattttttat tatgtatcaa attgtcttca 60
atataagtta caacttgatt aaagttgata gacatttgta tctatttaaa gacaaaaaaa 120
ttcttttatg tacaatatct tgtctagagt ctagcaaata tagtaccttt cattgcagga 180
tttctgctta atataacaag caaaaacaaa caactgaaaa aatataaacc aaagcaaacc 240
aaaccccccg ctcaactaca aatgtcaata ttgaatgaag cattaaaaga caaacataaa 300
gtaacttcag cttttatcta gcaatgcaga atgaatacta aaattagtgg caaaaaaaca 360
aacaacaaac aacaaacaaa acaaaacaaa caaaca 396


67
396
DNA
Homo sapien

67
acgcttttgt ccttcatttt aactgttatg tcatactgtt atgttgacat atttctttat 60
aagagaatag aggcaaaagt atagaactga ggatcatttg tatttttgag ttggaaatta 120
tgaaacttca ccatattatg atcatacata ttttgaagaa cagactgacc aaagctcacc 180
tgttttttgt gttaggtgct ttggctgaac ttgattccag cccccttttc cctttggtgt 240
tgtgtatgtc tcttcatttc ctctcaaatc ttcaactctt gccccatgtc tccttggcag 300
caggatgctg gcatctgtgt agtcctcata ctgtttactg ataacccaca aattcatttt 360
catggcagac ctaagctcag accctgcctt gtcctg 396


68
396
DNA
Homo sapien

68
acctgagtcc tgtcctttct ctctccccgg acagcatgag cttcaccact cgctccacct 60
tctccaccaa ctaccggtcc ctgggctctg tccaggcgcc cagctacggc gcccggccgg 120
tcagcagcgc ggccagcgtc tatgcaggcg ctgggggctc tggttcccgg atctccgtgt 180
cccgctccac cagcttcagg ggcggcatgg ggtccggggg cctggccacc gggatagccg 240
ggggtctggc aggaatggga ggcatccaga acgagaagga gaccatgcaa agcctgaacg 300
accgcctggc ctcttacctg gacagagtga ggagcctgga gaccgagaac cggaggctgg 360
agagcaaaat ccgggagcac ttggagaaga agggac 396


69
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


69
ntcncngnng ntgtggtnnt ttttttaatt tttatntttt cttttttttt ctngctagcn 60
cttncttttt ttggaattnc ggtncctttt tntntcnatt ttttngacaa aaanaacctn 120
ttntttnana ccanagnnng gnncacncnt nnaatntncc ccttttncgn tngggagctn 180
cncnttnnnc gccnacntca ntcgagacng tncttttnnn tnnancannn tnngtncgtt 240
gncngcnttn ntncannant nttccctatn nacntgnnnt cncncatnnt tggacnancn 300
cctagccttn ccatnntttn nttntttntn natnancctn gaaaacntcn gnntnttcnc 360
nncnttnccn cncncncctt cntatgtncn atgncn 396


70
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


70
tttttttttt ttttnttttt tttttttttt tttttttntt tttttttttt ttttttntnc 60
aannnntnaa cttttaanng gccnccngcn ccccaanggg gaccctgctt ttgnnggcta 120
aatgccnnaa aactttgggg nantnggtat naaaccccnc tttgcccnnc annttncngg 180
gggggggggg tttttgnngg ggaacangna naacnttttn ncnanggnat caccaaaaan 240
aaagcccnnc cctttttccn annggggggg ggngggggga aantcanccc ccanattgac 300
cttnatttca aaanggggct tataatcctg ggcntggann cttccctnta cccgggggtt 360
gnccacnttt tattanaggg gnangnggat ccccnt 396


71
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


71
gcatctagag ggccngttta ntctagaggn ccngnntaaa cnnnnncatc nacctncnnt 60
gcncctgctn gttgccnccc ntctgtgnct tgcnnnnccc nngagcgtnc cttnaccnnn 120
gaangtgcct nnnnnactga nnnnnncnna taanatgngg anantncgtc gncattntnt 180
natnnggggt gatgctattc tggggggtgg ggnggngnna tnnnatactn nggggacgtn 240
nnatnangag nnatntcnng nttntctnnt gntttntggg gggcnatnng nnntctntnn 300
ggactcntcg cncannnatc aatancttna ttcngtgtan ngtccgnccn tagnncngcn 360
ngtactnnan ngttgnnntc attactnttc gtnngg 396


72
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


72
tntttttttt tttctaaaac atnactnttt attnnnnang ntttntgaac ctctnngcnt 60
natggtgaga gtttgtctga ttaataanaa tnggannntt nannanangc ntgnncgcaa 120
ngatggcnnc nctgtatatc ccaccatccc attacactnt gaaccttttn tttgattaat 180
aaaaggaagg natgcgggga anggggaaag agaatgcttg aacattncca tgngnccttn 240
gacaaacttt ccaatggagg cnggaacnaa nnaccaccan ncaactcccc tttttgtaat 300
ttnnnaactt ncaacnncta nctntttatt ttggcntccc tggnngaaac agnctgtatn 360
annnnnaagn ccntgagaac atccctggnt nncnna 396


73
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


73
ntcaacntng actnctgtga ggnatggtgc tgggngcnta tgcngtgngn ttttggatac 60
naccttatgg acantngcnn tcccnnggaa ngatnataat ncttactgna gnnactnnaa 120
nnttccntnt cnaaaangtt naaaancatt ggatgtgcca caatgatgac agtttatttg 180
ctactcttga gtgctataat gatgaagatc ttanccacca ttatcttaac tgangcaccc 240
aanatggtga nttggggaac atatanagta cacctaagtt cacatgaagt tgtttnttcc 300
caggnnctaa agagcaagcc taactcaagc cattgncaca caggtgagac acctctattt 360
tgtacttctc acttttaagg gattagaaaa tagcca 396


74
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


74
cctttttttt tttttttact gngaatatat actttttatt tagtcatttt tgtttacaat 60
tgaaactctg ggaattcaaa attaacatcc ttgcccgtga gcttcttata gacaccanaa 120
aaagtttcaa ccttgtgttc cacattgttc tgctgtgctt tgtccaaatg aacctttatg 180
agccggctgc catctagttt gacgcggatt ctcttgccca caatttcgct tgggaagacc 240
aagtcctcaa ggatggcatc gtgcacagct gtcagagtac ggctcctggg acgcttttgc 300
ttattttttg tacggctttt tcgagttggc ttaggcagaa ttctcctctg agcgataaag 360
acgacatgct tcccactgaa ctttttctcc aattcg 396


75
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


75
tttttttttt tttntttttt tttttttttt tttttttnaa ntntaanggg ganggcccct 60
tttttttaaa ctngnccntt ttnctttcct tttttnaaaa ggaaaaaaaa anntttnttt 120
ttcnttnaaa aacccttttt cccacnaaca aaaaaaaccn ttccccntnc cttttnnnna 180
aaaaaaaggg gctnggnntt tccccttann caaaaaaccn tntccnnggg naaaaaantt 240
ntcnccgggg gggaaacnnn tgggggtgtn nccnaaattt gggggccntc ggaagggggg 300
nnccncncct aaagangtnt ttcaaaanaa aaacccccnt cctnttntaa aaanaaaana 360
aaanaangnn ngnntttttt ntcnttnncc ccccaa 396


76
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


76
acattcttca gaaatacagt gatgaaaatt cattttgaaa ctcaaatatt ttcattttgg 60
atattctcct gtttttatta aaccagngat tacncctggc cntccctnta aatgttctag 120
gaaggcatgt ctgttgtnnt ttnnnnaaaa nnaaattntt tttttttngn naaaccccaa 180
atcccanttt atcaggaagt tagncnaatg aaatggaaat tggntaatgg acaaaagcta 240
gcttgtaaaa aggaccaccc nnccacnngn ctttaccccc ttggttngtt gggggaaaaa 300
ccatnnttaa ccntntggnn aaaattgggn ncntaaagtt tncntggnna acagtncntn 360
cngtattnaa ttgncnttat nggaaaatcn gggatt 396


77
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


77
tttttttttt tttttttttt tttttttttt tatcaacatt tatatgcttt attgaaagtt 60
ganaanggca acagttaaat ncngggacnc cttacaattg tgtaaanaac atgcncanaa 120
acatatgcat ataactacta tacaggngat ntgcaaaaac ccctactggg aaatccattt 180
cattagttan aactgagcat ttttcaaagt attcaaccag ctcaattgaa anacttcagt 240
gaacaaggat ttacttcagc gtattcagca gctanatttc aaattacnca aagngagtaa 300
ctgngccaaa ttcttaaaat ttntttaggg gnggtttttg gcatgtacca gtttttatgt 360
aaatctatnt ataaaagtcc acacctcctc anacag 396


78
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


78
agctggcnaa aggngnatgn gctgcnangc gattangnnn ggtaacgtca nnggntnncc 60
agtgcangac nttgtaaaac gacggccaca tgaattgtaa tacgactcac tatngggcgn 120
attgggccgt gnaggatngt gntcacactc gaatgtatnc tggcngatnc ananngcttt 180
atngctnttg acggngnntn anccanctng ggctttaggg ggtatcccct cgcccctgct 240
tcnttgattt gcacgggcnn ctccganttc cttcataata ccngacgctt cnatccccta 300
gctcngacct ntcantntnt tcnntgggtt ntnnccgntc acngcttncc cgnangntat 360
aatctnggct cctttnggga tccattantc tttact 396


79
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


79
caccaaccaa aacctggcgc cgttggcatc gtagagtgaa cacaacccaa aaacgatacg 60
ccatctgttc tgccctggct gcctcagccc taccagcact ggtcatgtct aaaggncatc 120
gtattgagga agttcctgaa cttcctttgg tangttgaag ataaagctga aggctacaag 180
aagaccaang aagntgtttt gctccttaan aaacttanac gcctggaatg atatcaaaaa 240
ngctatgcct ctcagcgaat gagactggan angcaaaatg agaaaccntc nccgcatcca 300
gcgnaggggc cgtgcatctc tatnntgang atnntggnan cnttcaaggc cttcagaacc 360
tccctngaaa tnctctnctt taangaacca aactgn 396


80
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


80
tgtacatagg catcttattc actgcaccct gtcacaccca gcaccccccg ccccgcacat 60
tatttgaaag actgggaatt taatggttag ggacagtaaa tctacttctt tttccaggga 120
cgactgtccc ctctaaagtt aaagtcaata caagaaaact gtctattttt agcctaaagt 180
aaaggctgtg aagaaaattc attttacatt gggtagacag taaaaaacaa gtaaaataac 240
ttgacatgag cacctttaga tccttccctt catggggctt tgggcccaga atgacctttg 300
aggcctgtaa anggattgna atttcctata agctgtatag tggagggatt ggngggtcat 360
ttgagtaagc cctccaagat acnttcaata cctggg 396


81
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


81
gcagctgaag ttcagcaggt gctgaatcga ttctcctcgg cccctctcat tccacttcca 60
acccctccca ttattccagt actacctcag caatttgtgc cccctacaaa tgttagagac 120
tgtatacgcc ttcgaggtct tccctatgca gccacaattg aggacatcct gcatttcctg 180
ggggagttcg ccacagatat tcgtactcat ggggttcaca tggttttgaa tcaccagggn 240
ccgccatcag gagatgcctt tatccagatg aagtctgcgg acagancatt tatggctgca 300
cagaagtggc ataaaaaaaa catgaaggac agatatgttg aagttttcag tgtcagctga 360
nganagaaca ttgnngtann ngggggnact ttaaat 396


82
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


82
gactcagaaa tgtcagtctc atgaagttca aaagatcgag aatgtttgct atcttggtgg 60
agcagccgca gccaagcaag taacttgtaa aatgaggaat gccatcaccc ctcgagtgtc 120
catcccacat aacttggggt tagagcacaa gcgttcccag gaactactca ccttaccatc 180
ttggccgttt catttgcttc caccagttct ggaaagagan ggcctagaag ttcaaaaaaa 240
aagtaggaaa ngtgcttttg gagaaaatca cctgctcctc agaactgggc ttacaanctg 300
ngaagtacnc tatgtgccac ctaatcctca tatatgacct caagagacnc caataagcat 360
atttccacca cggaatgacc agtgctttgg gtaana 396


83
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


83
tttgatttaa ganatttatt atttttttaa aaaaagcaac ttccagggtt gtcattgtac 60
aggttttgcc cagtctccta tagcatggta tagtgataac tgatttttta taacaatgac 120
tcagaggcat tgaagatcca taactatctt ctgaattatc acagaaagaa gaaagttaga 180
agagtttaat gttaagtgta ttaaaaatca tattctaatt cttttaattt ggttatctga 240
gtatgataat ataggagagc tcagataaca aggaaaaggc attggggtaa gaacactcct 300
tcccacagga tggcattaac agactttttc tgcatatgct ttatatagtt gccaactaat 360
tcacctttta cncagcttna ttttttttta ctnggg 396


84
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


84
tttttacagc aatttttttt tattgatgtt taacctgtat acaaccatac ccattttaag 60
ngtacagaca aatgaatttt gacaaattca ttcactcatc taatcatcac tataaccatg 120
atacagattt ttatcactcc aaaagtccat cctgtgctct tttcaagtcc atcctcctca 180
tctgataccc caagccacca ttgttttgct ttctggaact acagttttgg gnttttagaa 240
tttcatatat ggtngaatca taccatttgn natttggggc tgacgncttt cctccaataa 300
tggatttgag aattatctac attttgcatg gatcctgggt tatttatacc aacnangggt 360
tattatgnaa aatnggacca caatttggng gcanta 396


85
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


85
cagtgaccgt gctcctaccc agctctgctc cacagcgccc acctgtctcc gcccctcggc 60
ccctcgcccg gctttgccta accgccacga tgatgttctc gggcttcaac gcagactacg 120
aggcgtcatc ctcccgctgc agcagcgcgt ccccggccgg ggatagcctc tcttactacc 180
actcacccgc agactccttc tccagcatgg gctcgcctgc aacgcgcagg acttctgcac 240
ggacctggcc gctccagtgc caacttcatt ccacggcact gcatctcgac canccggact 300
tgcannggtt ggggaanccg cccttgtttc tccgtggccc atctaanacc aaacccntca 360
ccttttcgga gnccccnccc ctccgntggg nttact 396


86
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


86
ttttnnactg aatgtttaat acatttgnag gaacagaaga aatgcagtan ggattaanat 60
tttataatta gacattaatg taacagatgn ttcatttttc aaagaagntn cccccttntc 120
cctatctttt tttaatcttc cttanagcaa taantagtaa ttactatatt tgtggacaag 180
ctgctccact gtgntggaca gtaattatta aatctttatg tttcacatca ttattacctt 240
ccanaattct accttcattt ccctgcacag gttcactgga ctggntcaca ancaaattgn 300
actccactca antanaagag cccaaagaaa ttagagtaac gncnantcct atgaattana 360
gacccaaaga tttnaggngn tgattagaaa cataan 396


87
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


87
atggaggcgc tggggaagct gaagcagttc gatgcctacc ccaagacttt ggaggacttc 60
cgggtcaaga cctgcggggg cgccaccgtg accattgtca gtggccttct catgctgcta 120
ctgttcctgt ccgagctgca gtattacctc accacggagg tgcatcctga gctctacgtg 180
gacaagtcgc ggggagataa actgaagatc aacatcgatg tactttttcc ncacatgcct 240
tgtgcctatc tgagtattga tgccatggat gtggccngag aacancagct ggatgnggaa 300
cacaacctgt ttaagccacc actagataaa gatgcatccc ngtgagctca nagctgagcg 360
gcatgagctt gngaaantcn aggtgaccgg gtttga 396


88
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


88
tccagagcag agtcagccag catgaccgag cgccgcgtcc ccttctcgct cctgcggggc 60
cccagctggg accccttccg cgactggtac ccgcatagcc gctcttcgac caggccttcg 120
ggctgccccg gctgccggag gagtggtcgc agtggttagg cggcagcagc tggccaggct 180
acgtgcgccc cctgcccccc gccgcatcga gagccccgca gtggccgcgc ccgctacagc 240
cgcgcngctc agccggcaac tcacancggg gctcggagat ccgggacact gcggaccgct 300
ngcgcgtgcc ctggatgtca ccactttngc ccggacaact gacggtnana caaggatggg 360
gggtgganan nccngtaanc caagaanggg naggac 396


89
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


89
gagagaacag taaacatcca gccttagcat ctctcangag tactgcagat cttcattagc 60
tatattcaca tggagnaatg ctattcaacc tatttctctt atcaaaacta attttgtatt 120
ctttgaccaa tgttcctaaa ttcactctgc ttctctatct caatcttttt cccctttctc 180
atctttcctc cttttttcag tttctaactt tcactggttc tttggaatgn tttttctttc 240
atctcttttc ttttacattt tggggtgtcc cctctctttt cttaccctct ttctncatcc 300
ttcttnttct tttgaattgg ctgcccttta tcntctcatc tgctgncatc ttcatttctc 360
ctccctcctn tttccnntca ttctactctc tcccnt 396


90
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


90
gggcgccggc gcgccccccc acccccgccc cacgtctcgt cgcgcgcgcg tccgctgggg 60
gcggggagcg gtcgggccgg cngcggtcgg ccggcggcag ggtggtgcgn tttcnttttn 120
nattnnccnc nttcttcttn nttnnncnnn ctnntanncn ntnncnttcn cnnnntttnc 180
tntntcttna ccnnnttttn taatcntctt ctncntnnnn tctcttnnat ntnttnctta 240
nttcctnnnn tttnttctnt cntttctcnc ctnnntctcn nnctcnncnc tcnncatttt 300
nntnttttnt nccttctnnt cttnnttctn ntnntnnttt nnnnttctnt tnntcatntt 360
ncctntntta ctntcanctt ntatnnncct cntttt 396


91
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


91
ntntcctnna tttttnnntc nncttttttt tnnaattttt ctttnttttn tttataaaaa 60
tcnncacnta aaacngcgga anaggggatt tnttnttngg gngtancncn nggccncaaa 120
naaccccaaa aatancccaa aatgcacagg nccngggnaa angaccnacn tgggtntttt 180
ntttntnaac aaggggggtt ttaaagggna tnggnatcaa agggnataaa ntttaaacct 240
ttganaaatt ttttaanagg cttgcccccc actttggncc ccnccccncn gnngggatcc 300
aatttttttt cnttggggct cccngncccn nannttccgg gttnntggnc nntcctnntt 360
tttttttttt tgccttcacc cntnccattn cntttt 396


92
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


92
ctntttnnnt ntttttttcc ccatcatcca naaatgggtt ttattctcag ccgagggaca 60
gcaggactgg taaaaactgt caggccacac ggttgcctgc acagcacccc catgcttggt 120
agggggtggg agggatggcg ggggctggnt gnccacaggc cgggcatgac aaggaggctc 180
actggaggtg gcacactttg gagtgggatg tcgggggaca ncttctttgg tanttgggcc 240
acaagattcc caaggatanc acnnnnactg attnccannc tanagncaag cggntggcca 300
tntgtangnn nttntntatn tgactattta tagattttta tanaacaggg naagggcata 360
ccncaaaagg gnccaanttt ttaccnccgg gcnccc 396


93
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


93
gctgccacag atctgttcct ttgtccgttt ttgggatcca caggccctat gtatttgaag 60
ggaaatgtgt atggctcaga tcctttttga aacatatcat acaggttgca gtcctgaccc 120
aagaacagtt ttaatggacc actatgagcc cagttacata aagaaaaagg agtgctaccc 180
atgttctcat ccttcagaag aatcctgcga acggagcttc agtaatatat cgtggcttca 240
catgtgagga agctacttaa cactagttac tctcacaatg aaggacctgn aatgaaaaat 300
ctgnttctaa ccnagtcctn tttanatttt agngcanatc cagaccancg ncggtgctcg 360
agtaattctt tcatgggacc tttggaaaac tttcag 396


94
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


94
tgccttaacc agtctctcaa gtgatgagac agtgaagtaa aattgagtgc actaaacgaa 60
taagattctg aggaagtctt atcttctgca gtgagtatgg cccaatgctt tctgnggcta 120
aacagatgta atgggaagaa ataaaagcct acgtgttggt aaatccaaca gcaagggaga 180
tttttgaatc ataataactc atanngtgct atctgtcagt gatgccctca gagctcttgc 240
tgntagctgg cagctgacgc ttctangata gttagnttgg aaatggtctt cataataact 300
acacaaggaa agtcanccnc cgggcttatg aggaattgga cttaataaat ttagngngct 360
tccnacctaa aatatatctt ttggaagtaa aattta 396


95
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


95
cctcccaccc ncttanttca tgagattcga naatgncact tntgtgctnt ttnctnnttn 60
tattctnacn atttctttct tggngcggna nnaatcccnt ttttnngggc gnctctcccn 120
ncttntnntt tcntggngct ntcccttttc nnnnnaaact tntacnnngt ttanaantnt 180
ttctgnangg gggnntccna aananttttt ccncctncct nattccnctc tnaannctcn 240
cnaattgttt cccccccccn ntagnntatt ttttctaaaa aattaactcc nacgganaaa 300
attttcccta aaatttcncc tccanatttn gaaaaaacnc gcccgganct nntntncgaa 360
tntnaatttt tnaaaaaaan ttattttcat cnggnn 396


96
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


96
cctgggtacc aaatttcttt atttgaagga atggtacaaa tcaaagaact taagtggatg 60
ttttggacaa cttatagaaa aggtaaagga aaccccaaca tgcatgcact gccttggcga 120
ccagggaagt caccccacgg ctatggggaa attagcccga ngcttaactt tcattatcac 180
tgcttccaag ggngtgcttg gcaaaaaaat attccgccaa ccaaatcggg cgctccatct 240
tgcccagttg gtnccgggnc cccaattctt ggatgctttc ncctcttntt ccggaatgng 300
ctcatgaant cccccaanng gggcattttg ccagnggccn tttngccatt cnagnnggcc 360
tgatccattt tttccaatgt aatgccnctt cattgn 396


97
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


97
ctcaccctcc tcntnnttnt canaatattg ngaacttnnt nctgntcgaa tcactggcat 60
taaagganca ctagctaatg gcactaaatt tacnnactan ggaaactttt ttataatant 120
gcaaaaacat ntnaaaaaga ntgnagttcg cccatttctg cttnggaaga nctcttcact 180
tntaancccn natgnngncc tttgggtcaa aanctccgcg attattacng ngttncccnc 240
tatttgncct tcctttntcc ccaangccnc anatttcnna actttnccnt naaatgcctt 300
tatttnatnn cntttcnacn ncttaanntt ccctttnaan aangatccct ncttcaaatn 360
ntttcccngt tcctngcatt ncccnnnnat ttctct 396


98
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


98
acagggacaa tgaagccttt gaagtgccag tctatgaaga ggccgtggtg ggactagaat 60
cccagtgccg cccccaagag ttggaccaac caccccctac agcactgttg tgataccccc 120
agcacctgan gaggaacaac ctaccatcca gaggggccag gaaaagccaa actggaacag 180
aggcgaatgg ctcagagggg tncatggcca agaaggaagc cctggaagaa cttcaatcac 240
cttcggtttc gggaccaccg gcttgtgtcc ctgttctgac tgcanaactt ggcgcngtnc 300
cccattanaa cctntgactc nncccttgct ataagnctgt tttggcccct gatgatgata 360
gggtttttat gangacactt gggcaccccc ttaatg 396


99
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


99
nttntttttc cgncnaaagg gcaagngttt ncatctttcc tgnccncnca ananngggtn 60
tntgtgcntt tnttttttcc caaaacccgg gtnggggaca ccttttgagg anccactnnt 120
cntccggggc nnnnttttag aaggngncta anaagcntct tgnnggggga aaaacatctt 180
tttgcncccn acataccccc aagggggggg ggtgtctggg agganactaa ngacttttnt 240
tttttnnccn caaanaactg anggccccca ttgctccccc cccantcttt aaaaaacccc 300
ttcaatttcc ttgncnggna aaaanggttg gnaaaaaang agngngcntc nnttncnttt 360
natggaaggn aaaaggtttt tggttgnaaa accccg 396


100
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


100
ctaacacggt gaaaccctgt ctctactaaa aatacaaaaa aattagccag gcgtggtggc 60
gggcacctgt agtcccagct gctcaggaag ctgaggcagg agaatggcgt gaacccagaa 120
ggcggagctt gcagtgagct gagatcgtgt cagtgcactc cagcctgggc gacagagcga 180
gactcccgct caaaaaaaaa aaaaaaaaga gaaaagaaaa agctgcagng agctgggaat 240
gggccctatc ccctccttgg ggatcaatga gacccctttt caaaanaaaa aaaaaaataa 300
tgngattttg gnaacatatg gcactggtgc ttcnnggaat tctgtttntn ggcatgnccc 360
cctntgactg nggaaaaatc cagcaggagg cccana 396


101
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


101
agttataact caacagttca tttatatgct gttcatttaa cagttcattt aaacagttca 60
ttataactgt ttaaaaatat atatgcttat agncaaaann tgttgtggcg nagttgttgc 120
cgcttatagc tgagcattat ttcttaaatt cttgaatgtt cttttggngg gntnctaaaa 180
ccgtatatga tccattttna tgggaaacng aattcntnnc attatcncac cttggaaata 240
cnnaacgtgg gggaaaaaaa tcattcccnc cntccaaaac tatacttctt ttatctngan 300
nttcttgntc ctgcncnggt ttngaatata nctgggcaaa nggntttncc aaatccntnt 360
acnntncttt gggaantanc ggcaantcnt cncttt 396


102
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


102
actatacata agaacangct cacatgggag gctggaggtg ggtacccagc tgctgtggaa 60
cgggtatgga caggtcataa acctagagtc agngtcctgt tggcctagcc catttcagca 120
ccctgccact tggagnggac ccctctactc ttcttagcgc ctaccctcat acctatctcc 180
ctnctcccat ctcctacgga ctggcgccaa atggctttcc tgccaatttt gggatcttct 240
ctggctctcc agcctgctta ctcctctatt tttaaagggc caaacaaatc ccttctcttt 300
ctcaaacaca gtaatgnggc actgacccta ccacacctca tgaagggggc ttgttgcttt 360
tatttgggcc cgatctgggg ggggcaaaat attttg 396


103
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


103
ttgtgttggg actgctgata ggaagatgtc ttcaggaaat gctaaaattg ggcaccctgc 60
cccaacttca aagccacagc tggtatgcca natggtcagg ttaaagatat caacctgctg 120
actacaaagg aaaatatggt ggggtcttct tttaccctct tgacttccct ttgngngccc 180
cccgaganca ttgctttccg ngatagggca aaanaaatta aaaaacttaa ctggccagtg 240
aatggggctt ctgnggatct ccttctggca ttacatnggc aatccctaaa aaacaagang 300
actgggaccc ataacattct tttgnatcaa ccgaagcccc cattgttang atatngggct 360
taaangctga tnaagcatct cgtccgggcn ttttat 396


104
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


104
aagggagggc gcgccaagac cttcccactc gngcacactg ggggcgccga cangacgcaa 60
cccagtccaa cttggatacc cttggnttta gttctcggac acttctttta tctctccgtc 120
gcaacttgtc aagttctcaa nactgtctct ctgngntatc ttttttcttc gctgctcttc 180
nncccccgac gtatttntca aaangtctgc aattgttgna tacntnganc tncaccactg 240
ttacnaggtc atnaatttcn cntcaactct ntnccncttg ttccctgata tntcggccgg 300
ngncnccaat tctgtatttt nctcntcaac gntctcactt ttncctcctc cnggccactt 360
tctccccttc cttattccgg cnttgtttgc cnccat 396


105
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


105
tcaatagcca gccagtgttc atttttatcc ttgagctttt agtaaaaact tcctggnttt 60
atttttagtc attgggtcat acagcactaa agtctgctat ttatggaaac taactttttt 120
gtttttaatc caggccaaca tgtatgtaaa ttaaattttt agataattga ttatctcttt 180
gtactacttg agatttgatt atgagatgtg catattgctt tgggaagagc tcgaggaagg 240
aaataattct ctcctttggt ttgaacctca actagataaa ccctaggaat tgttaactgc 300
acaagnattt tcattccaca aaacctgagg cagctctttt gccagagcgt tcctgnaccc 360
ccccacccca cttgccttgg gtctttanaa ngagcc 396


106
396
DNA
Homo sapien

106
gctgtgtagc acactgagtg acgcaatcaa tgtttactcg aacagaatgc atttcttcac 60
tccgaagcca aatgacaaat aaagtccaaa ggcattttct cctgtgctga ccaaccaaat 120
aatatgtata gacacacaca catatgcaca cacacacaca cacacccaca gagagagagc 180
tgcaagagca tggaattcat gtgtttaaag ataatccttt ccatgtgaag tttaaaatta 240
ctatatattt gctgatggct agattgagag aataaaagac agtaaccttt ctcttcaaag 300
ataaaatgaa aagcaattgc tcttttcttc ctaaaaaatg caaaagattt acattgctgc 360
caaatcattt caactgaaaa gaacagtatt gctttg 396


107
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


107
ttcacagaac anggtggttt attatttcaa tagcaaagag ctgaaaaatg tcgggtccca 60
taaaggagca gaacctgacc cagagcctgc agtacatttc caccccacag gggtgcaggc 120
tgggccaggc agggccaaag gcagcagaaa tgggagtaag agactgtgcc cactgagaag 180
ctctgctggg tgtgggcagg tgggcatgan atgatgatga tgtagtgtaa ggaccaggta 240
ggcaaaacct gtcaggnttg ntgaatgtca nagtggatcc aaaaggctga gggggtcgtc 300
anaaggccgg nggncccncc cttgcccgta tgggccttca aaaagtatgc ttgctcatcc 360
gttgtttncc ccanggagct gccanggana aggctn 396


108
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


108
gcctgctttt gatgatgtct acagaaaatg ctggctgagc tgaacacatt tgcccaattc 60
caggtgtgca cagaaaaccg agaatattca aaattccaaa tttttttctt aggagcaaga 120
agaaaatgtg gccctaaagg gggttagttg aggggtaggg ggtagtgagg atcttgattt 180
ggatctcttt ttatttaaat gtgaatttca acttttgaca atcaaagaaa agacttttgt 240
tgaaatagct ttactgcttc tcacgtgttt tggagaaaan natcanccct gcaatcactt 300
tttgnaactg ncnttgattt tcngcnncca agctatatcn aatatcgtct gngtanaaaa 360
tgncctggnc ttttgaanga atacatgngt gntgct 396


109
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


109
ggccgtaggc agccatggcg cccagcccgg aatggcatgg tcttgaagcc ccacttccac 60
aaggactggc agcggcgcgt ggccacgtgg ttcaaccagc cggcccggaa gatccgcaga 120
cgtaaggccc ggcaagccaa ggcgcgccgc atcgctccgc gccccgcgtc gggtcccatc 180
cggcccatcg tgcgctgccc acggttcggt accacacgaa gggcgcgccg gcgcggnttc 240
agcctggagg agctcagggt ggccggattt acaagaagng gccngacatc ngtattcttg 300
ggatncnnga agnggaacaa gtcacngagt ccttgcagcc acntcagcgg ntgatgacac 360
cgttcnaact catctnttcc caagaaacct cngnnc 396


110
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


110
nntgggctcc tnncantnat aataaaccng actcatacnc cacaaggaga tgaacaggan 60
tatgtncatn ctgacgcgga aacagngcan ggagctgagg aggngccaag atgagaccta 120
nnggccnngg tgggcgcatt cccggnggag ggggccacta aggantacga nnntcnagcg 180
gctcttgnng gcngncctcc tcacncctgn ntattcgatt gtcncnnatg ncntcctatn 240
atnntcanna ttctntnntn atctcntnta cnncntcncn ttcatgntta cngntccctc 300
tcnttctnac cnttntctgn anctcctttc tnnnnctttc atctntnttc ngctttcttt 360
ctnnaatcnt nntttaacnt nntctncttt ntnatt 396


111
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


111
taangancat nctggnttnt gcctnnccgn ctnattgant gttaaaggca attntgtggn 60
tgtcccagng aatgncggct nattttcttt ccacattgng cncattcact cctcccactc 120
ttggcatgtn gngacataag canggtacat aatngnaaaa atctgnattt ctgatgccan 180
angggtanan cntnttgnat ntcattccat tgatatacag ccactntttt atttttgatc 240
ancggccttc ggntcactgc ncanggtact tgacctcagt gtcactatta tgggntttgg 300
tttcnctctt ttncnggccn ttntntttcn cacnttncan cttncttnnt nnaaaannna 360
nncactctct cttgctctct ngatacnnng tctnaa 396


112
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


112
tcaacgtcac caattactgc catttagccc acgagctgcg tctcagctgc atggagagga 60
aaaaggtcca gattcgaagc atggatccct ccgccttggc aagcgaccga tttaacctca 120
tactggcaga taccaacagt gaccggctct tcacagtgaa cgatgttaaa gntggaggct 180
ccaagnatgg tatcatcaac ctgcaaagtc tgaagacccc tacgctcaag gtgttcatgc 240
acgaaaacct ctacttcacc aaccggaagg tgaattcggg gggctgggcc tcgctgaatc 300
acttggattc cacattctgc tatgcctcat gggactcgca gaacttcagg ctggccaccc 360
tgctcccacc atcactgntn gncaatantc acccag 396


113
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


113
nnnnttnnnn nggagcctta atttcagagt tttattgtat tgcactaaag gaacagcagg 60
atggntatac aattttctct cattcagttt tgaaaatctg tagtacctgc aaattcttaa 120
gaataccttt accaccagat tagaacagta agcataataa ccaatttctt aataagtaat 180
gtcttacaaa taaaaacaca tttaaaatag ctttaaatgc attcttcaca agtaattcag 240
catatatttt atatcatggt tacttatgct tangaattnn agcaggatnt ttattctttt 300
gatggaaata tgggaaaact ntattcatgc atatacangg ataatattca gcgaagggaa 360
aatcccgttt ttattttggn aatgattcat atataa 396


114
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


114
aaatgggaca acgtgattct tttgttttaa ataaatactn agaacacgga cttggctcct 60
acaagcattt ggactctaag gnttagaact ggagagtctt acccatgggc cccncncagg 120
gacgccacgg ttccctccca ccccgngatc aagacacgga atcngntggc gatngttgga 180
tcgcnatgtg ccccttatct atagccttcc cnggncatnt acangcagga tgcggntggg 240
anaactacaa ctgnaatntc tcnaacggtn atggtcccca ccgatnaaga ttctacctng 300
tcttttcntc ccctggagtg tgagtgnnng aggaagaagc ccttncctta catcaccttt 360
tgnacttctg aacaaganca anacnatggc cccccc 396


115
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


115
ccgcctggtt cggcccgcct gcctccactc ctgcctctac catgtccatc agggtgaccc 60
agaagtccta caaggtgtcc acctctggcc cccgggcctt cagcagccgc tcctacacga 120
gtgggcccgg ttcccgcatc agctcctcga gcttctcccg agtgggcagc agcaactttc 180
gcggtggcct ggcggcggct atggtggggc cagcggcatg ggaggcatca cccgcagtta 240
cggcaaccag agcctgctga gccccttgcc tggaggngga ccccaacatc aagccgngcg 300
cacccaggaa aaggagcaga ncaagaccct caacaacaag nttgcttctt catagacaag 360
ggaccggtcc ttgaacagca naacaagatg ntggag 396


116
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


116
atctcagttt actagctaag tgactttggg caagggattt aacctctcgt ccctcagttt 60
cctcctatgt aaaatgacaa ggataatagt accaacccaa tgtagattaa atgagtttac 120
gaagtgttag aatagtgctt ggcacattag tgctttacaa ctgctatttt gattgttgtt 180
gtgggctctc tcaaatgcat tgtctctaga tgccagtgac ccaggtcaaa atttaccttt 240
aaccaagctg catgtttccc agactgntgc acagtcctct accctgagan aaagcttcca 300
cccaaggata cttttacttt ctgctggaaa actgatgagc aanggcaaca ngggacactt 360
atcgccaact ggaaangaga aattcttcct tttgct 396


117
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


117
aaacattttt taataaaatt cctatagaaa gctcagtcat agggcaaata ctcagttctc 60
tttcccatat caccgaggat tgagagctcc caatattctt tggagaataa gcagtagttt 120
tgctggatgt tgccaggact cagagagatc acccatttac acattcaaac cagtagttcc 180
tattgcacat attaacatta cttgccccta gcaccctaaa tatatggnac ctcaacaaat 240
aacttaaaga tttccgtggg gcgcganacc atttcaattt gaactaatat ccttgaaaaa 300
aatcacatta ttacaagntt taataaatac nggaagaaga gctggcattt ttctaanatc 360
tgaattcnga cttggnttta ttccataaat acggtt 396


118
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


118
accnncacct gntnnntttt aacnattaca acttctttat atggcagttt ttactgggng 60
cctaacactc tctttactgn ctcaagngga agtccaaaca aatttcattt ttgtagtaaa 120
aaatctttat ttccaaaatg atttgttagc caaaagaact ataaaccacc taacaagact 180
ttggaagaaa gagacttgat gcttcttata aattccccat tgcanacaaa aaataacaat 240
ccaacaagag catggtaccc attcttacca ttaacctggn tttaannctc caaancnnga 300
tttaaaaatg accccactgg gcccaatcca acatganacc taggggggnt tgccttgatt 360
angaatcccc cttanggact ttatctnggc tganaa 396


119
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


119
atggccagct cactttaaat accacctcaa gactcatcga aatgaccgct ccttcatctg 60
tcctgcagaa ggttgtggga aaagcttcta tgtgctgcag aggctgaagg tgcacatgag 120
gacccacaat ggagagaagc cctttatgtg ccatgagtct ggctgtggta agcagtttac 180
tacagctgga aacctgaaga accaccggcg catccacaca ggagagaaac ctttcctttg 240
tgaagcccaa ngatgtggcc gtcctttgct gagtattcta ncttcgaaaa catctggngg 300
ntactcanga gagaaagcct cattantgcc antctgnggg aaaaccttct ntcagagngg 360
angcaggaat gtgcatatta aaaagctncc ttgnac 396


120
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


120
catgggtcag tcggtcctga gagttcgaag agggcacatt cccaaagaca ttcccagtca 60
tgaaatgtag aagactggaa aattaagaca ttatgtaaag gtagatatgg cttttagagt 120
tacattatgc ttggcatgaa taaggtgcca ggaaaacagt ttaaaattat acatcagcat 180
acagactgct gttagaaggt atgggatcat attaagataa tctgcagctc tactacgcat 240
ttattgttaa ttgagttaca nangncattc annactgagt ttatagancc atattgctct 300
atctctgngn agaacatttg attccattgn gaagaatgca gtttaaaata tctgaatgcc 360
atctagatgt attgtaccna aaggggaaaa ataaca 396


121
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


121
tttttttttt ttttttttaa aatcaagtta tgtttaataa acattaataa atgtttactt 60
aaaagggtta ataaacnttt actacatggc aaattatttt agctagaatg cttttggctt 120
caagncatan aaaccagatt cnaatgccct taaanaattt tnaaanatcc attgangggg 180
ataactgtaa tccccaaggg gaanagggtt gggtatgaca ggtacanggg gccagcccag 240
tnntnncana nncagactct taccntcttt ctgctgtgnc accctcaggc attggctcca 300
ttctcngggn tgcncatggg aagatggctt tggacntaac nacacccttt tgtncacgta 360
aaggccngat gcagggtcaa anagnttccn ccatnt 396


122
396
DNA
Homo sapien

122
gtcgacatgg ctgccctctg ggctcccaga acccacaaca tgaaagaaat ggtgctaccc 60
agctcaagcc tgggcctttg aatccggaca caaaaccctc tagcttggaa atgaatatgc 120
tgcactttac aaccactgca ctacctgact caggaatcgg ctctggaagg tgaagctaga 180
ggaaccagac ctcatcagcc caacatcaaa gacaccatcg gaacagcagc gcccgcagca 240
cccaccccgc accggcgact ccatcttcat ggccaccccc tgcggtggac ggttgaccac 300
cagccaccac atcatcccag agctgagctc ctccagcggg atgacgccgt ccccaccacc 360
tccctcttct tctttttcat ccttctgtct ctttgt 396


123
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


123
gccctttttt tttttttttt tttcctagtg ccaggtttat tccctcacat gggtggttca 60
catacacagc acanaggcac gggcaccatg gganagggca gcactcctgc cttctgaggg 120
gatcttggcc tcacggtgta anaagggana ggatggtttc tcttctgccc tcactagggc 180
ctagggaacc cagnagcaaa tcccaccacg ccttccatnt ctcagccaag ganaagccac 240
cttggtgacg tttagttcca accattatag taagtggana agggattggc ctggtcccaa 300
ccattacagg gtgaanatat aaacagtaaa ggaanataca gtttggatga ggccacagga 360
aggagcanat gacaccatca aaagcatatg caggga 396


124
396
DNA
Homo sapien

124
gaccattgcc ccagacctgg aagatataac attcagttcc caccatctga ttaaaacaac 60
ttcctccctt acagagcata caacagaggg ggcacccggg gaggagagca catactgtgt 120
tccaatttca cgcttttaat tctcatttgt tctcacacca acagtgtgaa gtgcgtggta 180
taatctccat ttcaaaacca aggaagcagc ctcagagtgg tcgagtgaca cacctcacgc 240
aggctgagtc cagagcttgt gctcctcttg attcctggtt tgactcagtt ccaggcctga 300
tcttgcctgt ctggctcagg gtcaaagaca gaatggtgga gtgtagcctc cacctgatat 360
tcaggctact cattcagtcc caaatatgta ttttcc 396


125
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


125
cccttttttt tttttttttt tttttttttt ttttttactt tgnaacaaaa atttattagg 60
attaagtcaa attaaaaaac ttcatgcncc nccncttgtc atatttacct gaaatgacaa 120
agttatactt agcttgagng naaaacttgn gccccaaaaa ttntgtttgg aaagcaaaaa 180
aataattgat gcncatagca gngggcctga tnccnccaca gngaatgttg tttaaggnct 240
aacaaacagg ggncancaaa gcatacatta cttttaagct ttgggnccaa ggaaaangtc 300
attccctacc tccttcaaaa gcaaactcat natagcctgg gcncctaggn ctggagcctn 360
ttttttcgag tctaanatga acatntggat ttcaan 396


126
396
DNA
Homo sapien

126
cgcgtcgact cgcaagtgga atgtgacgtc cctggagacc ctgaaggctt tgcttgaagt 60
caacaaaggg cacgaaatga gtcctcaggt ggccaccctg atcgaccgct ttgtgaaggg 120
aaggggccag ctagacaaag acaccctaga caccctgacc gccttctacc ctgggtacct 180
gtgctccctc agccccgagg agctgagctc cgtgcccccc agcagcatct gggcggtcag 240
gccccacgac ctggacacgc tggggctacg gctacagggc ggcatcccca acggctacct 300
ggtcctagac ctcagcatgc aagaggccct ctcggggacg ccctgcctcc taggacctgg 360
acctgttctc accgtcctgg cactgctcct agcctc 396


127
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


127
tttttttttt ttggnggtaa aatgcaaatg ttttaaaata tgtttatttt gtatgtttta 60
caatgaatac ttcagcaaag aaaataatta taatttcaaa atgcaatccc tggatttgat 120
aaatatcctt tataatcgat tacactaatc aatatctaga aatatacata gacaaagtta 180
gctaatgaat aaaataagta aaatgactac ataaactcaa tttcagggat gagggatcat 240
gcatgatcag ttaagtcact ctgccacttt ttaaaataat acgattcaca tttgcttcaa 300
tcacataaac attcattgca ggagttacac ggctaatcat tgaaaattat gatctttgtt 360
agcttaaaag aaaattcagt ttaatacaaa gacatt 396


128
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


128
gccctttttt ttttttttta aaggcaaata aaataagttt attgggatgt aaccccatca 60
taaattgagg agcatccata caggcaagct ataaaatctg gaaaatttaa atcaaattaa 120
attctgcttt taaaaaggtg ccttaagtta accaagcatt ttgataacac attcaaattt 180
aatatataaa aatagatgta tcctggaaga tataatgaan aacatgccat gtgtataaat 240
tcanaatacg ctttttacac aaagaactac aaaaagttac aaagacagcc ttcaggaacc 300
acacttagga aaagtgagcc gagcagcctt cacgcaaagc ctccttcaaa naagtctcac 360
aaagactcca gaaccagccg agtntgtgaa aaagga 396


129
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


129
gccctttttt tttttttttt ttttactcag acaggcaata tttgctcaca tttattctct 60
tgcatcgtaa atagtagcca actcacaaaa ataaagtata caanaatgta atatttttta 120
aaataagatt aacagtgtaa gaaggaaaat ctcaaaaaaa gcanatagac aatgtanaaa 180
attgaaatga aatcccacag taanaaaaaa aaaacanaaa agtgcctatt taanaattat 240
gctacatgtg gaacttaact agaccatttt aanaaagacc aatttctaat gcaaattttc 300
tgaggttttc anattttatt tttaaaatat gttatagcta catgttgtcn acncggccgc 360
tcgagtctan agggcccgtt taaacccgct gatcag 396


130
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


130
cgcccttttt tttttttttt tanngnacgt gnctttattt ctggatgata taaaanaaaa 60
aacttaaaaa acaccccaaa ccaaacacca atggatcccc aaagcgatgt gactccctct 120
tcccacccgg ataaatagag acttctgtat gtcagtctac cctcccgccc ccataacccc 180
ctctgctata nacatactct gggtatatat tactctactc ggcaatagac atctcccgaa 240
aatagaattc ctgccctgac acctgactct tccctggccg catcanacca cccgccactg 300
tagcacactg gtgtccttgc cccctgtggt cagggccatg ctgtcatccc acaanaaggc 360
cacatttgtc acatggctgc tgtgtccacc gtactt 396


131
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


131
gccctttttt tttttttttt tttttttttt ttcagtttac acaaaaacnc tttaattgac 60
agtatacnnt tttccaaaat atnttttngt aanaaaatgc aataattatt aactatagtt 120
tttacaaaca agtttntcan taaattccag tgtncttnaa accccnnncn annaaaacat 180
atatganccc ccagttcctg ggcaaactgt tgaacattca ctgcanacaa aaagaccanc 240
nccaaanagt catctgngnc ctccatgctg ngtttgcacc aaacctgagg gancagctag 300
ngaccgtgac aaaagctntg ctacagtttt actntngccc tntntgcctc ccccatnatg 360
tttccttggt ccctcantcc tgtnggagta agttcc 396


132
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


132
cgcgtcgacc gcggccgtag cagccgggct ggtcctgctg cgagccggcg gcccggagtg 60
gggcggcgnt atgtaccttc cacattgagt attcagaaag aagtgatctg aactctgacc 120
attctttatg gatacattaa gtcaaatata agagtctgac tacttgacac actggctcgg 180
tgagttctgc tttttctttt taatataaat ttattatgtt ggtaaattta gcttttggct 240
tttcactttg ctctcatgat ataagaaaat gtaggttttc tctttcagtt tgaattttcc 300
tattcagtaa aacaacatgc tagaaaacaa acttttggaa aggcattgta actatttttt 360
caaatagaac cataataaca agtcttgtct taccct 396


133
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


133
ntattacccc tcctggnnan ntggnnatan nctgcaaggn gatnnncccg nngaacttca 60
ctgatnnncc aatnaaaact gctttaaanc tgactgcaca tatgaattnt aatacttact 120
tngcgggagg ggtggggcag ggacagcaag ggggaggatt gggaanacaa tagacaggca 180
tgctggggat gcngcgggct ctatggcttc tgangcgnaa agaaccagct ggggctctag 240
ggggtatccc cacgcgccct gtagcngcnc attaaacgcg gcgggtgtgg nggttacttc 300
gcaaagngac cgatncactt gccagcgccc tagctgcccg ctcctttngc tttcttccct 360
tcctttctcg ccacnttnnc cggctntccc cgncaa 396


134
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


134
tttttttttt ttctgctttt tatatgttta aaaatctctc attctattgc tgctttattt 60
aaagaaagat tactttcttc cctacaagat ctttattaat tgtaaaggga aaatgaataa 120
ctttacaatg ganacacctg gcanacacca tcttaaccaa agcttgaagt taacataacc 180
agtaatagaa ctgatcaata tcttgtgcct cctgatatgg ngtactaana aaaacacaac 240
atcatgccat gatagtcttg ccaaaagtgc ataacctaaa tctaatcata aggaaacatt 300
anacaaactc aaattgaagg acattctaca aagtgccctg tattaaggaa ttattcanag 360
taaaggagac ttaaaagaca tggcaacaat gcagta 396


135
396
DNA
Homo sapien

135
gcgtcgacgc tggcagagcc acaccccaag tgcctgtgcc cagagggctt cagtcagctg 60
ctcactcctc cagggcactt ttaggaaagg gtttttagct agtgtttttc ctcgctttta 120
atgacctcag ccccgcctgc agtggctaga agccagcagg tgcccatgtg ctactgacaa 180
gtgcctcagc ttccccccgg cccgggtcag gccgtgggag ccgctattat ctgcgttctc 240
tgccaaagac tcgtgggggc catcacacct gccctgtgca gcggagccgg accaggctct 300
tgtgtcctca ctcaggtttg cttcccctgt gcccactgct gtatgatctg ggggccacca 360
ccctgtgccg gtggcctctg ggctgcctcc cgtggt 396


136
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


136
ttatgcttcc ggctcgtntg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 60
acagctatga ccatgattac gccaagctat ttaggtgaca ctatagaata ctcaagctat 120
gcatcaagct tggtaccgag ctcggatcca ctagtaacgg ccgccagtgt gctggaattc 180
gcggncgntc nantctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 240
tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 300
cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 360
tcattctatt ctggggggtg gggtggggca ggacan 396


137
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


137
tttttttttt ttctgctttg tacttgagtt tatttcacaa aaccacggag aaagatactg 60
aaatggagct ctttccagcc tccaagcaag gaggccccag cagccagtct ccagcccctt 120
gagccctttt tgttaggccc acacccaaaa gagganaacc agtgtgtgcg cgaaggtaca 180
tggcaaggca cttttgaaaa catcccagtt taccgnggtg aaattgaact tactctgaaa 240
cagatgaaaa gggacatgca aaattgctga gcacatggag gtgtttgtta gtaggtgaaa 300
atcatgtcct gggtataacc cagcttctcc aggttagggt gagccgccgt ctggatcagt 360
ggtggcgggc cacacaccag gatgagcgtg gacttc 396


138
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


138
cccttttttt ttttttttac aaatgagaaa aatgtttatt aagaaaacaa tttagcagct 60
ctcctttana attttacaga ctaaagcaca acccgaaggc aattacagtt tcaatcatta 120
acacactact taaggngctt gcttactcta caactggaaa gttgctgaag tttgtgacat 180
gccactgtaa atgtaagtat tattaaaaat tacaaattgt ttggtgatta ttttgatgac 240
ctcttgagca gcagctcccc ccaanaatgc ancaatggta tgtggctcac cagctccata 300
tcggcaaaat tcgtggacat aatcatcttt caccattaca gataaaccat attcctgaag 360
gaagccagtg agacaagact tcaactttcc tatatc 396


139
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


139
ccgccctttt tttttttttt ttcacaaaag cactttttat ttgaggcaaa nagaagtctt 60
gctgaaagga ttccagttcc aagcagtcaa aactcaaccg ttagnggcac tattttgacc 120
tggtanattt tgcttctctt tggtcanaaa agggtattca ggttgtactt tccccagcag 180
ggtaaaaaga agggcaaagc aaactggaan anacttctac tctactgaca gggctnttga 240
natccaacat caagctanac acnccctcgc tggccactct acaggttgct gtcccactgc 300
tgagtgacac aggccatact acatttgcaa ggaaaaaaat gaggcaanaa acacaggtat 360
aggtcacttg gggacgagca ggcaaccaca gcttca 396


140
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


140
tttttttttt tttttttttt tttttttctc atttaacttt tttaatgggn ctcaaaattn 60
tgngacaaat ttttggtcaa gttgtttcca ttaaaaagtn ctgattttaa aaactaataa 120
cttaaaactg ccncncccaa aaaaaaaaac caaaggggtc cacaaaacat tntcctttcc 180
ttntgaaggn tttacnatgc attgttatca ttaaccagtn ttttactact aaacttaaan 240
ggccaattga aacaaacagt tntganaccg ttnttccncc actgattaaa agnggggggg 300
caggtattag ggataatatt catttancct tntgagcttt ntgggcanac ttggngacct 360
tgccagctcc agcagccttn ttgtccactg ntttga 396


141
396
DNA
Homo sapien

141
acgccgagcc acatcgctca gacaccatgg ggaaggtgaa ggtcggagtc aacggatttg 60
gtcgtattgg gcgcctggtc accagggctg cttttaactc tggtaaagtg gatattgttg 120
ccatcaatga ccccttcatt gacctcaact acatggttta catgttccaa tatgattcca 180
cccatggcaa attccatggc accgtcaagg ctgagaacgg gaagcttgtc atcaatggaa 240
atcccatcac catcttccag gagcgagatc cctccaaaat caagtggggc gatgctggcg 300
ctgagtacgt cgtggagtcc actggcgtct tcaccaccat ggagaaggct ggggctcatt 360
tgcagggggg agccaaaagg gtcatcatct ctgccc 396


142
396
DNA
Homo sapien

142
acgcaggaga ggaagcccag cctgttctac cagagaactt gcccaggtca gaggtctgcg 60
tagaagccct tttctgagca tcctctcctc tcctcacacc tgccactgtc ctctgcgttg 120
ctgtcgaatt aaatcttgca tcaccatggt gcacttctgt ggcctactca ccctccaccg 180
ggagccagtg ccgctgaaga gtatctctgt gagcgtgaac atttacgagt ttgtggctgg 240
tgtgtctgca actttgaact acgagaatga ggagaaagtt cctttggagg ccttctttgt 300
gttccccatg gatgaagact ctgctgttta cagctttgag gccttggtgg atgggaagaa 360
aattgtagca gaattacaag acaagatgaa ggcccg 396


143
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


143
tttttttttt tttccatana aaataggatt tattttcaca tttaaggnga acacaaatcc 60
atgttccana aatgttttat gcataacaca tcatgagtag attgaatttc tttaacacac 120
anaaaaatca aagcctacca ggaaatgctt ccctccggag cacaggagct tacaggccac 180
ttntgttagc aacacaggaa ttcacattgt ctaggcacag ctcaagngag gtttgttccc 240
aggttcaact gctcctaccc ccatgggccc tcctcaaaaa cgacagcagc aaaccaacag 300
gcttcacagt aaccaggagg aaagatctca gngggggaac cttcacaaaa gccctgagtt 360
gtgtttcaaa agccaagctc tggggtctgn ggcctg 396


144
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


144
tttttttttt tttcgctctt tggtctgaca agaaaagagt tttaggtgtg tgaagtaggg 60
tgggaaaaaa ggtcagtttc aaattcagta acatatggta acactaagtt aggctgctgc 120
attcttttct ttgggtactt aagccagctg gcacttccac tttgtaacca attatattat 180
gatcaacaac taatcagtta gttcctcagc ttcaactgaa nagttcctga ttacctgatg 240
aaggacatac ttgctctggc ttcaattagc atgctgtcaa gcatccctct ccatgcttaa 300
catggcaaca caaaacccaa gagtccttct ntttttttca ttagccatga ataaacactc 360
acaaagggga agagtagaca ctgcttttag taaacg 396


145
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


145
tttttttttt tttttttcaa tggatccgtt agctttacta ctaanatctt gctganatca 60
nanaagggct tctgggcagg ctgagcactg ggggtgtgca acatggtaac tctgaataan 120
anaaaccctg agttttactg ggcaaanaaa naacaagngg taggtatgat ttctgaacct 180
ggaaatagcg aaaatgaagg aaattccaaa agcgcgtatt tccaaataat gacaggccag 240
caagaggaca ccaaacctnt anaaagaggt attntttctt ccagctactg atggctttgg 300
catcccacag gcacattcct ttggccttca ggatcttana tgcanatgtg ganagtcaag 360
aggtaggctg actctgagtc ttcagctaaa ttcttt 396


146
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


146
tttttttttt ttttcattag caaggaagga tttatttttt cttttgaggg gagggcggaa 60
cagccgggat ttttggaaca ctacctttgt ctttcacttt gttgtttgtg tgttaacacn 120
aataaatcan aagcgacttt aaatctccct tcgcaggact gtcttcacgt atcagngcan 180
acaanaaaac agtggcttta caaaaaanat gttcaagtag gctgcacttt gcctctgngg 240
gtgaggcaca ctgngggana nacaaggtcc cctgnaacca gaggngggaa ggacanagct 300
ggctgactcc ctgctctccc gcattctctc ctccatgtgt tttgaanagg gaagcaacat 360
gttgaggtct gatcatttct acccagggaa cctgtt 396


147
396
DNA
Homo sapien

147
acggggaagc caagtgaccg tagtctcatc agacatgagg gaatgggtgg ctccagagaa 60
agcagacatc attgtcagtg agcttctggg ctcatttgct gacaatgaat tgtcgcctga 120
gtgcctggat ggagcccagc acttcctaaa agatgatggt gtgagcatcc ccggggagta 180
cacttccttt ctggctccca tctcttcctc caagctgtac aatgaggtcc gagcctgtag 240
ggagaaggac cgtgaccctg aggcccagtt tgagatgcct tatgtggtac ggctgcacaa 300
cttccaccag ctctctgcac cccagccctg tttcaccttc agccatccca acagagatcc 360
tatgattgac aacaaccgct attgcacctt ggaatt 396


148
396
DNA
Homo sapien

148
acgtcccatg attgttccag accatgactc ttcctggttg tgggtttgtt acagagcagg 60
agaagcagag gttatgacag ttatgcagac tttccccctc ctttttctct tttctcttcc 120
ccttgctttt ccactgtttc ttcctgctgc cacctgggcc ttgaattcct gggctgtgaa 180
gacatgtagc agctgcaggg tttaccacac gtgggagggc agcccagtac tgtccctctg 240
ccttccccac tttgagaata tggcagcccc tttcattcct ggcttggggt aggggagacc 300
attgaagtag aagcctcaaa gcagactttt ccctttactg tgtgtactcc aggacgaaga 360
aggaagatca tgcttgatac ttagattggt tttccc 396


149
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


149
tttttttttt tttaaagagt cacattttat tcaatgccta tttgtacatg ttactagcaa 60
taaactcttt tatctttaat tttgagaagt tttacaaata cagcaaagca gaatgactaa 120
tagagccggt aaccaggaca cagatttgga aaaataggtc taattggttg ttacactgtg 180
tttatgtcat acatttcgct tatttttatc aaanaaaaat cagaatttat aaaatgttaa 240
ttaaaaggaa aacattctga gtaaatttag tcccgtgttt cttcctccaa atctntttgt 300
tctacactaa caggtcagga taagtatgga tggggaggct ggaaaaaggg catccttccc 360
catgcggtcc ccagagccac cctctccaag caggac 396


150
396
DNA
Homo sapien

150
acgcctctct tcagttggca cccaaacatc tggattggca aatcagtggc aagaagttcc 60
agcatctgga cttttcagaa ttgatcttaa gtctactgtc atttccagat gcattatttt 120
acaactgtat ccttggaaat atatttctag ggagaatatt attgaagaaa atgttaatag 180
cctgagtcaa atttcagcag acttaccagc atttgtatca gtggtagcaa atgaagccaa 240
actgtatctt gaaaaacctg ttgttccttt aaatatgatg ttgccacaag ctgcattgga 300
gactcattgc agtaatattt ccaatgtgcc acctacaaga gagatacttc aagtctttct 360
tactgatgta cacatgaagg aagtaattca gcagtt 396


151
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


151
acaaaatgcc cagcctacag agtctgagaa ggaaatttat aatcaggtga atgtagtatt 60
aaaagatgca gaaggcatct tggaggactt gcagtcatac agaggagctg gccacgaaat 120
acgagaggca atccagcatc cagcanatga gaagttgcaa gagaaggcat ggggtgcagt 180
tgttccacta gtaggcaaat taaagaaatt ttacgaattt tctcagaggt tagaagcagc 240
attaagaggt cttctgggag ccttaacaag taccccatat tctcccaccc agcatctana 300
gcgagagcag gctcttgcta aacagtttgc anaaattctt catttcacac tccggtttga 360
tgaactcaag atgacaaatc ctgccataca gaatga 396


152
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


152
acgcagcgct cggcttcctg gtaattcttc acctcttttc tcagctccct gcagcatggg 60
tgctgggccc tccttgctgc tcgccgccct cctgctgctt ctctccggcg acggcgccgt 120
gcgctgcgac acacctgcca actgcaccta tcttgacctg ctgggcacct gggtcttcca 180
ggtgggctcc agcggttccc agcgcgatgt caactgctcg gttatgggac cacaagaaaa 240
aaaagtagng gtgtaccttc agaagctgga tacagcatat gatgaccttg gcaattctgg 300
ccatttcacc atcatttaca accaaggctt tgagattgtg ttgaatgact acaagtggtt 360
tgcctttttt aagtataaag aagagggcag caaggt 396


153
396
DNA
Homo sapien

153
ccagagacaa cttcgcggtg tggtgaactc tctgaggaaa aacacgtgcg tggcaacaag 60
tgactgagac ctagaaatcc aagcgttgga ggtcctgagg ccagcctaag tcgcttcaaa 120
atggaacgaa ggcgtttgcg gggttccatt cagagccgat acatcagcat gagtgtgtgg 180
acaagcccac ggagacttgt ggagctggca gggcagagcc tgctgaagga tgaggccctg 240
gccattgccg ccctggagtt gctgcccagg gagctcttcc cgccactctt catggcagcc 300
tttgacggga gacacagcca gaccctgaag gcaatggtgc aggcctggcc cttcacctgc 360
ctccctctgg gagtgctgat gaagggacaa catctt 396


154
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


154
acagcaaacc tcctcacagc ccactggtcc tcaagagggg cnacntcttc acacatcanc 60
acaactacgc attgcctccc tncactcgga aggactatcc tgctgccaag agggtcaagt 120
tggacagtgt cagagtcctg agacagatca gcaacaaccg aaaatgcacc agccccaggt 180
cctcggacac cgaggagaat gtcaagaggc gaacacacaa cgtcttggag cgccagagga 240
ggaacgagct aaaacggagc ttttttgccc tgcgtgacca gatcccggag ttggaaaaca 300
atgaaaaggc ccccaaggta gttatcctta aaaaagccac agcatacatc ctgtccgtcc 360
aagcagagga gcaaaagctc atttctgaag aggact 396


155
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


155
tttttttttt tgaananaca ggtctttaat gtacggagtc tcacaaggca caaacaccct 60
caccaggacc aaataaataa ctccacggtt gcaggaaggc gcggtctggg gaggatgcgg 120
catctgagct ctcccagggc tggtgggcga gccgggggtc tgcagtctgt gaggggcctc 180
ctgggtgtgt ccgggcctct anagcgggtc cagtctccag gatggggatc gctcactcac 240
tctccgagtc ggagtagtcc gccacgaggg aggagccgan actgcagggg tgccgcgtgt 300
cgggggtgtc agctgcctcc tgggaggagc ctgctggcna caggggcttg tcctgacggc 360
tcccttcctg ccccctcggg ctgctgcact tggggg 396


156
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


156
gaaggggggc ngggcagggg cggaatgtan anattantgc catgattgaa gatttaagaa 60
acgtgagatt caggattttc accacatccc catttagtta gcttgctcgt ttggctggtg 120
caaatgccag atggattatg aacaatgaca gtaaattaat gcaacataat caggtaatga 180
tgccaagcgt atctggtgtt ccaggtattg tacctttacc ggaacaaatc agtaaatcca 240
caatccctgg cacctgttag gcagctatta acctagtaaa tgctccccca tcccatctca 300
atcagcaang acaatcaaaa acatttgctt tnagtggcag gaacactggt acatttttac 360
ttgctccaag ggctgtgcca acgctccctc tctctg 396


157
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


157
tttttttttt tttttgggga atgtaaatct tttattaaaa cagttgtctt tccacagtag 60
taaagctttg gcacatacag tataaaaaat aatcacccac cataattata ccaaattcct 120
nttatcaact gcatactaag tgttttcaat acaatttttt ccgtataaaa atactgggaa 180
aaattgataa ataacaggta ananaaagat atttctaggc aattactagg atcatttgga 240
aaaagtgagt actgnggata tttaaaatat cacagtaaca agatcatgct tgttcctaca 300
gtattgcggg ccanacactt aagtgaaagc anaagtgttt gggtgacttt cctacttaaa 360
attttggnca tatcatttca aaacatttgc atcttg 396


158
396
DNA
Homo sapien

158
tttccgaaga cgggcagctt cagagaagag gattattcgg gagattgctg gtgtggccca 60
tagactcttt ggcatagact ctttcgcagg cagccactct gagtgtggcc agttctataa 120
ccatccccaa actagctgga gcctgatgga taggaacggg tagtctgtcc tcttccccat 180
aaaaatgttc caaaaagtta tctccagaga gagtccctta tgaagacagt tgccaagctg 240
tattctcatt ctttaaacca atacccaggt cagggctagt tcacactagc actgttaggg 300
acatggtgtg gctagaaatg aattgagtgt gacttctccc tacaacccca ggcccaggga 360
taggaggagg cagaggggtg cctggagttt ctgcac 396


159
396
DNA
Homo sapien

159
tccgcgcgtt gggaggtgta gcgcggctct gaacgcgctg agggccgttg agtgtcgcag 60
gcggcgaggg cgcgagtgag gagcagaccc aggcatcgcg cgccgagaag gccgggcgtc 120
cccacactga aggtccggaa aggcgacttc cgggggcttt ggcacctggc ggaccctccc 180
ggagcgtcgg cacctgaacg cgaggcgctc cattgcgcgt gcgcgttgag gggcttcccg 240
cacctgatcg cgagacccca acggctggtg gcgtcgcctg cgcgtctcgg ctgagctggc 300
catggcgcag ctgtgcgggc tgaggcggag ccgggcgttt ctcgccctgc tgggatcgct 360
gctcctctct ggggtcctgg cggccgaccg agaacg 396


160
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


160
ggaaaccttc tcaactaaga gaacatcatt tctggcaaac tatttttgtt agctcacaat 60
atatgtcgta cactctacaa tgtaaatagc actganccac ancttacaga aggtaaaaag 120
angnataana acttccttta caaaanantt cctgttgttc ttaatactcc ccattgctta 180
tganaattnt ctatangtct ctcangantg ttcgcaccca tttcttttnt aacttctact 240
aaaaanccat ttacattgna nagtgtacna cntatatttg ngagctaaca aaaaatngtt 300
ttccnganat gatgttcttt tagtttnaga nggttcnnnc aanttnctac tccngcccgc 360
cactgnncnc cacatttnnn naattacacc ncacng 396


161
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


161
tttttgtttg attattttta ttataatgaa attaaactta tgactattac agtatgctca 60
gcttaaaaca tttatgagta ctgcaaggac taacagaaac aggaaaaatc ctactaaaaa 120
tatttgttga tgggaaatca ttgtgaaagc aaacctccaa atattcattt gtaagccata 180
agaggataag cacaaccata tgggaggaga taaccagtct ctcccttcat atatattctt 240
ttttatttct tggtatacct tcccaaaaca nanacattca acagtagtta gaatggccat 300
ctcccaacat tttaaaaaaa ctgcnccccc caatgggtga acaaagtaaa gagtagtaac 360
ctanagttca gctgagtaag ccactgtgga gcctta 396


162
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


162
tttttttttt tttttttttt tttttttttt ttnggggncc aaattttttt ntttgaagga 60
angggacaaa nnaaaaaact taaggggntg ttttggnncn acttanaaaa aagggaaagg 120
aaaccccaac atgcatgccc tnccttgggg accanggaan ncnccccncn ggtntgggga 180
aantaacccn aggnttaact ttnattatca ctgncnccca gggggggctt nnaaaaaaaa 240
nnttccccca anccaaantn gggnncnccc attttncnca anttggncnc cnggncnccc 300
nattttttga ngggtttcnc cngcncattn agggaanggg nntcaannaa accncncaaa 360
ngggggnnat ttttntcang ggccnatttg ngcnnt 396


163
396
DNA
Homo sapien

163
cactgtccgg ctctaacaca gctattaagt gctacctgcc tctcaggcac tctcctcgcc 60
cagtttctga ggtcagacga gtgtctgcga tgtcttcccg cactctattc ccccagcctc 120
tttctgcttt catgctcagc acatcatctt cctaggcagt ctcttcccca aagtctcacc 180
ttttcttcca atagaaaatt ccgcttgacc tttggtgcac tgcccacttc ccagctccac 240
tggcccaagt ctgagccgga ggcccttgtt ttgggggcgg ggggagagtt ggatgtgatt 300
gcccttgaag aacaaggctg acctgagagg ttcctggcgc cctgaggtgg ctcagcacct 360
gcccagggta ggcctggcat gaggggttag gtcagc 396


164
396
DNA
Homo sapien

164
gacacgcggc ggtgtcctgt gttggccatg gccgactacc tgattagtgg gggcacgtcc 60
tacgtgccag acgacggact cacagcacag cagctcttca actgcggaga cggcctcacc 120
tacaatgact ttctcattct ccctgggtac atcgacttca ctgcagacca ggtggacctg 180
acttctgctc tgaccaagaa aatcactctt aagaccccac tggtttcctc tcccatggac 240
acagtcacag aggctgggat ggccatagca atggcgctta caggcggtat tggcttcatc 300
caccacaact gtacacctga attccaggcc aatgaagttc ggaaagtgaa gaaatatgaa 360
cagggattca tcacagaccc tgtggtcctc agcccc 396


165
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


165
tttttttttt tttttttttt ttttttcang ggncactgag gctttttatt ttgancncaa 60
aaccnccggg gatctancct gnggccnccc cggaaatnac ncnaggctca catnactnta 120
aacncttggg ggaaagggag gcaaaaaaaa caatgacttg ggccaattnc ncnactgcaa 180
agntananct gccaacaggg ctccagggag cttggnttnt gtaaaanttn taaggaagcg 240
gnncnaactc cncggggggg gggcnctaac tancagggac ccctgcaagn gttggncggg 300
ggcctcaacc tgcctgagct nacncaaggg gnggggtntn tntanccaac aggggaccna 360
agggcttgcc tncccacagn ttacttggcc aagggg 396


166
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


166
ttttttcaaa ttcagagcat ttttattaaa agaacaaaat attaaggcac aaaatacatc 60
aatttttcaa atgaaaaccc ttcaaacggt tatgtcctac attcaacgaa acttcttcca 120
aattacggaa taatttaact ttttaaaata naaaaataca agttcttaaa tgcctaaaat 180
ttctccccaa ataaatgttt tcttagtttt aatgaagtct cttcatgcag tactgagctc 240
caatattata atgtncactt ccttaaaaat ctagttttgc cacttatata cattcaatat 300
gtttaaccag tatattaacc agtatattaa ccaatatgtt aaacttcttt taagtataag 360
gcttggtatt ttgtattgct tattgcatgc tttgat 396


167
396
DNA
Homo sapien

167
tggcggcagc ggcggtggcg gtggctgagc agaggacccg gcgggcggcc tcgcgggtca 60
ggacacaatg tttgcacgag gactgaagag gaaatgtgtt ggccacgagg aagacgtgga 120
gggagccctg gccggcttga agacagtgtc ctcatacagc ctgcagcggc agtcgctcct 180
ggacatgtct ctggtgaagt tgcagctttg ccacatgctt gtggagccca atctgtgccg 240
ctcagtcctc attgccaaca cggtccggca gatccaagag gagatgacgc aggatgggac 300
gtggcgcaca gtggcacccc aggctgcaga gcgggcgccg ctcgaccgct tggtctccac 360
ggagatcctg tgccgtgcag cgtgggggca agaggg 396


168
396
DNA
Homo sapien

168
taggatggta agagtattat aaggattggt acaaggcatg atgagtcctt ttgcttttag 60
gcttttgact tctggtttta gactttcttt agcttctgtt gttagacaac attgtgcaag 120
cttggttttt ataagtttgc atggattaaa ctgaacttaa tgaaattgtc cctcccccca 180
aattctcagc acaattttta ggcccacaag gagtcaagca cctcaaggag atcttcagtt 240
tgaacttggt gtagacacag ggatactgat gaatcaatat tcaaattagc tgttacctac 300
ttaagaaaga gaggagacct tggggatttc gaggaagggt tcataaggga gattttagct 360
gagaaatacc atttgcacag tcaatcactt ctgacc 396


169
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


169
tttttttttt tttcanaatt aaattcttta atacaaaatg cttttttttt tttaaaanat 60
atctgtattt ctttgncgtt gttnaaaaat aaatatgtnc tacggaatat ntcnaaaaac 120
tgcnctaaaa acaaanacgn gatgttaata tcttttcccc ncaattntta cggataaaca 180
gtanccccna taaataaatg atancnaatn ttaaaattaa aaaagganan anatttagta 240
tgnaaaattc tctatttttt cttggtttgg ttttncntat aaaaaacana atagcaatgt 300
ntnttttatc anaatcccnt ntntncctaa acnttttttt ttttntttnc ccccnaatnc 360
aagnngccaa anatntntnt agnatgnana tgtntn 396


170
396
DNA
Homo sapien

170
tgagaagtac catgccgctt ctgcagagga acaggcaacc atcgaacgca acccctacac 60
catcttccat caagcactga aaaactgtga gcctatgatt gggctggtac ccatcctcaa 120
gggaggccgt ttctaccagg tccctgtacc cctacccgac cggcgtcgcc gcttcctagc 180
catgaagtgg atgatcactg agtgccggga taaaaagcac cagcggacac tgatgccgga 240
gaagctgtca cacaagctgc tggaggcttt ccataaccag ggccccgtga tcaagaggaa 300
gcatgacttg cacaagatgg cagaggccaa ccgtgccctg gcccactacc gctggtggta 360
gagtctccag gaggagccca gggccctctg cgcaag 396


171
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


171
ggtcctcgtc gtggtgagcg cagccactca ggctggtcct gggggtgggg ctgtagggga 60
aagtgctaaa gccgctgagt gaagtaagaa ctctgctaga gaggaaaatg ggcttgcttt 120
catcatcatc ctnctcagct ggtggggtca agtgggaagt tctgtcactg ggatctggtt 180
cagtgtctca agaccttgcc ccaccacgga aagccttttt cacntacccc aaaggacttg 240
gagagatgtt agaagatggn tctnaaanat tcctctgcna atntgttttt agctatcaag 300
tggcttcccc ccttaancag gnaaaacatg atcagcangt tgctcggatg gaaaaactan 360
cttggtttgn naaaaaanct ggaggcttga caatgg 396


172
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


172
agccttgggc caccctcttg gagcatctgg ctgtcgaatt cttgtgaccc tgttacacac 60
actggagaga atgggcagaa gtcgtggtgt tgcagccctg tgcattgggg gtgggatggg 120
aatagcaatg tgtgttcaga gagaatgaat tgcttaaact ttgaacaacc tcaatttctt 180
tttaaactaa taaagtacta ggttgcaata tgtgaaaaaa aaaaaaaaag ggcggccgnt 240
cnantntana gggcccnttn aaacccgttg atcaacctcg actgtgcctt ctagttgcca 300
gccatctgtt gttngcccct cccccgtgnc tttcttgacc ttgaaagggg ccccncccct 360
gtctttccta anaaaaanga agaantnncc ttccnt 396


173
396
DNA
Homo sapien

misc_feature
(1)...(396)
n = A,T,C or G


173
aagcatgtgg atatgtttag ctacgtttac tcacagccag cgaactgaca ttaaaataac 60
taacaaacag attcttttat gtgatgctgg aactcttgac agctataatt attattcaga 120
aatgactttt tgaaagtaaa agcagcataa agaatttgtc acaggaaggc tgtctcagat 180
aaattatggt aaaattttgc aggggacann ctttttaaga cttgcacaat tnccggatcc 240
tgcnctgact ttggaaaagg catatatgtn ctagnggcat gganaatgcc ccatactcat 300
gcatgcaaat taaacaacca agtttgaatc tttttggggg ngngctatnc tttaacccng 360
tacnggcntt attatntaan gnccctgnnn cntgtg 396


174
924
DNA
Homo sapiens

174
cctgacgacc cggcgacggc gacgtctctt ttgactaaaa gacagtgtcc agtgctccag 60
cctaggagtc tacggggacc gcctcccgcg ccgccaccat gcccaacttc tctggcaact 120
ggaaaatcat ccgatcggaa aacttcgagg aattgctcaa agtgctgggg gtgaatgtga 180
tgctgaggaa gattgctgtg gctgcagcgt ccaagccagc agtggagatc aaacaggagg 240
gagacacttt ctacatcaaa acctccacca ccgtgcgcac cacagagatt aacttcaagg 300
ttggggagga gtttgaggag cagactgtgg atgggaggcc ctgtaagagc ctggtgaaat 360
gggagagtga gaataaaatg gtctgtgagc agaagctcct gaagggagag ggccccaaga 420
cctcgtggac cagagaactg accaacgatg gggaactgat cctgaccatg acggcggatg 480
acgttgtgtg caccagggtc tacgtccgag agtgagtggc cacaggtaga accgcggccg 540
aagcccacca ctggccatgc tcaccgccct gcttcactgc cccctccgtc ccaccccctc 600
cttctaggat agcgctcccc ttaccccagt cacttctggg ggtcactggg atgcctcttg 660
cagggtcttg ctttctttga cctcttctct cctcccctac accaacaaag aggaatggct 720
gcaagagccc agatcaccca ttccgggttc actccccgcc tccccaagtc agcagtccta 780
gccccaaacc agcccagagc agggtctctc taaaggggac ttgagggcct gagcaggaaa 840
gactggccct ctagcttcta ccctttgtcc ctgtagccta tacagtttag aatatttatt 900
tgttaatttt attaaaatgc ttta 924


175
3321
DNA
Homo sapiens

175
atgaagattt tgatacttgg tatttttctg tttttatgta gtaccccagc ctgggcgaaa 60
gaaaagcatt attacattgg aattattgaa acgacttggg attatgcctc tgaccatggg 120
gaaaagaaac ttatttctgt tgacacggaa cattccaata tctatcttca aaatggccca 180
gatagaattg ggagactata taagaaggcc ctttatcttc agtacacaga tgaaaccttt 240
aggacaacta tagaaaaacc ggtctggctt gggtttttag gccctattat caaagctgaa 300
actggagata aagtttatgt acacttaaaa aaccttgcct ctaggcccta cacctttcat 360
tcacatggaa taacttacta taaggaacat gagggggcca tctaccctga taacaccaca 420
gattttcaaa gagcagatga caaagtatat ccaggagagc agtatacata catgttgctt 480
gccactgaag aacaaagtcc tggggaagga gatggcaatt gtgtgactag gatttaccat 540
tcccacattg atgctccaaa agatattgcc tcaggactca tcggaccttt aataatctgt 600
aaaaaagatt ctctagataa agaaaaagaa aaacatattg accgagaatt tgtggtgatg 660
ttttctgtgg tggatgaaaa tttcagctgg tacctagaag acaacattaa aacctactgc 720
tcagaaccag agaaagttga caaagacaac gaagacttcc aggagagtaa cagaatgtat 780
tctgtgaatg gatacacttt tggaagtctc ccaggactct ccatgtgtgc tgaagacaga 840
gtaaaatggt acctttttgg tatgggtaat gaagttgatg tgcacgcagc tttctttcac 900
gggcaagcac tgactaacaa gaactaccgt attgacacaa tcaacctctt tcctgctacc 960
ctgtttgatg cttatatggt ggcccagaac cctggagaat ggatgctcag ctgtcagaat 1020
ctaaaccatc tgaaagccgg tttgcaagcc tttttccagg tccaggagtg taacaagtct 1080
tcatcaaagg ataatatccg tgggaagcat gttagacact actacattgc cgctgaggaa 1140
atcatctgga actatgctcc ctctggtata gacatcttca ctaaagaaaa cttaacagca 1200
cctggaagtg actcagcggt gttttttgaa caaggtacca caagaattgg aggctcttat 1260
aaaaagctgg tttatcgtga gtacacagat gcctccttca caaatcgaaa ggagagaggc 1320
cctgaagaag agcatcttgg catcctgggt cctgtcattt gggcagaggt gggagacacc 1380
atcagagtaa ccttccataa caaaggagca tatcccctca gtattgagcc gattggggtg 1440
agattcaata agaacaacga gggcacatac tattccccaa attacaaccc ccagagcaga 1500
agtgtgcctc cttcagcctc ccatgtggca cccacagaaa cattcaccta tgaatggact 1560
gtccccaaag aagtaggacc cactaatgca gatcctgtgt gtctagctaa gatgtattat 1620
tctgctgtgg atcccactaa agatatattc actgggctta ttgggccaat gaaaatatgc 1680
aagaaaggaa gtttacatgc aaatgggaga cagaaagatg tagacaagga attctatttg 1740
tttcctacag tatttgatga gaatgagagt ttactcctgg aagataatat tagaatgttt 1800
acaactgcac ctgatcaggt ggataaggaa gatgaagact ttcaggaatc taataaaatg 1860
cactccatga atggattcat gtatgggaat cagccgggtc tcactatgtg caaaggagat 1920
tcggtcgtgt ggtacttatt cagcgccgga aatgaggccg atgtacatgg aatatacttt 1980
tcaggaaaca catatctgtg gagaggagaa cggagagaca cagcaaacct cttccctcaa 2040
acaagtctta cgctccacat gtggcctgac acagagggga cttttaatgt tgaatgcctt 2100
acaactgatc attacacagg cggcatgaag caaaaatata ctgtgaacca atgcaggcgg 2160
cagtctgagg attccacctt ctacctggga gagaggacat actatatcgc agcagtggag 2220
gtggaatggg attattcccc acaaagggag tgggaaaagg agctgcatca tttacaagag 2280
cagaatgttt caaatgcatt tttagataag ggagagtttt acataggctc aaagtacaag 2340
aaagttgtgt atcggcagta tactgatagc acattccgtg ttccagtgga gagaaaagct 2400
gaagaagaac atctgggaat tctaggtcca caacttcatg cagatgttgg agacaaagtc 2460
aaaattatct ttaaaaacat ggccacaagg ccctactcaa tacatgccca tggggtacaa 2520
acagagagtt ctacagttac tccaacatta ccaggtgaaa ctctcactta cgtatggaaa 2580
atcccagaaa gatctggagc tggaacagag gattctgctt gtattccatg ggcttattat 2640
tcaactgtgg atcaagttaa ggacctctac agtggattaa ttggccccct gattgtttgt 2700
cgaagacctt acttgaaagt attcaatccc agaaggaagc tggaatttgc ccttctgttt 2760
ctagtttttg atgagaatga atcttggtac ttagatgaca acatcaaaac atactctgat 2820
caccccgaga aagtaaacaa agatgatgag gaattcatag aaagcaataa aatgcatgct 2880
attaatggaa gaatgtttgg aaacctacaa ggcctcacaa tgcacgtggg agatgaagtc 2940
aactggtatc tgatgggaat gggcaatgaa atagacttac acactgtaca ttttcacggc 3000
catagcttcc aatacaagca caggggagtt tatagttctg atgtctttga cattttccct 3060
ggaacatacc aaaccctaga aatgtttcca agaacacctg gaatttggtt actccactgc 3120
catgtgaccg accacattca tgctggaatg gaaaccactt acaccgttct acaaaatgaa 3180
gacaccaaat ctggctgaat gaaataaatt ggtgataagt ggaaaaaaga gaaaaaccaa 3240
tgattcataa caatgtatgt gaaagtgtaa aatagaatgt tactttggaa tgactataaa 3300
cattaaaaga gactggagca t 3321


176
487
DNA
Homo sapiens

176
gaaatacttt ctgtcttatt aaaattaata aattattggt ctttacaaga cttggataca 60
ttacagcaga catggaaata taattttaaa aaatttctct ccaacctcct tcaaattcag 120
tcaccactgt tatattacct tctccaggaa ccctccagtg gggaaggctg cgatattaga 180
tttccttgta tgcaaagttt ttgttgaaag ctgtgctcag aggaggtgag aggagaggaa 240
ggagaaaact gcatcataac tttacagaat tgaatctaga gtcttccccg aaaagcccag 300
aaacttctct gcagtatctg gcttgtccat ctggtctaag gtggctgctt cttccccagc 360
catgagtcag tttgtgccca tgaataatac acgacctgtt atttccatga ctgctttact 420
gtatttttaa ggtcaatata ctgtacattt gataataaaa taatattctc ccaaaaaaaa 480
aaaaaaa 487


177
3999
DNA
Homo sapiens

177
caagattcca catttgatgg ggtgactgac aaacccatct tagactgctg tgcctgcgga 60
actgccaagt acagactcac attttatggg aattggtccg agaagacaca cccaaaggat 120
taccctcgtc gggccaacca ctggtctgcg atcatcggag gatcccactc caagaattat 180
gtactgtggg aatatggagg atatgccagc gaaggcgtca aacaagttgc agaattgggc 240
tcacccgtga aaatggagga agaaattcga caacagagtg atgaggtcct caccgtcatc 300
aaagccaaag cccaatggcc agcctggcag cctctcaacg tgagagcagc accttcagct 360
gaattttccg tggacagaac gcgccattta atgtccttcc tgaccatgat gggccctagt 420
cccgactgga acgtaggctt atctgcagaa gatctgtgca ccaaggaatg tggctgggtc 480
cagaaggtgg tgcaagacct gattccctgg gacgctggca ccgacagcgg ggtgacctat 540
gagtcaccca acaaacccac cattccccag gagaaaatcc ggcccctgac cagcctggac 600
catcctcaga gtcctttcta tgacccagag ggtgggtcca tcactcaagt agccagagtt 660
gtcatcgaga gaatcgcacg gaagggtgaa caatgcaata ttgtacctga caatgtcgat 720
gatattgtag ctgacctggc tccagaagag aaagatgaag atgacacccc tgaaacctgc 780
atctactcca actggtcccc atggtccgcc tgcagctcct ccacctgtga caaaggcaag 840
aggatgcgac agcgcatgct gaaagcacag ctggacctca gcgtcccctg ccctgacacc 900
caggacttcc agccctgcat gggccctggc tgcagtgacg aagacggctc cacctgcacc 960
atgtccgagt ggatcacctg gtcgccctgc agcatctcct gcggcatggg catgaggtcc 1020
cgggagaggt atgtgaagca gttcccggag gacggctccg tgtgcacgct gcccactgag 1080
gaaacggaga agtgcacggt caacgaggag tgctctccca gcagctgcct gatgaccgag 1140
tggggcgagt gggacgagtg cagcgccacc tgcggcatgg gcatgaagaa gcggcaccgc 1200
atgatcaaga tgaaccccgc agatggctcc atgtgcaaag ccgagacatc acaggcagag 1260
aagtgcatga tgccagagtg ccacaccatc ccatgcttgc tgtccccatg gtccgagtgg 1320
agtgactgca gcgtgacctg cgggaagggc atgcgaaccc gacagcggat gctcaagtct 1380
ctggcagaac ttggagactg caatgaggat ctggagcagg tggagaagtg catgctccct 1440
gaatgcccca ttgactgtga gctcaccgag tggtcccagt ggtcggaatg taacaagtca 1500
tgtgggaaag gccacgtgat tcgaacccgg atgatccaaa tggagcctca gtttggaggt 1560
gcaccctgcc cagagactgt gcagcgaaaa aagtgccgca tccgaaaatg ccttcgaaat 1620
ccatccatcc aaaagctacg ctggagggag gcccgagaga gccggcggag tgagcagctg 1680
aaggaagagt ctgaagggga gcagttccca ggttgtagga tgcgcccatg gacggcctgg 1740
tcagaatgca ccaaactgtg cggaggtgga attcaggaac gttacatgac tgtaaagaag 1800
agattcaaaa gctcccagtt taccagctgc aaagacaaga aggagatcag agcatgcaat 1860
gttcatcctt gttagcaagg gtacgagttc cccagggctg cactctagat tccagagtca 1920
ccaatggctg gattatttgc ttgtttaaga caatttaaat tgtgtacgct agttttcatt 1980
tttgcagtgt ggttcgccca gtagtcttgt ggatgccaga gacatccttt ctgaatactt 2040
cttgatgggt acaggctgag tggggcgccc tcacctccag ccagcctctt cctgcagagg 2100
agtagtgtca gccaccttgt actaagctga aacatgtccc tctggagctt ccacctggcc 2160
agggaggacg gagactttga cctactccac atggagaggc aaccatgtct ggaagtgact 2220
atgcctgagt cccagggtgc ggcaggtagg aaacattcac agatgaagac agcagattcc 2280
ccacattctc atctttggcc tgttcaatga aaccattgtt tgcccatctc ttcttagtgg 2340
aactttaggt ctcttttcaa gtctcctcag tcatcaatag ttcctgggga aaaacagagc 2400
tggtagactt gaagaggagc attgatgttg ggtggctttt gttctttcac tgagaaattc 2460
ggaatacatt tgtctcaccc ctgatattgg ttcctgatgc ccccccaaca aaaataaata 2520
aataaattat ggctgcttta tttaaatata aggtagctag tttttacacc tgagataaat 2580
aataagctta gagtgtattt ttcccttgct tttgggggtt cagaggagta tgtacaattc 2640
ttctgggaag ccagccttct gaactttttg gtactaaatc cttattggaa ccaagacaaa 2700
ggaagcaaaa ttggtctctt tagagaccaa tttgcctaaa ttttaaaatc ttcctacaca 2760
catctagacg ttcaagtttg caaatcagtt tttagcaaga aaacattttt gctatacaaa 2820
cattttgcta agtctgccca aagccccccc aatgcattcc ttcaacaaaa tacaatctct 2880
gtactttaaa gttattttag tcatgaaatt ttatatgcag agagaaaaag ttaccgagac 2940
agaaaacaaa tctaagggaa aggaatatta tgggattaag ctgagcaagc aattctggtg 3000
gaaagtcaaa cctgtcagtg ctccacacca gggctgtggt cctcccagac atgcatagga 3060
atggccacag gtttacactg ccttcccagc aattataagc acaccagatt cagggagact 3120
gaccaccaag ggatagtgta aaaggacatt ttctcagttg ggtccatcag cagtttttct 3180
tcctgcattt attgttgaaa actattgttt catttcttct tttataggcc ttattactgc 3240
ttaatccaaa tgtgtaccat tggtgagaca catacaatgc tctgaataca ctacgaattt 3300
gtattaaaca catcagaata tttccaaata caacatagta tagtcctgaa tatgtacttt 3360
taacacaaga gagactattc aataaaaact cactgggtct ttcatgtctt taagctaagt 3420
aagtgttcag aaggttcttt tttatattgt cctccacctc catcattttc aataaaagat 3480
agggcttttg ctcccttgtt cttggaggga ccattattac atctctgaac tacctttgta 3540
tccaacatgt tttaaatcct taaatgaatt gctttctccc aaaaaaagca caatataaag 3600
aaacacaaga tttaattatt tttctacttg gggggaaaaa agtcctcatg tagaagcacc 3660
cacttttgca atgttgttct aagctatcta tctaactctc agcccatgat aaagttcctt 3720
aagctggtga ttcctaatca aggacaagcc accctagtgt ctcatgtttg tatttggtcc 3780
cagttgggta cattttaaaa tcctgatttt ggagacttaa aaccaggtta atggctaaga 3840
atgggtaaca tgactcttgt tggattgtta ttttttgttt gcaatgggga atttataaga 3900
agcatcaagt ctctttctta ccaaagtctt gttaggtggt ttatagttct tttggctaac 3960
aaatcatttt ggaaataaag attttttact acaaaaatg 3999


178
1069
DNA
Homo sapiens

178
aaaaaagatg aataaatgaa taagagagat gaataaacaa atttacatta catgtgatag 60
ttatcatggt atggccttca tgacaagatg gatgagaata tcactgatag gatattagcc 120
ttctttcata tctttatatt gaaatatggg ctttacttca atttgaaggt ctttcatgaa 180
caataaaaga gagtagaagg actgtctgag aaggcaggag acatataaaa cagatgactg 240
aaagactgac tagctcctgg aaagggaaac atttggaaca tccagagtaa gggcaaatgg 300
gcttctacca gcacaacaaa gagcctccag gtggcaacat ggaagcaggt tatcagagaa 360
aataaatgtg caaattcctt atttacaatg actcacttaa ccccacaaac atgtttcact 420
gctgccttcc ccagttgtcg cttatgtact gttgttacct ttcagttaca tgcctttgat 480
cctaaaattc tctacttttg gtgccttatc agttctttgc aatctgcctg tggttatcag 540
cacttaaagc acaattttga aggggaaaaa aatgataatc accttagtcc caaagaaata 600
atttgtcaaa ctgccttatt agtattaaaa acagacacac tgaatgaagt agcatgatac 660
gcatatatcc tactcagtat cattggcctt ttatcaaatg gggaaactat acttttgtat 720
tacatagttt tagaaatcga aagttagaga ctctttataa gtaatgtcaa ggaacagtaa 780
tttaaaaaca aagttctaac aaatatattg tttgcttaat cacaatgccc tcaacttgta 840
tttgaataac taaataggac atgtcttcct tggagctgtg ggcattagtt cagaagcact 900
acctgcatct taattttcaa aacttaagtt ttattagcaa atcctcttct ctgtaagact 960
tagctatgaa gtggtatatt ttttccaaat atttttctga aaacatttgt tgttgtaact 1020
gcacaataaa agtccagttg caattaaaaa aaaaaaaaaa aaaaaaaaa 1069


179
1817
DNA
Homo sapiens

179
tgctattctg ccaaaagaca atttctagag tagttttgaa tgggttgatt tcccccactc 60
ccacaaactc tgaagccagt gtctagctta ctaaaaaaag agttgtatat aatatttaag 120
atgctgagta tttcatagga aagctgaatg ctgctgtaaa gtgctcttta agtctttttt 180
ttttttaatc cccttctaat gaatgaaact aggggaattt caggggacag agatgggatt 240
tgttgtatga taaactgtat gtagttttta gtctttctgt tttgagaagc agtggttggg 300
gcatttttaa gatggctggc tactcttgtt ttccctcatg ataataaatt tgtcataact 360
cagtaacatg aacttgcccc tagaggtagt tgttaataat tttgaaatat taaggtcttg 420
ccaagcttct gatgattcac acctgtacta ctgattatta agcaggacag actgagcttt 480
ctgttgcaaa taccttggag gagaaagtaa tttctaaata tacagagagg taacttgact 540
atatatgttg catcctgtgc ctcccttcat attaatattt gataaagatt ttaatttatg 600
taaaacttct aaagcagaat caaagctcct cttggggaaa tggcaagtct ttaggatagg 660
caagaccctg tatgaatagt accaaagcat taccgcatgg tagagaacac actcgattaa 720
aaatgttaag ctatctgaaa aataaaatgt gcaagtcttc aggatggcac aaaacaaagg 780
ttaatgcttc ttggggcaca tttcttagag ggcttgctga gtgtgtaaat ataatcgact 840
tttgtttgtg ttacatgact tctgtgactt cattgaaaat ctgcacaatt cagtttcagc 900
tctggattac ttcagttgac ctttgtgaag gtttttatct gtgtagaatg ggtgtttgac 960
ttgttttagc ctattaaatt tttattttct ttcactctgt attaaaagta aaacttacta 1020
aaagaaaaga ggtttgtgtt cacattaaat ggttttggtt tggcttcttt tagtcaggct 1080
ttctgaacat tgagatatcc tgaacttaga gctcttcaat cctaagattt tcatgaaaag 1140
cctctcactt gaacccaaac cagagtactc ttactgcctc ttttctaaat gttcaggaaa 1200
agcattgcca gttcagtctt ttcaaaatga gggagaaaca tttgcctgcc ttgtaataac 1260
aagactcagt gcttattttt taaactgcat tttaaaaatt ggatagtata ataacaataa 1320
ggagtaagcc accttttata ggcaccctgt agttttatag ttcttaatct aaacatttta 1380
tatttccttc ttttggaaaa aacctacatg ctacaagcca ccatatgcac agactataca 1440
gtgagttgag ttggctctcc cacagtcttt gaggtgaatt acaaaagtcc agccattatc 1500
atcctcctga gttatttgaa atgatttttt ttgtacattt tggctgcagt attggtggta 1560
gaatatacta taatatggat catctctact tctgtattta tttatttatt actagacctc 1620
aaccacagtc ttctttttcc ccttccacct ctctttgcct gtaggatgta ctgtatgtag 1680
tcatgcactt tgtattaata tattagaaat ctacagatct gttttgtact ttttatactg 1740
ttggatactt ataatcaaaa cttttactag ggtattgaat aaatctagtc ttactagaaa 1800
aaaaaaaaaa aaaaaaa 1817


180
2382
DNA
Homo sapiens

180
acttttattg gaagcagcag ccacatccct gcatgatttg cattgcaata caaccataac 60
cgggcagcca ctcctgagtg ataaccagta taacataaac gtagcagcct caatttttgc 120
ctttatgacg acagcttgtt atggttgcag tttgggtctg gctttacgaa gatggcgacc 180
gtaacactcc ttagaaactg gcagtcgtat gttagtttca cttgtctact ttatatgtct 240
gatcaatttg gataccattt tgtccagatg caaaaacatt ccaaaagtaa tgtgtttagt 300
agagagagac tctaagctca agttctggtt tatttcatgg atggaatgtt aattttatta 360
tgatattaaa gaaatggcct tttattttac atctctcccc tttttccctt tcccccttta 420
ttttcctcct tttctttctg aaagtttcct tttatgtcca taaaatacaa atatattgtt 480
cataaaaaat tagtatccct tttgtttggt tgctgagtca cctgaacctt aattttaatt 540
ggtaattaca gcccctaaaa aaaacacatt tcaaataggc ttcccactaa actctatatt 600
ttagtgtaaa ccaggaattg gcacactttt tttagaatgg gccagatggt aaatatttat 660
gcttcacggt ccatacagtc tctgtcacaa ctattcagtt ctgctagtat agcgtgaaag 720
cagctataca caatacagaa atgaatgagt gtggttatgt tctaataaaa cttatttata 780
aaaacaaggg gaggctgggt ttagcctgtg ggccatagtt tgtcaaccac tggtgtaaaa 840
ccttagttat atatgatctg cattttcttg aactgatcat tgaaaactta taaacctaac 900
agaaaagcca cataatattt agtgtcatta tgcaataatc acattgcctt tgtgttaata 960
gtcaaatact tacctttgga gaatacttac ctttggagga atgtataaaa tttctcaggc 1020
agagtcctgg atataggaaa aagtaattta tgaagtaaac ttcagttgct taatcaaact 1080
aatgatagtc taacaactga gcaagatcct catctgagag tgcttaaaat gggatcccca 1140
gagaccatta accaatactg gaactggtat ctagctactg atgtcttact ttgagtttat 1200
ttatgcttca gaatacagtt gtttgccctg tgcatgaata tacccatatt tgtgtgtgga 1260
tatgtgaagc ttttccaaat agagctctca gaagaattaa gtttttactt ctaattattt 1320
tgcattactt tgagttaaat ttgaatagag tattaaatat aaagttgtag attcttatgt 1380
gtttttgtat tagcccagac atctgtaatg tttttgcact ggtgacagac aaaatctgtt 1440
ttaaaatcat atccagcaca aaaactattt ctggctgaat agcacagaaa agtattttaa 1500
cctacctgta gagatcctcg tcatggaaag gtgccaaact gttttgaatg gaaggacaag 1560
taagagtgag gccacagttc ccaccacacg agggcttttg tattgttcta ctttttcagc 1620
cctttacttt ctggctgaag catccccttg gagtgccatg tataagttgg gctattagag 1680
ttcatggaac atagaacaac catgaatgag tggcatgatc cgtgcttaat gatcaagtgt 1740
tacttatcta ataatcctct agaaagaacc ctgttagatc ttggtttgtg ataaaaatat 1800
aaagacagaa gacatgagga aaaacaaaag gtttgaggaa atcaggcata tgactttata 1860
cttaacatca gatcttttct ataatatcct actactttgg ttttcctagc tccataccac 1920
acacctaaac ctgtattatg aattacatat tacaaagtca taaatgtgcc atatggatat 1980
acagtacatt ctagttggaa tcgtttactc tgctagaatt taggtgtgag attttttgtt 2040
tcccaggtat agcaggctta tgtttggtgg cattaaattg gtttctttaa aatgctttgg 2100
tggcactttt gtaaacagat tgcttctaga ttgttacaaa ccaagcctaa gacacatctg 2160
tgaatactta gatttgtagc ttaatcacat tctagacttg tgagttgaat gacaaagcag 2220
ttgaacaaaa attatggcat ttaagaattt aacatgtctt agctgtaaaa atgagaaagt 2280
gttggttggt tttaaaatct ggtaactcca tgatgaaaag aaatttattt tatacgtgtt 2340
atgtctctaa taaagtattc atttgataaa aaaaaaaaaa aa 2382


181
2377
DNA
Homo sapiens

181
atctttatgc aagacaagag tcagccatca gacactgaaa tatattatga tagattatga 60
agaattttct ctgtagaatt atattcttcc tggaacctgg tagagtagat tagactcaaa 120
ggctttttct tccttttctt actcctgttt tttccactca ctcttcccaa gagatttcct 180
aaagcttcaa gcttaataag cctaatagtg aaaaataact gaatttaatg gtataatgaa 240
gttcttcatt tccagacatc tttaattgat cttaaagctc atttgagtct ttgcccctga 300
acaaagacag acccattaaa atctaagaat tctaaatttt cacaactgtt tgagcttctt 360
ttcattttga aggatttgga atatatatgt tttcataaaa gtatcaagtg aaatatagtt 420
acatgggagc tcaatcatgt gcagattgca ttctgttatg ttgactcaat atttaattta 480
caactatcct tatttatatt gacctcaaga actccatttt atgcaatgca gaccactgag 540
atatagctaa cattctttca aataattttc cttttctttt ataattcctc tatagcaaat 600
ttttatgtat aactgattat acatatccat atttatattt cattgattcc aagacatcac 660
tttttcaatt taacatctct gaaattgtga catttcttgc aactgttggc acttcagatg 720
cagtgtttaa aattatgctt gaataaatat tacactaatc caactttacc taaatgttta 780
tgcatctagg caaattttgt tttcttataa agatttgaga gcccatttat gacaaaatat 840
gaaggcgaaa tttaaggaca actgagtcac gcacaactca acatggagcc taactgatta 900
tcagctcaga tcccgcatat cttgagttta caaaagctct ttcaggtccc catttatact 960
ttacgtgagt gcgaatgatt tcagcaaacc ctaacttaac taacaagaat gggtaggtat 1020
gtctacgttt cattaacaaa tttttattat ttttattcta ttatatgaga tccttttata 1080
ttatcatctc acttttaaac aaaattaact ggaaaaatat tacatggaac tgtcatagtt 1140
aggttttgca gcatcttaca tgtcttgtat caatggcagg agaaaaatat gataaaaaca 1200
atcagtgctg tgaaaaacaa ctttcttcta gagtcctctt actttttatt cttctttatc 1260
atttgtgggt ttttccccct tggctctcac tttaacttca agcttatgta acgactgtta 1320
taaaactgca tatttaaatt atttgaatta tatgaaataa ttgttcagct atctgggcag 1380
ctgttaatgt aaacctgaga gtaataacac tactctttta tctacctgga atacttttct 1440
gcataaaatt tatctttgta agctaactct attaatcagg tttcttctag cctctgcaac 1500
ctacttcagt tagaattgtc taatactgct ctattaatca ggtttctacc ctctacaacc 1560
tacttcagtt aaaattgtct aatacagcaa tatttaaaaa aaaaacactg caattgtcaa 1620
ggatggaaaa tgtgtgattt gtgtaaacaa tttttaccaa ctttacattt tcctacagat 1680
aaatgtgaaa ttttgataag aagtctacgc aatgacaagt acggtacata aattttatta 1740
agaatattga gtataaagta ctttaattct aaattataag aaaatataca tttgcacata 1800
ttaatataga aattcatttt gtgtatattt aacatagctt ttaaactatt ttacattagc 1860
tacttcatta tggtttcttg aacttctgaa aaaaattaga aatgtattaa acttatcagt 1920
aacataaaaa cttattttgt ttcacctaac gaatactgcg tttgtaaaaa taaatttaat 1980
atagaatata tttttaaatt aaatatttga atataaaata gctctaagaa agaagcaaat 2040
tatcactgaa catatttctt attatttctg gctttgaatt atacgtaact taaattgtct 2100
taaatgatac agaatattgg agaatatgat actttcacat aatatactat gaacctgttc 2160
atataactct gattgactac taacttctgt tttatgtatt tattaaagag ctgacactgt 2220
agtttgtggt gagatgttta tttttctaac agagcttata acagttagga caaggcattt 2280
aattaatgca tcattctgtt tagtagtagg tgttaatcaa tatgaaattc tctgttttaa 2340
aataaaaatg taaaaatcta aaaaaaaaaa aaaaaaa 2377


182
1370
DNA
Homo sapiens

182
tgtgagcatg gtattttgtc tcggaagaaa aaaatatggg tcaggcgcaa agtaagccca 60
ccccactggg aactatgtta aaaaaaaatt tcaagattta agggagatta cggtgttact 120
atgacaccag aaaaacttag aactttgtgt gaaatagact ggctaacatt agaggtgggt 180
tggctatcag aagaaagcct ggagaggtcc cttgtttcaa aggtatggca caaggtaacc 240
tgtaagccaa agcacccgga ccagtttcta tacatagaca gttacagctg gtttagaccc 300
cttccccctc tccccacagt agttaagaga acagcagcat aagcagctgg cagaggcaag 360
gaaagaccag cagagagaaa aaaaggccat ctataccaat tttaagttaa tttagactga 420
acaagggctt attaatagca aaggataatt gaaatcacaa acttataagg gtttcaacaa 480
aagtgaagtt tgctaaaagt taacagtgta acatgtatta tggtaacttc taatcttgtg 540
gccttagaca gtctagtcaa aacacataaa gaaagtttgc tttaaaaaaa caatggttat 600
cttcaaaaat aaaggggaga ggcagaattt atataaaaag agttatatga taaattcttg 660
tcctgaaata aattaactgg ttgtttaaag aaaagaatgt ttgtaataag tcaaaaagtt 720
aaaacatgtt taaaaaattg tctgcaaaag tcataaaaga aaaaatttta ttaaaaaaat 780
tttaagcaaa aaatgttgta taatttaaaa gtaataaggc ctcctgtgta ctattaagac 840
agatgcaaat tcctggttga aatggatcaa atattccatc tgcacattaa acaaaagcaa 900
ttgttatgct tgtgcacatg gcaggccaga ggccctgatt gtcccccttc cactaaggtg 960
gtcctctagt cgaccaggcg tggactgcat ggtagctctt ttccaggatt ctacagcctg 1020
gagtaataag tcatgccaag ctctctctgc tatatcccaa agtctctgcg ggtcagcccc 1080
caagggccat gcagcttctg tctcccaaca ctaagttcac ttcgtgtctc tcacggcaga 1140
gaggaaactt agtattcctt ggagacctga agggatgcag tgagcttaag aattttcaag 1200
agcttatcaa tcagtcagcc cttgttcatc cccgagtgga tgtgtggtgg tattgtggtg 1260
gacctttact gggcactctg ccaaataact agtgtggcac ttgtgcttta gtccatttgg 1320
ctatcccttt caccctggca tttcatcaac caaaaaaaaa aaaaaaaaaa 1370


183
2060
DNA
Homo sapiens

misc_feature
(1)...(2060)
n=A,T,C or G


183
gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60
gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120
gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180
gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240
tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300
cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360
tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420
ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480
agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540
tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600
tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660
cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720
caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780
catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840
tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900
cttctccaga gcaggtgact caggagagga caaggtgaga gcccagcacc ttatggtcta 960
gtctcagaag tcacacgcca tcatttctgc aatgtcattt tggggttcca ggtcagctgt 1020
atcactgtgg gaggtgagta tatagatgtc ctagaccatt caggctgcta tgacagaaca 1080
ccatgaactg agtggctcat gaacaacaga aatttcccac agttctgtag gctgggaaat 1140
ccaagatcaa ggtggcagca ggttcagcgt ctgctaagct cctgcttttc atggattgca 1200
tcttctcact gtgtcctcac gtgatggaca gagcaaatga gctctcaggc actagtccca 1260
gccatgagga ctctgctttc atgactcatc actccgcaaa ggcccacctc catcagaaga 1320
cagctgctaa ctgcagctgc catcctccaa gacgggagac acagaattgg gggacatata 1380
cattgagatc tgaaaggcct ggacagcaac aggtggggat cgtgggggca tcttggaggg 1440
tggctgccgc agtaacattt ctgacccatg ctttctgctt gcactcatct cctgcctttg 1500
atcttcatta tctcargcag tccccacaac gactgtatct aggagttcat tttaccctca 1560
ttttacagat gaaacgtctc agagggtaat gtgcttgccc agtgtctcac aaatgcaaag 1620
tcactgaggt aggatttcaa cctaggtcca atcatctctg cagcattagg ggttcaccat 1680
tgccatagac ttaactgtgt cccccaaaat ttgtatgttg aagccctacc agcctccccc 1740
ccccaatgtg ctgatgtttg gagaaagggc ctttgggagg taattaggtt tagatgagat 1800
catgagggtg ggactctcat aatggcatta atgccatcag gtgaagagat accagagacc 1860
ttgtgtcctc tctctctgca atgtgaggac acagtgagaa ggcagctgtc tgcaagctgg 1920
gaagagagta ctgaccagga acttaatcag agggcatctt gatcttggac ttcccagcct 1980
ccagaactct gaaaagttaa tgnctattat ttaagccacg cagtctatgg aattttgtta 2040
gagccaaccc caagcttact 2060


184
3079
DNA
Homo sapiens

184
ggcacaaagt tgggggccgc gaagatgagg ctgtccccgg cgcccctgaa gctgagccgg 60
actccggcac tgctggccct ggcgctgccc ctggccgcgg cgctggcctt ctccgacgag 120
accctggaca aagtgcccaa gtcagagggc tactgtagcc gtatcctgcg cgcccagggc 180
acgcggcgcg agggctacac cgagttcagc ctccgcgtgg agggcgaccc cgacttctac 240
aagccgggaa ccagctaccg cgtaacactt tcagctgctc ctccctccta cttcagagga 300
ttcacattaa ttgccctcag agagaacaga gagggtgata aggaagaaga ccatgctggg 360
accttccaga tcatagacga agaagaaact cagtttatga gcaattgccc tgttgcagtc 420
actgaaagca ctccacggag gaggacccgg atccaggtgt tttggatagc accaccagcg 480
ggaacaggct gcgtgattct gaaggccagc atcgtacaaa aacgcattat ttattttcaa 540
gatgagggct ctctgaccaa gaaactttgt gaacaagatt ccacatttga tggggtgact 600
gacaaaccca tcttagactg ctgtgcctgc ggaactgcca agtacagact cacattttat 660
gggaattggt ccgagaagac acacccaaag gattaccctc gtcgggccaa ccactggtct 720
gcgatcatcg gaggatccca ctccaagaat tatgtactgt gggaatatgg aggatatgcc 780
agcgaaggcg tcaaacaagt tgcagaattg ggctcacccg tgaaaatgga ggaagaaatt 840
cgacaacaga gtgatgaggt cctcaccgtc atcaaagcca aagcccaatg gccagcctgg 900
cagcctctca acgtgagagc agcaccttca gctgaatttt ccgtggacag aacgcgccat 960
ttaatgtcct tcctgaccat gatgggccct agtcccgact ggaacgtagg cttatctgca 1020
gaagatctgt gcaccaagga atgtggctgg gtccagaagg tggtgcaaga cctgattccc 1080
tgggacgctg gcaccgacag cggggtgacc tatgagtcac ccaacaaacc caccattccc 1140
caggagaaaa tccggcccct gaccagcctg gaccatcctc agagtccttt ctatgaccca 1200
gagggtgggt ccatcactca agtagccaga gttgtcatcg agagaatcgc acggaagggt 1260
gaacaatgca atattgtacc tgacaatgtc gatgatattg tagctgacct ggctccagaa 1320
gagaaagatg aagatgacac ccctgaaacc tgcatctact ccaactggtc cccatggtcc 1380
gcctgcagct cctccacctg tgacaaaggc aagaggatgc gacagcgcat gctgaaagca 1440
cagctggacc tcagcgtccc ctgccctgac acccaggact tccagccctg catgggccct 1500
ggctgcagtg acgaagacgg ctccacctgc accatgtccg agtggatcac ctggtcgccc 1560
tgcagcatct cctgcggcat gggcatgagg tcccgggaga ggtatgtgaa gcagttcccg 1620
gaggacggct ccgtgtgcac gctgcccact gaggaaatgg agaagtgcac ggtcaacgag 1680
gagtgctctc ccagcagctg cctgatgacc gagtggggcg agtgggacga gtgcagcgcc 1740
acctgcggca tgggcatgaa gaagcggcac cgcatgatca agatgaaccc cgcagatggc 1800
tccatgtgca aagccgagac atcacaggca gagaagtgca tgatgccaga gtgccacacc 1860
atcccatgct tgctgtcccc atggtccgag tggagtgact gcagcgtgac ctgcgggaag 1920
ggcatgcgaa cccgacagcg gatgctcaag tctctggcag aacttggaga ctgcaatgag 1980
gatctggagc aggtggagaa gtgcatgctc cctgaatgcc ccattgactg tgagctcacc 2040
gagtggtccc agtggtcgga atgtaacaag tcatgtggga aaggccacgt gattcgaacc 2100
cggatgatcc aaatggagcc tcagtttgga ggtgcaccct gcccagagac tgtgcagcga 2160
aaaaagtgcc gcatccgaaa atgccttcga aatccatcca tccaaaagcc acgctggagg 2220
gaggcccgag agagccggcg gagtgagcag ctgaaggaag agtctgaagg ggagcagttc 2280
ccaggttgta ggatgcgccc atggacggcc tggtcagaat gcaccaaact gtgcggaggt 2340
ggaattcagg aacgttacat gactgtaaag aagagattca aaagctccca gtttaccagc 2400
tgcaaagaca agaaggagat cagagcatgc aatgttcatc cttgttagca agggtacgag 2460
ttccccaggg ctgcactcta gattccagag tcaccaatgg ctggattatt tgcttgttta 2520
agacaattta aattgtgtac gctagttttc atttttgcag tgtggttcgc ccagtagtct 2580
tgtggatgcc agagacatcc tttctgaata cttcttgatg ggtacaggct gagtggggcg 2640
ccctcacctc cagccagcct cttcctgcag aggagtagtg tcagccacct tgtactaagc 2700
tgaaacatgt ccctctggag cttccacctg gccagggagg acggagactt tgacctactc 2760
cacatggaga ggcaaccatg tctggaagtg actatgcctg agtcccaggg tgcggcaggt 2820
aggaaacatt cacagatgaa gacagcagat tccccacatt ctcatctttg gcctgttcaa 2880
tgaaaccatt gtttgcccat ctcttcttag tggaacttta ggtctctttt caagtctcct 2940
cagtcatcaa tagttcctgg ggaaaaacag agctggtaga cttgaagagg agcattgatg 3000
ttgggtggct tttgttcttt cactgagaaa ttcggaatac atttgtctca cccctgatat 3060
tggttcctga tgccccagc 3079


185
3000
DNA
Homo sapiens

185
gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60
gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120
gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180
gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240
tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300
cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360
tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420
ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480
agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540
tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600
tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660
cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720
caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780
catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840
tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900
cttctccaga gcaggtgact caggagagga caaggtgaga gccacagcac cttatggtct 960
agtctcagaa gtcacacgcc atcatttctg caatgtcatt ttggggttcc aggtcagctg 1020
tatcactgtg ggaggtgagt atatagatgt cctagaccat tcaggctgct atgacagaac 1080
accatgaact gagtggctca tgaacaacag aaatttccca cagttctgta ggctgggaaa 1140
tccaagatca aggtggcagc aggttcagcg tctgctaagc tcctgctttt catggattgc 1200
atcttctcac tgtgtcctca cgtgatggac agagcaaatg agctctcagg cactagtccc 1260
agccatgagg actctgcttt catgactcat cactccgcaa aggcccacct ccatcagaag 1320
acagctgcta actgcagctg ccatcctcca agacgggaga cacagaattg ggggacatat 1380
acattgagat ctgaaaggcc tggacagcaa caggtgggga tcgtgggggc atcttggagg 1440
gtggctgccg cagtaacatt tctgacccat gctttctgct tgcactcatc tcctgccttt 1500
gatcttcatt atctcaggca gtccccacaa cgactgtatc taggagttca ttttaccctc 1560
attttacaga tgaaacgtct cagagggtaa tgtgcttgcc cagtgtctca caaatgcaaa 1620
gtcactgagg taggatttca acctaggtcc aatcatctct gcagcattag gggttcacca 1680
ttgccataga cttaactgtg tcccccaaaa tttgtatgtt gaagccctac cagcctcccc 1740
cccccaatgt gctgatgttt ggagaaaggg cctttgggag gtaattaggt ttagatgaga 1800
tcatgagggt gggactctca taatggcatt aatgccatca ggtgaagaga taccagagac 1860
cttgtgtcct ctctctctgc aatgtgagga cacagtgaga aggcagctgt ctgcaagctg 1920
ggaagagagt actgaccagg aacttaatca gagggcatct tgatcttgga cttcccagcc 1980
tccagaactc tgaaaagtta atgtctatta tttaagccac gcagtctatg gaattttgtt 2040
agagccaacc caagcttact aagataatca gtatgctgca ctttctataa atgtaatttt 2100
tacatttata aaaacaaaac aagagatttg ctgctctata acaactgtac ctacattgta 2160
gatggaataa caaatctaca tacagattta gtaatctcta tgtagatata gaacatagtg 2220
tatctaatag agacatagtg tctgtggtct gatgttaatt ttaggaatta gccgtcactg 2280
attgggcctt gtccaggtat tcttctccct tgtcctggct ctgtaaccta gttatccttg 2340
tctttgctaa cccataacca actattgtat caggactatt atgccactac agatgatgca 2400
gtttgggttt actgtttctc accatttaga caatacttca tcaaatatat ttctgtatga 2460
ctttagtgat atcagttttt gattcattcc tgcatagatc tgggcaaatt gtagacctta 2520
ggaggtgtat tcaccatcca gttctctgga actgcttatg acatttttct ctgagctttc 2580
ttgtcccaaa aggagccttc ctaaaatagt ctttaagtgc ctttaaaaag agaaagagaa 2640
attaagagaa aaaaaacccc aaactcattc ctttactctg atgtgacagt cctcccagga 2700
cactgcagtg gcctgagttt tgctgttaat ttcattcact tatgtttggg ctatgtaaat 2760
tctgcctaga gctggaatgt cattatgtaa agaaatattt tttgtttata ttctttaata 2820
gtaccagtaa tgtatatctt attcagcttc gagaatataa ttgggttgtt tataaaaacc 2880
acacatcatc aaactcacat tgtaacgatt atttcacttt tcaaaaaaaa tggcattaga 2940
aaaacttgaa tgatgttagt tatcttaaag aagtgtgtac tatgtttaaa aaaaaaaaaa 3000


186
807
PRT
Homo sapiens

186
Met Arg Leu Ser Pro Ala Pro Leu Lys Leu Ser Arg Thr Pro Ala Leu
5 10 15
Leu Ala Leu Ala Leu Pro Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu
20 25 30
Thr Leu Asp Lys Val Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu
35 40 45
Arg Ala Gln Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg
50 55 60
Val Glu Gly Asp Pro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val
65 70 75 80
Thr Leu Ser Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile
85 90 95
Ala Leu Arg Glu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly
100 105 110
Thr Phe Gln Ile Ile Asp Glu Glu Glu Thr Gln Phe Met Ser Asn Cys
115 120 125
Pro Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg Ile Gln
130 135 140
Val Phe Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Ile Leu Lys
145 150 155 160
Ala Ser Ile Val Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser
165 170 175
Leu Thr Lys Lys Leu Cys Glu Gln Asp Ser Thr Phe Asp Gly Val Thr
180 185 190
Asp Lys Pro Ile Leu Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Arg
195 200 205
Leu Thr Phe Tyr Gly Asn Trp Ser Glu Lys Thr His Pro Lys Asp Tyr
210 215 220
Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile Gly Gly Ser His Ser
225 230 235 240
Lys Asn Tyr Val Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val
245 250 255
Lys Gln Val Ala Glu Leu Gly Ser Pro Val Lys Met Glu Glu Glu Ile
260 265 270
Arg Gln Gln Ser Asp Glu Val Leu Thr Val Ile Lys Ala Lys Ala Gln
275 280 285
Trp Pro Ala Trp Gln Pro Leu Asn Val Arg Ala Ala Pro Ser Ala Glu
290 295 300
Phe Ser Val Asp Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met
305 310 315 320
Gly Pro Ser Pro Asp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys
325 330 335
Thr Lys Glu Cys Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro
340 345 350
Trp Asp Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys
355 360 365
Pro Thr Ile Pro Gln Glu Lys Ile Arg Pro Leu Thr Ser Leu Asp His
370 375 380
Pro Gln Ser Pro Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gln Val
385 390 395 400
Ala Arg Val Val Ile Glu Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn
405 410 415
Ile Val Pro Asp Asn Val Asp Asp Ile Val Ala Asp Leu Ala Pro Glu
420 425 430
Glu Lys Asp Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Asn Trp
435 440 445
Ser Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Asp Lys Gly Lys Arg
450 455 460
Met Arg Gln Arg Met Leu Lys Ala Gln Leu Asp Leu Ser Val Pro Cys
465 470 475 480
Pro Asp Thr Gln Asp Phe Gln Pro Cys Met Gly Pro Gly Cys Ser Asp
485 490 495
Glu Asp Gly Ser Thr Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro
500 505 510
Cys Ser Ile Ser Cys Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val
515 520 525
Lys Gln Phe Pro Glu Asp Gly Ser Val Cys Thr Leu Pro Thr Glu Glu
530 535 540
Met Glu Lys Cys Thr Val Asn Glu Glu Cys Ser Pro Ser Ser Cys Leu
545 550 555 560
Met Thr Glu Trp Gly Glu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met
565 570 575
Gly Met Lys Lys Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly
580 585 590
Ser Met Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met Met Pro
595 600 605
Glu Cys His Thr Ile Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser
610 615 620
Asp Cys Ser Val Thr Cys Gly Lys Gly Met Arg Thr Arg Gln Arg Met
625 630 635 640
Leu Lys Ser Leu Ala Glu Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln
645 650 655
Val Glu Lys Cys Met Leu Pro Glu Cys Pro Ile Asp Cys Glu Leu Thr
660 665 670
Glu Trp Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys Gly Lys Gly His
675 680 685
Val Ile Arg Thr Arg Met Ile Gln Met Glu Pro Gln Phe Gly Gly Ala
690 695 700
Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg Ile Arg Lys Cys
705 710 715 720
Leu Arg Asn Pro Ser Ile Gln Lys Pro Arg Trp Arg Glu Ala Arg Glu
725 730 735
Ser Arg Arg Ser Glu Gln Leu Lys Glu Glu Ser Glu Gly Glu Gln Phe
740 745 750
Pro Gly Cys Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cys Thr Lys
755 760 765
Leu Cys Gly Gly Gly Ile Gln Glu Arg Tyr Met Thr Val Lys Lys Arg
770 775 780
Phe Lys Ser Ser Gln Phe Thr Ser Cys Lys Asp Lys Lys Glu Ile Arg
785 790 795 800
Ala Cys Asn Val His Pro Cys
805


187
892
DNA
Homo sapiens

187
tttattgatg tttcaacagg cacttattca aataagttat atatttgaaa acagccatgg 60
taagcatcct tggcttctca cccattcctc atgtggcatg ctttctagac tttaaaatga 120
ggtaccctga atagcactaa gtgctctgta agctcaagga atctgtgcag tgctacaaag 180
cccacaggca gagaaagaac tcctcaagtg cttgtggtca gagactaggt tccatatgag 240
gcacacctat gatgaaggtc ttcacctcca gaaggtgaca ctgttcagag atcctcattt 300
cctggagagt gggagaaaat ccctcctttg ggaaatccct tttcccagca gcagagccca 360
cctcattgct tagtgatcat ttggaaggca ctgagagcct tcaggggctg acagcagaga 420
aatgaaaatg agtacagttc agatggtgga agaagcatgg cagtgacatc ttccatgctc 480
tttttctcag tgtctgcaac tccaaagatc aaggccataa cccaggagac catcaacgga 540
agattagttc tttgtcaagt gaatgaaatc caaaagcacg catgagacca atgaaagttt 600
ccgcctgttg taaaatctat tttcccccaa ggaaagtcct tgcacagaca ccagtgagtg 660
agttctaaaa gatacccttg gaattatcag actcagaaac ttttattttt tttttctgta 720
acagtctcac cagacttctc ataatgctct taatatattg cacttttcta atcaaagtgc 780
gagtttatga gggtaaagct ctactttcct actgcagcct tcagattctc atcattttgc 840
atctattttg tagccaataa aactccgcac tagcaaaaaa aaaaaaaaaa aa 892


188
1448
DNA
Homo sapiens

misc_feature
(1)...(1448)
n = A,T,C or G


188
tgtgactcac atttctttta ctgtgacaca ataatgtgat cctaaaactg gcttatcctt 60
gagtgtttac aactcaaaca actttttgaa tgcagtagtt tttttttttt aaaaacaaac 120
ttttatgtca aatttttttt cttagaagta gtcttcatta ttataaattt gtacaccaaa 180
aggccatggg gaactttgtg caagtacctc atcgctgagc aaatggagct tgctatgttt 240
taatttcaga aaatttcctc atatacgtag tgtgtagaat caagtctttt aataattcat 300
tttttcttca taatatttac tcaaagttaa gcttaaaaat aagttttatc ttaaaatcat 360
atttgaagac agtaagacag taaactattt taggaagtca acccccattg cactctgtgg 420
cagttattct ggtaaaaata ggcaaaagtg acctgaatct acaatggtgt cccaaagtaa 480
ccaagtaaga gagattgtaa atgataaacc gagctttaaa ggataaagtg ttaataaaga 540
aaggaagctg ggcacatgtc aaaaagggag atcgaaatgt taggtaatca tttagaaagg 600
acagaaaata tttaaagtgg ctcataggta atgaatattt ctgacttaga tgtaaatcca 660
tctggaatct ttacatcctt tgccagctga aacaagaaag tgaagggaca atgatatttc 720
atggtcagtt tattttgtaa gagacagaag aaattatatc tatacattac cttgtagcag 780
cagtacctgg aagccccagc ccgtcacaga agtgtggagg ggggctcctg actagacaat 840
ttccctagcc cttgtgattt gaagcatgaa agttctggca ggttatgagc agcactaggg 900
ataaagtatg gttttatttt ggtgtaattt aggtttttca acaaagccct tgtctaaaat 960
aaaaggcatt attggaaata tttgaaaact agaaaatgat ggataaaagg gctgataaga 1020
aaatttctga ctgtcagtag aagtgagata agatcctcag aggaaacagt aagaagggat 1080
aatcattaag atagtaaaac aggcaaagca gaatcacatg tgcncacaca catacacatg 1140
taaacattgg aatgcataag ttttaatatt ttagcgctat cagtttctaa atgcattaat 1200
tactaactgc cctctcccaa gattcattta gttcaaacag tatccgtaaa ctaggaataa 1260
tgccacatgc attcaatggg atcttttaag tactcttcag tttgttccaa gaaatgtgcc 1320
tactgaaatc aaattaattt gtattcaatg tgtacttcaa gactgctaat tgtttcatct 1380
gaaagcctac aatgaatcat tgttcamcct tgaaaaataa aattttgtaa atcaaaaaaa 1440
aaaaaaaa 1448


189
460
DNA
Homo sapiens

189
ttttgggagc acggactgtc agttctctgg gaagtggtca gcgcatcctg cagggcttct 60
cctcctctgt cttttggaga accagggctc ttctcagggg ctctagggac tgccaggctg 120
tttcagccag gaaggccaaa atcaagagtg agatgtagaa agttgtaaaa tagaaaaagt 180
ggagttggtg aatcggttgt tctttcctca catttggatg attgtcataa ggtttttagc 240
atgttcctcc ttttcttcac cctccccttt tttcttctat taatcaagag aaacttcaaa 300
gttaatggga tggtcggatc tcacaggctg agaactcgtt cacctccaag catttcatga 360
aaaagctgct tcttattaat catacaaact ctcaccatga tgtgaagagt ttcacaaatc 420
cttcaaaata aaaagtaatg acttaaaaaa aaaaaaaaaa 460


190
481
DNA
Homo sapiens

190
aggtggtgga agaaactgtg gcacgaggtg actgaggtat ctgtgggagc taatcctgtc 60
caggtggaag taggagaatt tgatgatggt gcagaggaaa ccgaagagga ggtggtggcg 120
gaaaatccct gccagaacca ccactgcaaa cacggcaagg tgtgcgagct ggatgagaac 180
aacaccccca tgtgcgtgtg ccaggacccc accagctgcc cagcccccat tggcgagttt 240
gagaaggtgt gcagcaatga caacaagacc ttcgactctt cctgccactt ctttgccaca 300
aagtgcaccc tggagggcac caagaagggc cacaagctcc acctggacta catcgggcct 360
tgcaaataca tccccccttg cctggactct gagctgaccg aattccccct gcgcatgcgg 420
gactggctca agaacgtcct ggtcaccctg tatgagaggg atgaggacaa caaccttctg 480
a 481


191
489
DNA
Homo sapiens

misc_feature
(1)...(489)
n = A,T,C or G


191
atataaatta gactaagtgt tttcaaataa atctaaatct tcagcatgat gtgttgtgta 60
taattggagt agatattaat taagtcccct gtataatgtt ttgtaatttt gcaaaacata 120
tcttgagttg tttaaacagt caaaatgttt gatattttat accagcttat gagctcaaag 180
tactacagca aagcctagcc tgcatatcat tcacccaaaa caaagtaata gcgcctcttt 240
tattattttg actgaatgtt ttatggaatt gaaagaaaca tacgttcttt tcaagacttc 300
ctcatgaatc tntcaattat aggaaaagtt attgtgataa aataggaaca gctgaaagat 360
tgattaatga actattgtta attcttccta ttttaatgaa tgacattgaa ctgaattttt 420
tgtctgttaa atgaacttga tagctaataa aaagncaact agccatcaaa aaaaaaaaaa 480
aaaaaaaaa 489


192
516
DNA
Homo sapiens

192
acttcaaagc cagctgaagg aaagaggaag tgctagagag agcccccttc agtgtgcttc 60
tgacttttac ggacttggct tgttagaagg ctgaaagatg atggcaggaa tgaaaatcca 120
gcttgtatgc atgctactcc tggctttcag ctcctggagt ctgtgctcag attcagaaga 180
ggaaatgaaa gcattagaag cagatttctt gaccaatatg catacatcaa agattagtaa 240
agcacatgtt ccctcttgga agatgactct gctaaatgtt tgcagtcttg taaataattt 300
gaacagccca gctgaggaaa caggagaagt tcatgaagag gagcttgttg caagaaggaa 360
cttcttactg ctttagatgg ctttagcttg gaagcaatgt tgacaatata ccagctccac 420
aaaatctgtc acagcagggc ttttcaacac tgggagttaa tccaggaaga tattcttgat 480
actggaaatg acaaaaatgg aaaggaagaa gtcata 516


193
1409
DNA
Homo sapiens

193
tgattctttt ccaaaacttt tagccatagg gtcttttata gacagggata gtaaaatgaa 60
aattgagaaa tataagatga aaaggaatgg taaaaatatc ttttaggggg cttttaattg 120
gtgatctgaa atcttgggag aagctgttct tttcaggcct gaggtgctct tgactgtcgc 180
ctgcgcactg tgtaccccga gcaacattct aagggtgtgc tttcgccttg gctaactcct 240
ttgacctcat tcttcatata gtagtctagg aaaaagttgc aggtaattta aactgtctag 300
tggtacatag taactgaatt tctattccta tgagaaatga gaattattta tttgccatca 360
acacatttta tactttgcat ctccaaattt attgcggcga gacttgtcca ttgtgaaagt 420
tagagaacat tatgtttgta tcatttcttt cataaaacct caagagcatt tttaagccct 480
tttcatcaga cccagtgaaa actaaggata gatgtttttt aactggaggt ctcctgataa 540
ggagaacaca atccaccatt gtcatttaag taataagaca ggaaattgac cttgacgctt 600
tcttgttaaa tagatttaac aggaacatct gcacatcttt tttccttgtg cactatttgt 660
ttaattgcag tggattaata cagcaagagt gccacattat aactaggcaa ttatccattc 720
ttcaagactt agttattgtc acactaattg atcgtttaag gcataagatg gtctagcatt 780
aggaacatgt gaagctaatc tgctcaaaaa gatcaacaaa ttaatattgt tgctgatatt 840
tgcataattg gctgcaatta tttaatgttt aattgggttg atcaaatgag attcagcaat 900
tcacaagtgc attaatataa acagaactgg ggcacttaaa atgataatga ttaacttata 960
ttgcatgttc tcttcctttc acttttttca gtgtctacat ttcagaccga gtttgtcagc 1020
ttttttgaaa acacatcagt agaaaccaag attttaaaat gaagtgtcaa gacgaaggca 1080
aaacctgagc agttcctaaa aagatttgct gttagaaatt ttctttgtgg cagtcattta 1140
ttaaggattc aactcgtgat acaccaaaag aagagttgac ttcagagatg tgttccatgc 1200
tctctagcac aggaatgaat aaatttataa cacctgcttt agcctttgtt ttcaaaagca 1260
caaaggaaaa gtgaaaggga aagagaaaca agtgactgag aagtcttgtt aaggaatcag 1320
gttttttcta cctggtaaac attctctatt cttttctcaa aagattgttg taagaaaaaa 1380
tgtaagmcaa aaaaaaaaaa aaaaaaaaa 1409


194
441
DNA
Homo sapiens

194
cagatttcgg tagccatctc cctccaaata tgtctctttc tgctttctta gtgcccatta 60
tttccccttc tcctttcttc tgtcactgcc atctccttct tggtcttccc attgttcttt 120
aactggccgt aatgtggaat tgatatttac attttgatac ggtttttttc ttggcctgtg 180
tacgggattg cctcatttcc tgctctgaat tttaaaatta gatattaaag ctgtcatatg 240
gtttcctcac aaaagtcaac aaagtccaaa caaaaatagt ttgccgtttt actttcatcc 300
attgaaaaag gaaattgtgc ctcttgcagc ctaggcaaag gacatttagt actatcgatt 360
ctttccaccc tcacgatgac ttgcggttct ctctgtagaa aagggatggc ctaagaaata 420
caactaaaaa aaaaaaaaaa a 441


195
707
DNA
Homo sapiens

195
cagaaaaata tttggaaaaa atataccact tcatagctaa gtcttacaga gaagaggatt 60
tgctaataaa acttaagttt tgaaaattaa gatgcaggta gagcttctga actaatgccc 120
acagctccaa ggaagacatg tcctatttag ttattcaaat acaagttgag ggcattgtga 180
ttaagcaaac aatatatttg ttagaacttt gtttttaaat tactgttcct tgacattact 240
tataaagagt ctctaacttt cgatttctaa aactatgtaa tacaaaagta tagtttcccc 300
atttgataaa aggccaatga tactgagtag gatatatgcg tatcatgcta cttcattcag 360
tgtgtctgtt tttaatacta ataaggcagt ttgacagaaa ttatttcttt gggactaagg 420
tgattatcat ttttttcccc ttcaaaattg tgctttaagt gctgataacc acaggcagat 480
tgcaaagaac tgataaggca acaaaagtag agaattttag gatcaaaggc atgtaactga 540
aaggtaacaa cagtacataa gcgacaactg gggaaggcag cagtgaaaca tgtttgtggg 600
gttaagtgag tcattgtaaa taaggaattt gcacatttat tttctgtcga cgcggccgcc 660
actgtgctgg atatctgcag aattccacca cactggacta gtggatc 707


196
552
DNA
Homo sapiens

misc_feature
(1)...(552)
n = A,T,C or G


196
tggccagcca gcctgatgtg gatggcttcc ttggggtggt gcttccctca agcccgaatt 60
ngtggacatc atcaatgcca aacaatgagc cccatccatt ttccctaccc ttcctgccaa 120
gccagggant aagcagccca gaagcccagt aactgccctt tccctgcata tgcttttgat 180
ggtgtcatnt gctccttcct gtggcctcat ccaaactgta tnttccttta ctgtttatat 240
nttcaccctg taatggttgg gaccaggcca atcccttntc cacttactat aatggttgga 300
actaaacgtc accaaggtgg cttntccttg gctgaganat ggaaggcgtg gtgggatttg 360
ctnctgggtt ccctaggccc tagtgagggc agaagagaaa ccatcctntc ccttnttaca 420
ccgtgaggcc aagatcccct cagaaggcag gagtgctgcc ctntcccatg gtgcccgtgc 480
ctntgtgctg tgtatgtgaa ccacccatgt gagggaataa acctggcact aggaaaaaaa 540
aaaaaaaaaa aa 552


197
449
DNA
Homo sapiens

misc_feature
(1)...(449)
n = A,T,C or G


197
ctccagagac aacttcgcgg tgtggtgaac tctctgagga aaaacacgtg cgtggnanca 60
agtgactgag acctanaaat ccaagcgttg gaggtcctga ggccagccta agtcgcttca 120
aaatggaacg aaggcgtttg cggggttcca ttcagagccg atacatcagc atgagtgtgt 180
ggacaagccc acggagactt gtggagctgg cagggcagag cctgctgaag gatgaggccc 240
tggccattgc ccgccctgga gttgctgccc agggagctct tcccgccact cttcatggca 300
gcctttgacg ggagacacag ccagaccctg aaggcaatgg tgcaggcctg gcccttcacc 360
tgcctccctc tgggagtgct gatgaaggga caacatcttc acctggagac cttcaaagct 420
gtgcttgatg gacttgatgt gctccttgc 449


198
606
DNA
Homo sapiens

198
tgagtttgcc cccttacccc catcccagtg aatatttgca attcctaaag acgtgttttg 60
attgtcacac ctgggtgggg aacatgctac tggcatctaa tgcatagagg gcagtaatgc 120
tgctaaacat ctttcaacgc acaggacaga gccccacaaa agagaattat ctagccccaa 180
atgtccataa cactgctgtt gagaaaacct accgcaggat cttactgggc ttcataggta 240
agcttgcctt tgttctggct tctgtagata tataaaataa agacactgcc cagtccctcc 300
ctcaacgtcc cgagccaggg ctcaaggcaa ttccaataac agtagaatga acactaaata 360
ttgatttcaa aatctcagca actagaagaa tgaccaacca tcctggttgg cctgggactg 420
tcctagtttt agcattgaaa gtttcaggtt ccaggaaagc cctcaggcct gggctgctgg 480
tcaccctagc agctgaggga ctcttcaata cagaattagt ctttgtgcac tggagatgaa 540
tatactttaa tttgtaacat gtgaaaacat ctataaacat ctactgaagc ctgttcttgt 600
ctgcac 606


199
369
DNA
Homo sapiens

misc_feature
(1)...(369)
n = A,T,C or G


199
ggcaactttt tgcggattgt tcttgcttnc aggctttgcg ctgcaaatcc agtgctacca 60
gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg tgaattgcac 120
ggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg ggatcatgta 180
ccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt accagtcctt 240
ctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc ctctttgtaa 300
cgggccaagg cccaagaaaa ggggaagttc tgcctcggcc ctcangccat ggctccgcac 360
caccatcct 369


200
55
PRT
Homo sapiens

200
Met Tyr Arg Asn Trp Ser Gly Cys Phe Gly Leu Gln Val Thr Leu Cys
5 10 15
His Thr Phe Glu Thr Arg Asp Leu Ser Arg Leu Ser Ser Asp Ser Gln
20 25 30
Pro Thr Ser Asn Val Ser Gln Ser Ile Ser His Lys Val Leu Ser Phe
35 40 45
Ser Gly Val Ile Val Thr Pro
50 55


201
67
PRT
Homo sapiens

201
Met Gln Leu Leu Ser Pro Asn Thr Lys Phe Thr Ser Cys Leu Ser Arg
5 10 15
Gln Arg Gly Asn Leu Val Phe Leu Gly Asp Leu Lys Gly Cys Ser Glu
20 25 30
Leu Lys Asn Phe Gln Glu Leu Ile Asn Gln Ser Ala Leu Val His Pro
35 40 45
Arg Val Asp Val Trp Trp Tyr Cys Gly Gly Pro Leu Leu Gly Thr Leu
50 55 60
Pro Asn Asn
65


202
73
PRT
Homo sapiens

202
Met Thr Pro Glu Lys Leu Arg Thr Leu Cys Glu Ile Asp Trp Leu Thr
5 10 15
Leu Glu Val Gly Trp Leu Ser Glu Glu Ser Leu Glu Arg Ser Leu Val
20 25 30
Ser Lys Val Trp His Lys Val Thr Cys Lys Pro Lys His Pro Asp Gln
35 40 45
Phe Leu Tyr Ile Asp Ser Tyr Ser Trp Phe Arg Pro Leu Pro Pro Leu
50 55 60
Pro Thr Val Val Lys Arg Thr Ala Ala
65 70


203
2008
DNA
Homo sapiens

203
ctccagagac aacttcgcgg tgtggtgaac tctctgagga aaaacacgtg cgtggtaaca 60
agtgactgag acctagaaat ccaagcgttg gaggtcctga ggccagccta agtcgcttca 120
aaatggaacg aaggcgtttg cggggttcca ttcagagccg atacatcagc atgagtgtgt 180
ggacaagccc acggagactt gtggagctgg cagggcagag cctgctgaag gatgaggccc 240
tggccattgc ccgccctgga gttgctgccc agggagctct tcccgccact cttcatggca 300
gcctttgacg ggagacacag ccagaccctg aaggcaatgg tgcaggcctg gcccttcacc 360
tgcctccctc tgggagtgct gatgaaggga caacatcttc acctggagac cttcaaagct 420
gtgcttgatg gacttgatgt gctccttgcc caggaggttc gccccaggag gtggaaactt 480
caagtgctgg atttacggaa gaactctcat caggacttct ggactgtatg gtctggaaac 540
agggccagtc tgtactcatt tccagagcca gaagcagctc agcccatgac aaagaagcga 600
aaagtagatg gtttgagcac agaggcagag cagcccttca ttccagtaga ggtgctcgta 660
gacctgttcc tcaaggaagg tgcctgtgat gaattgttct cctacctcat tgagaaagtg 720
aagcgaaaga aaaatgtact acgcctgtgc tgtaagaagc tgaagatttt tgcaatgccc 780
atgcaggata tcaagatgat cctgaaaatg gtgcagctgg actctattga agatttggaa 840
gtgacttgta cctggaagct acccaccttg gcgaaatttt ctccttacct gggccagatg 900
attaatctgc gtagactcct cctctcccac atccatgcat cttcctacat ttccccggag 960
aaggaagagc agtatatcgc ccagttcacc tctcagttcc tcagtctgca gtgcctgcag 1020
gctctctatg tggactcttt atttttcctt agaggccgcc tggatcagtt gctcaggcac 1080
gtgatgaacc ccttggaaac cctctcaata actaactgcc ggctttcgga aggggatgtg 1140
atgcatctgt cccagagtcc cagcgtcagt cagctaagtg tcctgagtct aagtggggtc 1200
atgctgaccg atgtaagtcc cgagcccctc caagctctgc tggagagagc ctctgccacc 1260
ctccaggacc tggtctttga tgagtgtggg atcacggatg atcagctcct tgccctcctg 1320
ccttccctga gccactgctc ccagcttaca accttaagct tctacgggaa ttccatctcc 1380
atatctgcct tgcagagtct cctgcagcac ctcatcgggc tgagcaatct gacccacgtg 1440
ctgtatcctg tccccctgga gagttatgag gacatccatg gtaccctcca cctggagagg 1500
cttgcctatc tgcatgccag gctcagggag ttgctgtgtg agttggggcg gcccagcatg 1560
gtctggctta gtgccaaccc ctgtcctcac tgtggggaca gaaccttcta tgacccggag 1620
cccatcctgt gcccctgttt catgcctaac tagctgggtg cacatatcaa atgcttcatt 1680
ctgcatactt ggacactaaa gccaggatgt gcatgcatct tgaagcaaca aagcagccac 1740
agtttcagac aaatgttcag tgtgagtgag gaaaacatgt tcagtgagga aaaaacattc 1800
agacaaatgt tcagtgagga aaaaaagggg aagttgggga taggcagatg ttgacttgag 1860
gagttaatgt gatctttggg gagatacatc ttatagagtt agaaatagaa tctgaatttc 1920
taaagggaga ttctggcttg ggaagtacat gtaggagtta atccctgtgt agactgttgt 1980
aaagaaactg ttgaaaaaaa aaaaaaaa 2008


204
923
DNA
Homo sapiens

204
tgagtttgcc cccttacccc catcccagtg aatatttgca attcctaaag acgtgttttg 60
attgtcacac ctgggtgggg aacatgctac tggcatctaa tgcatagagg gcagtaatgc 120
tgctaaacat ctttcaacgc acaggacaga gccccacaaa agagaattat ctagccccaa 180
atgtccataa cactgctgtt gagaaaacct accgcaggat cttactgggc ttcataggta 240
agcttgcctt tgttctggct tctgtagata tataaaataa agacactgcc cagtccctcc 300
ctcaacgtcc cgagccaggg ctcaaggcaa ttccaataac agtagaatga acactaaata 360
ttgatttcaa aatctcagca actagaagaa tgaccaacca tcctggttgg cctgggactg 420
tcctagtttt agcattgaaa gtttcaggtt ccaggaaagc cctcaggcct gggctgctgg 480
tcaccctagc agctgaggga ctcttcaata cagaattagt ctttgtgcac tggagatgaa 540
tatactttaa tttgtaacat gtgaaaacat ctataaacat ctactgaagc ctgttctgtc 600
tgcaccgaca ttttcattga gtacggattc ttcctaccag atacagctgc tctacaactt 660
tcgagggctg gtataaaact agcttttacc tatttttaaa aattacatga atagtaaaaa 720
cttggattaa cccagtattc gggtattttc aatttccttg ggagcttaga ggacggacaa 780
ataaaaagat tatttcaaca tcaaatatat gctattgttt acatatgaag ataaccacat 840
atatgtataa attcaccgtt actttttagc aatactataa aatccaacag aaaaaaatag 900
catttactaa aaaaaaaaaa aaa 923


205
1619
DNA
Homo sapiens

205
ggcaactttt tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc agtgctacca 60
gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg tgaattgcac 120
ggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg ggatcatgta 180
ccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt accagtcctt 240
ctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc ctctttgtaa 300
cgggccaagg cccaagaaaa ggggaagttc tgcctcggcc ctcaggccag ggctccgcac 360
caccatcctg ttcctcaaat tagccctctt ctcggcacac tgctgaagct gaaggagatg 420
ccaccccctc ctgcattgtt cttccagccc tcgcccccaa ccccccacct ccctgagtga 480
gtttcttctg ggtgtccttt tattctgggt agggagcggg agtccgtgtt ctcttttgtt 540
cctgtgcaaa taatgaaaga gctcggtaaa gcattctgaa taaattcagc ctgactgaat 600
tttcagtatg tacttgaagg aaggaggtgg agtgaaagtt cacccccatg tctgtgtaac 660
cggagtcaag gccaggctgg cagagtcagt ccttagaagt cactgaggtg ggcatctgcc 720
ttttgtaaag cctccagtgt ccattccatc cctgatgggg gcatagtttg agactgcaga 780
gtgagagtga cgttttctta gggctggagg gccagttccc actcaaggct ccctcgcttg 840
acattcaaac ttcatgctcc tgaaaaccat tctctgcagc agaattggct ggtttcgcgc 900
ctgagttggg ctctagtgac tcgagactca atgactggga cttagactgg ggctcggcct 960
cgctctgaaa agtgcttaag aaaatcttct cagttctcct tgcagaggac tggcgccggg 1020
acgcgaagag caacgggcgc tgcacaaagc gggcgctgtc ggtggtggag tgcgcatgta 1080
cgcgcaggcg cttctcgtgg ttggcgtgct gcagcgacag gcggcagcac agcaccttgc 1140
acgaacaccc gccgaaactg ctgcgaggac accgtgtaca ggagcgggtt gatgaccgag 1200
ctgaggtaga aaaacgtctc cgagaagggg aggaggatca tgtacgcccg gaagtaggac 1260
ctcgtccagt cgtgcttggg tttggccgca gccatgatcc tccgaatctg gttgggcatc 1320
cagcatacgg ccaatgtcac aacaatcagc cctgggcaga cacgagcagg agggagagac 1380
agagaaaaga aaaacacagc atgagaacac agtaaatgaa taaaaccata aaatatttag 1440
cccctctgtt ctgtgcttac tggccaggaa atggtaccaa tttttcagtg ttggacttga 1500
cagcttcttt tgccacaagc aagagagaat ttaacactgt ttcaaacccg ggggagttgg 1560
ctgtgttaaa gaaagaccat taaatgcttt agacagtgta aaaaaaaaaa aaaaaaaaa 1619