(en)Taught is how to make a growth hormone transcription factor that is useful for the control of gene expression and growth rate in organisms.

1.ApplicationNumber: US-3243801-A
1.PublishNumber: US-2004186068-A1
2.Date Publish: 20040923
3.Inventor: HURLEY DAVID L.

4.Inventor Harmonized: HURLEY DAVID L(US)

5.Country: US
6.Claims:
(en)Taught is how to make a growth hormone transcription factor that is useful for the control of gene expression and growth rate in organisms.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATION
[0001] The invention described in the current application was also disclosed in provisional application 60/258,237, filed Dec. 25, 2000, from which priority is claimed.



STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Research support which led to the making of the present invention was provided in part by funding from the National Science Foundation under Grant No. IBN-9600805. Accordingly, the federal government may possess certain statutory rights to the invention.
FIELD OF THE INVENTION
[0003] The invention relates to a growth hormone transcription factor.
DESCRIPTION OF RELATED ART
[0004] Cell-specific gene expression leads to the exclusive production of secreted hormones by selected cell types in the anterior pituitary. These hormones are produced in differentiated cells through selective processes of transcriptional and translational control. In addition, each cell type displays a distinctive pattern of differentiation during development, and specific neuroendocrine regulation.
[0005] Transcriptional control of the growth hormone (GH) gene resides primarily in the promoter region that contains multiple transcription factor response elements. Pit-1, also known as GHF-1, is a prototypical POU-domain protein which was isolated by virtue of its ability to activate transcription of the GH gene by binding to the GH promoter at two sites. Pit-1 was also found to regulate transcription of the prolactin and TSH beta-subunit genes. Expression of all these genes is pituitary-specific, implying a central role of Pit-1 as a transcription factor controlling pituitary development and differentiation. The important role of Pit-1 in the regulation of anterior pituitary differentiation was confirmed by the identification of Pit-1 mutations as the causes of congenital hypopituitary dwarfism in the Snell dwarf mouse and in humans.
[0006] Because Pit-1 promotes transcription of three distinct hormones, other transcription factors must establish the specificity of GH production in somatotrophs. One such protein was isolated by virtue of its binding to an 18 base pair (bp) Z-box response element (ZRE) conserved among mammals in the GH promoter. The protein that bound to the ZRE contained 15 consensus sequences for DNA-binding zinc fingers, and was named Zn-15. It was also shown that in synergy with Pit-1, Zn-15 activates GH transcription 100-fold above basal levels. The importance of Zn-15 in the physiological regulation of GH gene expression was shown when mutations in the ZRE in the GH promoter abrogated pituitary expression of a reporter gene in transgenic animals. See Lipkin et al., Genes Devel. 7, 1674.
[0007] Although Zn-15 plays an important synergistic role in GH transcriptional control, only the C-terminal portion appears to be necessary for this synergism. Zinc fingers IX, X and XI have been shown to bind to the ZRE in the GH promoter, but DNA binding elements recognized by the remaining 12 zinc fingers of rat Zn-15 have not been found. Some of these fingers in the rat are separated by long linker regions of 20 or more amino acids, a structural feature evidently determinative of DNA binding or transcriptional activation. Zn-15 protein also can bind to a subset of thyroid response elements using fingers IX-XI, and may bind to RNA as well as DNA using the zinc fingers in the N-terminus of the protein. In order to effect cell type-specific regulation of GH, appropriate use must be made of the linkers and zinc fingers which do not bind the ZRE. See Tuggle and Trenkle, Domes. Anim. Endocrinol. 13, 1.
BRIEF SUMMARY OF THE INVENTION
[0008] The invention relates to a growth hormone transcription factor that is useful for the control of gene expression and growth rate in organisms.



DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention is useful, inter alia, for the control of cell-type specific regulation of GH in mammals, including, importantly, the mouse. Pituitary differentiation has been extensively studied in the mouse, where both transgenic and spontaneous GH-deficient mutants are available. To make a representative embodiment of the present invention, a mouse cDNA, hereinafter called mouse Zn-16 or mZn-16, was isolated. Expression of mouse Zn-16 mRNA was detected in 1 day old normal and GH-deficient Ames dwarf (Prop-1<df−/−>) mouse pituitary samples, as well as in the murine pre-somatotrophic GHFT1-5 cell line, see Lew et al., Genes Development 7, 683, consistent with a developmental role for Zn-16.
[0010] To demonstrate an aspect of the invention, comparison between the GH-altered Ames dwarf (Prop-1<df−/−>), Snell dwarf (Pit-1<dw J −/−), little (GHRHR, <lit−/−>) transgenic GHRH excess (MT, GHRF, Bri 11) and normal littermate mouse pituitaries showed predicted changes in GH mRNA, as well as other changes in hormone products that had not been previously evaluated or reported. Such quantitative analysis suggests that changes in the regulation of factors functioning in the GH homeostatic regulatory system are consistent with a role for Zn-16 in GH transcriptional control.
[0011] Animals, Cell Culture and Isolation of RNA. Normal and Ames dwarf mice maintained in a breeding colony were sacrificed by carbon dioxide euthanasia. The day of birth (by 1400 hrs) was termed day 1. Adult rat pituitaries were obtained by the same procedure. Pituitaries were collected on dry ice, then stored at −70° C. until extracted. GHFT1-5 cells were obtained from Dr. Pamela Mellon (Univ. of California, San Diego), and cultured as described, see Lew et al., supra. Pituitaries and cells were first homogenized (Tissue Tearor, BioSpec, Bartlesville Okla.) in extraction buffer. RNA extraction was performed using the guanidinium-isothiocyanate:phenol:chloroform method as modified by the manufacturer (RNAzol B or Ultraspec, Biotecx, Houston, Tex.). Purity and concentration were checked by UV spectrophotometry (GeneQuant, Pharmacia, Piscataway N.J.).
[0012] Reverse Transcription. The RNA pellet was briefly dried before resuspension in 0.1 μM oligo-dT for subsequent reverse transcription of poly-A RNA using the SuperScript II Preamplification System (Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. The reaction was carried out in a 20 μl volume containing 200 units of MMLV reverse transcriptase at 45° C. for 60 minutes, then the enzyme was denatured by heating at 65° C. for 15 minutes. Hybridized RNA was removed by digestion with 2 units of E. coli RNase H. The volume was then brought to 50 μl with diethylpyrocarbonate-treated water and stored at −20° C. UV absorbance at 260 nm was measured on a GeneQuant spectrophotometer (Pharmacia, Piscataway, N.J.) in a 50 μl cuvette.
[0013] Primer Design. The sequence of rat Zn-15 (accession number L23077) was obtained from GenBank. Prospective primer pairs were computer designed (Right Primer 1.2, BioDisk, San Francisco Calif. or Oligo 4.0, National Biosciences, Plymouth Minn.) using the following criteria: location with respect to zinc fingers, match of T m between the primers, possible secondary structure within the primers, self-hybridization, and hybridization between primers. Primers were synthesized, quantified by UV spectrophotometry (Pharmacia GeneQuant), and stored desiccated at −70° C.
[0014] PCR Amplification. Standard PCR conditions for cDNA amplification included each dNTP at a concentration of 0.2 mM and 1.25 U of Taq polymerase in a final volume of 50 μl. Amplifications were performed in a Model TC-1 thermal cycler (Perkin Elmer-Applied Biosystems, Foster City, Calif.). Amplification reactions were initiated with a hot-start using wax beads (Perkin Elmer) to separate the primers, MgCl 2 , and dNTPs (Lower master mix) from the DNA template and Taq polymerase (Upper master mix). Thermal cycling conditions consisted of initial denaturation for 60 s at 94° C.; followed by a 3-step profile (94° C. for 60 s; 54° C. for 45 s; and 72° C. for 90 s) for the desired number of cycles, and a terminal extension step of 72° C. for 5 min. For some reactions, a final, non-template dependent extension was carried out at 60° C. for 30 min.
[0015] Sequencing Methods, Strategy and Analysis. PCR products from amplification reactions were cloned according to the manufacturer's instructions (TA Cloning Kit, InVitrogen, San Diego Calif.). Ligation reactions were incubated at 14° C. overnight, and then transformed into E. coli INValphaF cells (InVitrogen). Colonies with putative inserts were cultured overnight in 2×YT or TB containing 100 μg/ml ampicillin (Sigma). Bacteria were lysed and plasmid DNA was isolated with a commercial DNA binding matrix (PERFECTprep Plasmid DNA kit, 5 Prime->3 Prime, Boulder Colo.). Restriction digestion with EcoRI followed by agarose gel electrophoresis was used to confirm the presence of inserts prior to sequencing. DNA sequencing reactions were performed using 400 ng of plasmid template and fluorescent dye-labeled dideoxy terminators with AmpliTaq FS DNA polymerase according to the manufacturer's protocol (PRISM Ready Reaction DyeDeoxy Terminator kit, Applied Biosystems, Inc., Foster City Calif.). Thermal cycler conditions were 30 sec. at 96° C., 15 sec. at 50° C., and 4 min. at 60° C. for a total of 25 cycles. Unreacted fluorescent dye-labeled dideoxy terminators were removed from the sequencing reactions using size exclusion gel columns (CentriSep, Princeton Separations, Inc., Adelphia N.J.). Sequencing reactions were electrophoresed on an automated sequencer (Applied Biosystems Model 373A) using a 36 cm well-to-read 6% acrylamide gel (Sooner Scientific, Garvin Okla.) at 28 watts constant power for 10 hours. Data collected on a Macintosh computer were evaluated for ambiguities and “clear” sequence length using Factura 1.2.Or6 software (Applied Biosystems, Inc.). Sequences were aligned using GeneWorks 2.45 software (Oxford BioMolecular, San Diego Calif.) and remaining ambiguities in the electropherograms were resolved from overlapping information, manual inspection, or resequencing. Nucleotide and amino acid sequence data were analyzed, aligned and compared with reported sequences using GeneWorks and Statistical Analysis of Protein Sequences. See Brendel et al., Proc. Natl. Acad. Sci. USA 89, 2002.
[0016] Ribonuclease Protection Assay. Total RNA extracted from pituitaries or cells was subjected to ribonuclease protection assay using non-radioactive probe synthesis and detection according to the manufacturer's protocol (RPA II/BrightStar systems, Ambion, Austin Tex.). A 651 nt probe from positions 840 to 1490 of mZn-15 was employed, and a 250 nt probe for mouse beta-actin was used as a control. Chemiluminescence after substrate treatment was visualized on x-ray film (Fuji RX) and then quantified using constant intensity illumination (FotoDyne, Madison Wis.), a CCD video camera (Hamamatsu C2400) or scanner (Nikon) and computer assisted image analysis (Gel-Pro Analyzer, Media Cybernetics).
[0017] Mouse Zn-16 cDNA Isolation and Characterization. In order to obtain a probe for mouse (m) Zn-16, mouse pituitary RNA was reverse transcribed using an oligo-dT primer and then amplified using gene-specific oligonucleotide primers which annealed to zinc fingers 1× and XI of rat (r) Zn-15, the region that binds to the GH promoter. Cloned products near the expected size of 506 bp in the rat were found to have a similar DNA sequence, and were used as probes in hybridization screens of a mouse pituitary cDNA library previously constructed in the isolation of mLIM-3. See Seidah et al., DNA 13, 1163. However, no positive colonies were found through several rounds of hybridization. Therefore, the majority of the coding region of mZn-16 cDNA was isolated by amplification using primers specific for different zinc fingers of rZn-15, and the 5′ and 3′ ends were isolated using rapid amplification of cDNA ends.
[0018] Six portions of mZn-16 of at least 1500 bp were independently amplified, cloned and sequenced. Sequences were obtained in both directions from multiple clones of each fragment, and then aligned to assemble the entire cDNA sequence of 6879 nucleotides. The full-length mZn-16 cDNA sequence was assembled from overlapping regions of at least 1500 bp.
[0019] The open reading frame in the mZn-16 cDNA as shown in SEQ ID:NO 1 encodes a polypeptide as shown in SEQ ID:NO 2. The mZn-16 amino acid sequence of 2292 amino acids has several additional aa that are not present in rZn-15, particularly in the N-terminus. There are four in-frame methionine residues upstream from the initial methionine in the rat, for additional translational start sites not reported in the rat. A feature of the protein that has been identified based on computer analysis are several regions (aa 830-845, 1550-1567, 1999-2016) encoding consensus eukaryotic nuclear localization signals, see Robbins et al., Cell 64, 615.
[0020] Importantly, multiple zinc fingers of the Cys2His2-type, see Berg and Shi, Science 271, 1081, similar to those found in rZn-15, are present in mZn-16. There is 97% amino acid identity between the zinc fingers of rZn-15 and mZn-16. However, the four differences in mZn-16 in finger V change three proline residues. This region of mZn-16 is now predicted to contain two consensus zinc fingers, designated as Va and Vb in FIG. 2B, and finger Va now agrees at all positions with the zinc finger consensus sequence. Changed residues in fingers II, IX, X and XIII are conservative substitutions, and substitutions in regions outside the fingers would not be predicted to have any impact on zinc coordination or DNA binding, see Berg and Shi, Science 271, 1081. As found in rZn-15, there are extended linker regions between the zinc fingers in mZn-16, particularly in the C-terminal half of the protein. The largest region of consecutive difference between mouse Zn-16 and rat Zn-15 is found in the linker between fingers VII and VIII (aa 845 to 860 in mZn-16), where only two of the 14 consecutive residues are similar.
[0021] Mouse Zn-16 mRNA expression in Normal and Ames dwarf pituitaries. Zn-16 expression in normal and GH-deficient Ames dwarf (Prop-1<df−/−>) mouse pituitaries was studied at the day of birth (postnatal day one). Total RNA was isolated, reverse transcribed using oligo-dT priming, then amplified using primers in fingers IX-XI for 30 cycles. The amount of 503 bp product of these amplifications was then determined using image analysis of ethidium bromide-stained gels. Samples in which no pituitary cDNA was added as control showed no amplification. The average relative image intensity of the bands was determined for normal and Ames dwarf mice (n=3). At one day of age, there was no significant difference in the expression of Zn-16 between normal and Ames dwarf pituitary.
[0022] Mouse Zn-16 mRNA Expression in GHFT1-5 pre-somatotroph cells. The expression of Zn-16 mRNA in Ames dwarf pituitaries suggested that Zn-16 might be expressed in the mouse cell line GHFT1-5, which is derived by immortalization of pituitary cells with a Pit-1 promoter-driven large T antigen construct. These cells have been characterized as pre-somatotrophs, but they do not express Pit-1 protein or GH mRNA. Probes were produced to measure the expression of mZn-15 mRNA in total RNA using ribonuclease protection assays (RPA). Total RNA was isolated either from ca. 30 pooled mouse pituitaries or 107 GHFT1-5 cells, and used for RPA for either Zn-16 or actin mRNA in each sample. Actin mRNA was assayed to serve as a control for each sample in a 5 μg RNA aliquot. The size of the protected fragment for Zn-16 was the same in both pituitary and GHFT1-5 samples, and was of the size predicted from the length of homologous probe sequences (651 nt). In comparing the pituitary and GHFT1-5 samples, the detectable amounts of mZn-16 were different, suggesting that Zn-16 expression levels varied between normal pituitary and GHFT1-5 samples.
[0023] The further description found immediately below shows Zn-16 function in the transcriptional regulatory control of pituitary GH expression.
[0024] Animal care and use. Male mice 3-4 months old were used in this study. Mice were euthanized with carbon dioxide anesthesia, then individual pituitaries were removed using washed instruments and stored in single tubes at −70° C.
[0025] RNA preparation. Total RNA was extracted from individual mouse pituitaries using a modified phenol/chloroform/guanidinium protocol (Ultraspec; Biotecx, Houston, Tex.). The RNA pellet was briefly dried before resuspension in 0.1 μM oligo-dT for subsequent reverse transcription of poly-A RNA using the SuperScript II Preamplification System (Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. The volume was brought to 50 μl with diethylpyrocarbonate-treated water for storage at −20° C. UV absorbance at 260 nm was measured on a GeneQuant spectrophotometer (Pharmacia, Piscataway, N.J.) in a 50 μl cuvette.
[0026] PCR. Primers were selected for specificity for mouse mRNAs, high annealing temperature, and absence of secondary structure using the computer programs Right Primer (BioDisk, San Francisco, Calif.) and Oligo 5.0 (National Biosciences, Plymouth, Minn.). Standard cDNA amplification reactions included each dNTP at a concentration of 0.2 mM and 1.25 U of Taq polymerase in a final volume of 50 μl. The fluorescent dye-labeled dUTPs ([F]dUTP) used for labeling were [R110], [R6G], or [TAMRA] (ABI; Foster City, Calif.). [F]dUTPs were diluted for a constant addition volume of 0.1 μl per reaction. Amplifications were performed in a Model TC-1 thermal cycler (ABI). Amplification reactions were initiated with a hot-start using AmpliWax PCR Gem 50 beads (ABI). Thermal cycling conditions consisted of initial denaturation for 60 s at 94° C.; followed by a 3-step profile (94° C. for 60 s; 54° C. for 45 s; and 72° C. for 90 s) for the desired number of cycles, and a terminal extension step of 72° C. for 5 min. For some reactions, a final, non-template dependent extension was carried out at 60° C. for 30 min. After amplification, unincorporated primers and dNTPs were removed by centrifugation through Centricon-50 or Microcon-30 filters (Amicon, Beverly, Mass.) filters. Products were analyzed on 12 cm well-to-read (WTR) gels composed of 10% Long Ranger (FMC BioProducts, Rockland, Me.) with a 373A instrument using GeneScan 1.2 software. Electrophoresis was performed with power limiting at 12W in 0.5×TBE buffer. Samples were mixed with a ROX-dye labeled size marker (ROX-500, ABI) and a sucrose- or Ficoll-bromophenol blue dye solution before loading on the gel. Results were statistically analyzed using the computer programs Excel (Microsoft, Redmond, Wash.) and SuperANOVA (Abacus Concepts, Berkeley, Calif.).
[0027] Comparison of transcript abundance. In order to compare product intensities among a large number of amplification reactions, the fluorescence detection and sizing capacity of an automated sequencing instrument was used. Amplifications were performed in the presence of fluorescence-labeled dUTP, which was detectable during electrophoresis. Fluorescence labeled products were examined for molecular weight compared to the standards present in each lane as an internal control, and for peak intensity from the processed electropherograms. From peak heights and the calculated efficiency, the transcript abundance was determined as related by the equation in Gilliland et al., Proc. Natl. Acad. Sci. USA 87, 2725. For normal littermates and GH-affected mice, pituitaries were amplified for GH, Zn-16 and Pit-1 abundance. The results, expressed as percentage of normal littermate expression, were:
mouse type GH Zn-16 Pit-1 Ames dwarf 0 4.5 0 Snell dwarf 0 5.9 0.2 Little 1.3 5.9 2.9 GHRH giant 242.4 198.2 199.8

[0028] Simple regression correlation tests showed that there was a significant correlation (p<0.001) for Pit-1 vs. GH (F 1.17 , 26.82) and Zn-16 vs. GH (F 1.17 =12.48). This correlation from an in vivo model implicates Zn-16 function in the transcriptional regulatory control of pituitary GH expression.
[0029] Further embodiments of aspects of the invention are described below.
[0030] In an embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has undergone surgery. The cells within the hollow fiber express GH regulated by the Zn-16 which is induced by the physician (e.g., with tetracycline) as needed to promote healing. In a preferred embodiment, localized administration of GH expression is provided by implantation of the hollow fiber unit during the surgery, where compatible with needed removal processes. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.
[0031] In another embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian IQ cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has experienced severe burns. The cells within the hollow fiber express GH regulated by the Zn-16 which is de-induced by the physician (e.g., with tetracycline) after promoting healing. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.
[0032] In yet another embodiment of an aspect of the invention, a Zn-16-expressing construct with inducible control by exogenous factors (e.g., tetracycline) is stably transfected into mammalian cells (e.g., GC cells). The transfected cells are placed within a permeable membrane for immunological protection (“hollow fiber”). The hollow fiber containing the transfected cells is implanted into the kidney capsule of a patient who has experienced muscle wasting, as is observed, for example, in later-stage HIV-infected patients. The cells within the hollow fiber express GH regulated by the Zn-16 which is subject to control by the physician (e.g., with tetracycline) for the optimal control of healing and the lessening of the pace of muscle wasting. Such an embodiment of the invention is useful for enhancing GH production, desired in particular during the healing process, promoting tissue regeneration and lessening the need for invasive repetitive injections.
[0033] In certain embodiments of the invention, Zn-16 is administered to a patient. As used in this application, “administration” includes the implantation of a construct or a host cell containing a Zn-16 nucleic acid sequence or, more generally, encoding the Zn-16 polypeptide, for instance as described above. That is, administration of Zn-16 is effected also by the administration to a patient, or the implantation into a patient, of a construct encoding the Zn-16 polypeptide.
[0034] In an embodiment of an aspect of the invention, Zn-16 is used to alter gene expression in a patient who has a pituitary tumor. In a preferred embodiment, Zn-16 and somatostatin are co-administered to the patient. Upon administration, the somatostatin binds its receptor, is internalized, and the Zn-16 alters gene expression in such a way as to ameliorate the effects of the tumor.
[0035] In another embodiment of an aspect of the invention, Zn-16 is used to control expression of proteins whose expression is otherwise driven by other zinc finger proteins. In a particular embodiment, Zn-16 is administered to a patient suffering from a cancer that is characterized by the overexpression of proteins whose expression is driven by zinc finger proteins other than Zn-16. The administered Zn-16 blocks the overexpression driven by other zinc finger proteins and slows tumor growth.
[0036] In another embodiment, Zn-16 is affixed to a surface. The presence of heavy metals is detected by their binding to the affixed Zn-16. Methods of the detection of subtle changes in binding, such as surface plasmon resonance or methods capable of detection of small changes in potential difference across a very limited space, enable the detection of very low concentrations of heavy metals when the metals bind Zn-16. In a particular embodiment, Zn-16 is preloaded with one heavy metal, and displacement of that heavy metal is measured.
[0037] In another embodiment, Zn-16 is used as a heavy-metal responsive factor that indicates contamination levels. In such an embodiment, Zn-16-driven expression of a reporter gene changes when there is a change in the heavy metal concentration. Changes in the concentration of not only zinc but also other heavy metals such as mercury and lead are detected.
[0038] In yet another embodiment, the heavy-metal-binding property of Zn-16 is exploited to ameliorate the adverse effect on a patient of heavy metal exposure. By virtue of its heavy-metal-binding property, Zn-16 serves as a sink that binds excess heavy metal that is toxic to key tissues in the patient, such as liver and kidney.
[0039] In another embodiment, Zn-16 is pre-administered to a human to prevent the adverse effect on the human of anticipated heavy metal exposure. A human anticipating participation in cleanup of heavy metal environmental contamination, for example from a spill of stored liquids or from radioactive fallout, self-pre-administers Zn-16, which binds environmental heavy metals to lessen harm to the human. The Zn-16 is a reservoir for the metals which otherwise pose a danger to the human.
[0040] In another embodiment of an aspect of the invention, Zn-16 is administered to a human patient who has diabetes or, more generally, experiences dysregulation of expression or availability of insulin or insulin-like growth factor. The administered Zn-16 regulates the expression of GH to alter levels of insulin and insulin-like growth factor and ameliorates the patient's condition.
[0041] In an embodiment of an aspect of the invention, Zn-16 is used in a method of controlling the expression of gene products at a multiplicity of loci in the genome of an organism, said method comprising: the site-directed or random mutagenesis of Zn-16 to encode an altered polypeptide, said altered polypeptide possessing an affinity for a transcriptional regulatory factor different from the affinity of the polypeptide of Zn-16 for said transcriptional regulatory factor; the formation of an assemblage of said transcriptional regulatory factor with said altered polypeptide; and the direction of said expression at said loci by said assemblage. In a particular embodiment, the altered polypeptide binds a multiplicity of transcriptional regulatory factors to form the assemblage. In a particularly preferred embodiment, the method controls the expression of gene products throughout the entire genome of the organism, allows specific targeting of gene promoters, and creates a controllable platform to assemble other transcriptional regulatory factors.
[0042] In another embodiment, Zn-16 is modified to have temperature-responsive zinc binding. This modified Zn-16 is useful as a temperature-dependent regulator of gene expression.
[0043] In another embodiment, a cell or tissue sample is taken from a patient. Nucleic acids from the patient are extracted from the sample. Hybridization of the patient's nucleic acids to the nucleic acid encoding Zn-16 is measured. Detection of a difference between the hybridization of the patient's sample and the hybridization of a control sample is useful in the diagnosis of dwarfism, gigantism, hypothyroidism, and other disorders of metabolism.
[0044] The invention is not limited to the exact details of operation, or to the exact compositions, methods, procedures, or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art. As the invention would therefore be limited only by the full scope which could reasonably, legally and equitably be accorded any of the appended claims, the foregoing examples are provided merely by way of illustration of the breadth of the present invention, which exceeds any and all of these examples.



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2


1
6879
DNA
Mus musculus

1

atgttataca accagccaga ccagaaatat gatgaagaga atcttccaat accaaattct 60

ctacgttgtg agctcttact tgttttgaaa actcagtggc cctttgatcc agaattttgg 120

gattggaaaa ctttgaagcg ccagtgtctt gctctcatgg gagaagaggc gtctattgtg 180

tcctcaattg atgaactgaa tgacagtgaa gtctatgaga aagtagacta ccagggtgaa 240

aggggagaca catctgtgaa tggcctttct gctgctggac ttggtactga ttctggcctg 300

ctgatggata ctggtgatga aaagcagaag aagaaagaga taaaagaatt aaaagatagg 360

gggtttatat ctgctaggtt taggaattgg caagcctaca tgcagtattg tttgctctgt 420

gacaaagaat tccttggaca cagaatagta cggcatgctc aaaaacatta caaagatggg 480

atttacagct gtcccatatg tgcaaagaat tttaattcta aagactcgtt tgtccctcat 540

gttaccctgc atgttaaaca gtctagtaaa gagagactag cagctatgaa gccattaaga 600

agattgggaa ggcctcctaa aatcacagcc acccatgaaa atcaaaagac taatattaat 660

actgtggcta aacaggaaca gcgacccata aaaaagaata gtctttattc aacagatttc 720

atagtgttta atgacaacga tggttcagat gatgaaaatg acgacaagga caagtcttat 780

gagcccgagg tgatccctgt ccagaaacca gtacctgtta atgagtttaa ttgtcctgtg 840

accttttgta aaaagggctt taagtacttc aaaaatttaa ttgctcatgt gaaaggccat 900

aaggatagtg aagatgccaa acgctttctt gaaatgcaaa gcaagaaagt catttgccag 960

tactgtagaa ggcactttgt aagcgtcact catctcaatg atcacttaca aatgcactgt 1020

ggcagtaagc catatatatg tatacagatg aaatgtaagg ctggttttaa tagttacgca 1080

gagctgttag cccaccgaaa ggagcatcaa gtctttagag caaagtgctt atttccaaaa 1140

tgtggcagaa ttttttccca agcttattta ctgtatgatc atgaggccca acattataat 1200

acgtacacgt gcaagttcac aggttgtggt aaggtgtacc gttctcagag cgagatggag 1260

aagcaccagg atggccacag tcatcctgaa acagggctgc ctcctgaaga ccagcttcag 1320

ccatctggaa atgatgtgaa tccggactca ggagcgacgg ctgcaggagg aaggtccgag 1380

aacagcattg acaagaacct gggttcaaac agaagtgcag attgggagaa aaacagagca 1440

gagccagctg tgactaaaca cggccagatc tctgccgctg aactcaggca agctaacata 1500

ccattgtcaa atggtctgga aacccgtgat aatactactg ttcttcggac caatgaagta 1560

gctgtgtcca tcaaggtgtc tgtcaaccat ggggtagagg gtgactttgg aaagcaagaa 1620

aacctaacca tggaaggcac tggtgagccg ctgatcacag atgtgcataa accaggtata 1680

ggtgctgggg tccagttatg tcatccaggt ttccaagaaa agaaaggtca cgagtgcctg 1740

aacgaagccc agaattcttt atcaaactca gaatcactga agatggatga ccttaaccca 1800

caaagcttag aaagacaggt gaacactctg atgacctttt ctgtacaaaa tgaggcagga 1860

cttgaagaca attcacaaat ttgcaagttt gaatgtggag gtgatgttaa aacctcatcc 1920

agcctttatg atttacctct taagacacta gaaagtatca catttgttca gtcacagccc 1980

gacctaagca gtccgttggg atctccatca gtacctccaa aagctccagg tcagaagttc 2040

agctgccagg ttgagggatg cactcgaaca tataactctt cacagagtat tggaaaacac 2100

atgaagacag cacaccctga ccaatatgct gcttttaaac tgcagcgcaa gacgaaaaaa 2160

ggtcagaaat ctaacaactt aaatacacca aatcatggaa agtgtgttta ttttttgcca 2220

tcacaagtga gcagctctaa tcatgctttt tttacaccac agaccaaagc caatgggaac 2280

cctgcctgtt cagcccaggt gcagcatgtc tcgccttcca ttttcccagc tcatttagca 2340

agtgtatcag ctccattgtt accctcagtg gaaagtgtcc taagtccaaa tataccttct 2400

caggataaac atggacaaga tggcatatta tgttcacaaa tggaaaattt gtcttatgct 2460

cccttgccag cacaaatgga agatctaacc aagacagttt tgcctttgaa tattgacagc 2520

ggctcagatc cgtttcttcc tttacccaca gaaaatagct ctctcttctc ttcaccagca 2580

gacagtgaga ataattctgt tttttcccaa ctggaaaata gtacaaatca ttatccctcc 2640

cagacggatg gaaacataaa ttcctctttt ctgaaaggag gcagcagtga aaatggagtt 2700

tttccttccc aagtaagttc tgcagatgac ttcagtagca ccagtgccca accgtctaca 2760

cctaagaaag tgaaaaaaga ccgtggtcga ggcccaaatg ggaaggaaag aaaacccaag 2820

cacaacaaaa gggctaaatg gcctgcgatt atcagggatg ggaaattcat ctgtagcagg 2880

tgttacaggg ctttcaccaa ccccaggtcc ctgggtggac acctgtctaa aaggtcttac 2940

tgcaaaccac tggatggagc agaaatagca caggaacttc tacagaccaa cagacagcct 3000

tccctcctag ctagcatgat tctctccaca agtgcagtaa atatgcaaca gccgcaacag 3060

tctaacttta atccagaaac atgctttaaa gacccatcat tcctgcaact tcttaatgtg 3120

gaaaatcgtc caaccttttt accaagtaca tttccaagat gtgacgtgag taactttaat 3180

gccagtgtta gtcaggaagg cagtgaaatt attaagcagg ctttagaaac tgctggcatt 3240

cccagcacgt ttgagagtgc cgaaatgctt tctcaggttg ttccaatagg cagtgtctcc 3300

gatgcagcac aagtcagtgc agcggggatg ccagggccac ctgtgacacc cttgttacag 3360

actgtttgcc acccaaacac ctcaccatca aaccagaatc aaacgccaaa ttccaaaacc 3420

ctcaaagaat gtaacagttt gcctctcttt acaacaaatg atttactgct aaagactatt 3480

gaaaatggct tgtgctccaa ttcattcagt agttctactg aaccaccaca aaattttacc 3540

aataatagtg cacatgtttc tgttataagt gggcctcaga atacaagatc cagtcatttg 3600

aataaaaaag gaaatagtgc atctaagaag agaaaaaaag ttgctcctgc agtaagtgta 3660

tctaatactt cccaaaatgt gctaccaact gatttaccag tgggccttcc atcgaagaat 3720

cttacagtcc ctgataccaa cacacggtca gacatgaccc cagattgtga acctcgggct 3780

ttggtggaaa atctcacaca gaaattaaat aacattgaca atcatttgtt tataactgat 3840

gtaaaagaga actgtaaagc cagtcttgag ccccatacaa tgttaacccc tttaacatta 3900

aaaacggaaa acggcgattc ccgaatgatg cctttgagtt catgcacacc agtgaattct 3960

gatttgcaga tttctgaaga taatgttatt cagaactttg agaagactct tgaaattatt 4020

aaaactgcta tgaattctca aatacttgag gtaaaaagtg gatctcaggg tactggtgag 4080

acaacacaga atgctcagat aaattacagc atgcaacttc cctcagtaaa ctctatccca 4140

gatagcaagc tgcctgatgc ttctcagtgc tcctctttcc taactgtaat gccaacaaag 4200

tctgaagcat tacataagga ggatcaaata caggacattt tagagggttt gcaaaactta 4260

aaactagaaa atgacacttc tgctccagct tcccagagta tgctaatgaa caaatcagta 4320

gcactgtccc ctactcctac taaatcaact ccaaatattg tagtccagcc agtacccgaa 4380

gtgatacatg ttcagcttaa tgacagagtt aataagccct ttgtgtgtca aaaccaaggc 4440

tgtaactaca gtgctatgac aaaggatgcc ctgtttaaac actatggtaa aatccatcag 4500

tatactccag agatgattct tgaaattaag aagaatcaat taaaatttgc tccatttaaa 4560

tgtgtagtac cttcatgtac caaaacattt acaagaaatt ctaatctccg ggcacactgt 4620

cagttggtgc atcattttac aatagaagaa atggtaaagc taaaaataaa aaggccctat 4680

ggaagaaaat ctcagagtga aaatttgtca tctccacaga ataatcaagt gaagaaacag 4740

ccatccatgg ccgaggaaac aaaaactgag tcacaaccag ccttcaaggt accagcagca 4800

acaggtgatg ctgcacttgc taatgcaaca gtaatcccag aaaaacaact tgcagaaaaa 4860

aaaagtcctg agaaaccaga aagttcttca cagcctgtca catcttctgc tgaacaatat 4920

aatgcaaatc ttgcaaacct aaaaaccaaa ggaaggaaaa ataagaggca tagaaaagaa 4980

aaggaagaaa aacgggaaaa gaatccagtt tcccaggcct ttgaacttcc aacaaaatac 5040

agttcgtaca gaccttactg ctgtgtccac cagggatgct ttgctgcttt tacaatacag 5100

caaaacttga ttcttcatta ccaggctgta cataaatcaa atcttcctac attttctgca 5160

gaggttcaag aggaaagtga agctgttaaa gaaagtgaag aaactgaacc gaaacaatca 5220

atgaaagaat ttaggtgtca ggtgagtgac tgttctagga ttttccaagc aattactggc 5280

ctaatacagc actacatgaa acttcatgaa atgacccccg aggaaattga aagcatgact 5340

gctgctgtgg atgttggcaa atttccatgt gatcagttgg agtgtaagtt gtcttttaca 5400

acatacctga gctatgttgt tcatcttgag gtagaccatg gaattggaac aaggacaagt 5460

aaggcagaag aagatggcat atacaagtgt gactgtgagg gctgtgacag gatatatgcc 5520

actcggtcta atcttctccg acacatcttt aataaacata atgacaagca taaagcccat 5580

ctgattcggc caagaaaatt aactggccag gaaaatatat caagtaaggc aaaccaagaa 5640

aaatcaaagt ctaaacatcg gacaacaaaa cccaacagat ccgggaaaga cggaatgaaa 5700

atgccaaaga caaagcgaaa gaaaaaaagt aatttagaaa acaagagcgc aaaagtagtg 5760

cagattgagg aaaataagcc ttattctcta aagcgtggga agcacgtgta ttccataaag 5820

gctaggaatg atgccttggc agagtgtaca agcaaatttg tgacacagta tccatgtatg 5880

ataaaagggt gtacttcagt cgttacaagt gaaagcaata tcatcagaca ttataagtgt 5940

cataagttgt ccagggcatt tacatcacaa caccgcaaca ttcttattgt ctttaagcga 6000

tatggcaacc cacaaggaag ggaaatctct gagcaagaag atgaaaagaa tgataagaaa 6060

ggtcctgatt catctgtttt agagaaaaat gataactcgg aaccagctgc tgctccacag 6120

gaagaaggta gaaaaggtga aaaggatgag atggatgagt taacagaatt atttattaca 6180

aagttaataa atgaagacag cacaaatgca gaaaaccaag gcaataccac tttaaaggga 6240

aataacgaat ttcaggagca tgattcctgc acatcagaaa gacaaaagcc tggtaatttg 6300

aagagagttt ataaagaaaa aaacactgtg cagagtaaga aacggaagat tgataaaact 6360

gagccagaag tatccttggt ggtaaataat acacggaaag aggaagagcc tgccgtagca 6420

gttcagacca ctgaggagca tcctgcatcc tttgactgga gctccttcaa gcctatggga 6480

tttgaagcat cctttctgaa gtttcttgaa gagtctgcag tgaagcagaa gaaaaatagt 6540

gacagagacc attcaaacag tggaagtaaa agaggatccc attccagctc cagaagacat 6600

gttgataagg ctgctgtggc tggtagcagt catgtgtgtt cctgtaaaga cagtgaaatc 6660

tttgtacagt ttgccaaccc ctcaaagctt cagtgcagtg agaatgtaaa aattgtttta 6720

gacaagactc ttaaagatcg ctctgagctt gtcctaaaac agcttcagga aatgaaacct 6780

actgtcagtc taaaaaaact tgaagtacta tccaatagtc cagataggac tgttttaaaa 6840

gaaatcagta taggtaaagc cacgggcaga gggcagtac 6879



2
2293
PRT
Mus musculus

2

Met Leu Tyr Asn Gln Pro Asp Gln Lys Tyr Asp Glu Glu Asn Leu Pro
1 5 10 15

Ile Pro Asn Ser Leu Arg Cys Glu Leu Leu Leu Val Leu Lys Thr Gln
20 25 30

Trp Pro Phe Asp Pro Glu Phe Trp Asp Trp Lys Thr Leu Lys Arg Gln
35 40 45

Cys Leu Ala Leu Met Gly Glu Glu Ala Ser Ile Val Ser Ser Ile Asp
50 55 60

Glu Leu Asn Asp Ser Glu Val Tyr Glu Lys Val Asp Tyr Gln Gly Glu
65 70 75 80

Arg Gly Asp Thr Ser Val Asn Gly Leu Ser Ala Ala Gly Leu Gly Thr
85 90 95

Asp Ser Gly Leu Leu Met Asp Thr Gly Asp Glu Lys Gln Lys Lys Lys
100 105 110

Glu Ile Lys Glu Leu Lys Asp Arg Gly Phe Ile Ser Ala Arg Phe Arg
115 120 125

Asn Trp Gln Ala Tyr Met Gln Tyr Cys Leu Leu Cys Asp Lys Glu Phe
130 135 140

Leu Gly His Arg Ile Val Arg His Ala Gln Lys His Tyr Lys Asp Gly
145 150 155 160

Ile Tyr Ser Cys Pro Ile Cys Ala Lys Asn Phe Asn Ser Lys Asp Ser
165 170 175

Phe Val Pro His Val Thr Leu His Val Lys Gln Ser Ser Lys Glu Arg
180 185 190

Leu Ala Ala Met Lys Pro Leu Arg Arg Leu Gly Arg Pro Pro Lys Ile
195 200 205

Thr Ala Thr His Glu Asn Gln Lys Thr Asn Ile Asn Thr Val Ala Lys
210 215 220

Gln Glu Gln Arg Pro Ile Lys Lys Asn Ser Leu Tyr Ser Thr Asp Phe
225 230 235 240

Ile Val Phe Asn Asp Asn Asp Gly Ser Asp Asp Glu Asn Asp Asp Lys
245 250 255

Asp Lys Ser Tyr Glu Pro Glu Val Ile Pro Val Gln Lys Pro Val Pro
260 265 270

Val Asn Glu Phe Asn Cys Pro Val Thr Phe Cys Lys Lys Gly Phe Lys
275 280 285

Tyr Phe Lys Asn Leu Ile Ala His Val Lys Gly His Lys Asp Ser Glu
290 295 300

Asp Ala Lys Arg Phe Leu Glu Met Gln Ser Lys Lys Val Ile Cys Gln
305 310 315 320

Tyr Cys Arg Arg His Phe Val Ser Val Thr His Leu Asn Asp His Leu
325 330 335

Gln Met His Cys Gly Ser Lys Pro Tyr Ile Cys Ile Gln Met Lys Cys
340 345 350

Lys Ala Gly Phe Asn Ser Tyr Ala Glu Leu Leu Ala His Arg Lys Glu
355 360 365

His Gln Val Phe Arg Ala Lys Cys Leu Phe Pro Lys Cys Gly Arg Ile
370 375 380

Phe Ser Gln Ala Tyr Leu Leu Tyr Asp His Glu Ala Gln His Tyr Asn
385 390 395 400

Thr Tyr Thr Cys Lys Phe Thr Gly Cys Gly Lys Val Tyr Arg Ser Gln
405 410 415

Ser Glu Met Glu Lys His Gln Asp Gly His Ser His Pro Glu Thr Gly
420 425 430

Leu Pro Pro Glu Asp Gln Leu Gln Pro Ser Gly Asn Asp Val Asn Pro
435 440 445

Asp Ser Gly Ala Thr Ala Ala Gly Gly Arg Ser Glu Asn Ser Ile Asp
450 455 460

Lys Asn Leu Gly Ser Asn Arg Ser Ala Asp Trp Glu Lys Asn Arg Ala
465 470 475 480

Glu Pro Ala Val Thr Lys His Gly Gln Ile Ser Ala Ala Glu Leu Arg
485 490 495

Gln Ala Asn Ile Pro Leu Ser Asn Gly Leu Glu Thr Arg Asp Asn Thr
500 505 510

Thr Val Leu Arg Thr Asn Glu Val Ala Val Ser Ile Lys Val Ser Val
515 520 525

Asn His Gly Val Glu Gly Asp Phe Gly Lys Gln Glu Asn Leu Thr Met
530 535 540

Glu Gly Thr Gly Glu Pro Leu Ile Thr Asp Val His Lys Pro Gly Ile
545 550 555 560

Gly Ala Gly Val Gln Leu Cys His Pro Gly Phe Gln Glu Lys Lys Gly
565 570 575

His Glu Cys Leu Asn Glu Ala Gln Asn Ser Leu Ser Asn Ser Glu Ser
580 585 590

Leu Lys Met Asp Asp Leu Asn Pro Gln Ser Leu Glu Arg Gln Val Asn
595 600 605

Thr Leu Met Thr Phe Ser Val Gln Asn Glu Ala Gly Leu Glu Asp Asn
610 615 620

Ser Gln Ile Cys Lys Phe Glu Cys Gly Gly Asp Val Lys Thr Ser Ser
625 630 635 640

Ser Leu Tyr Asp Leu Pro Leu Lys Thr Leu Glu Ser Ile Thr Phe Val
645 650 655

Gln Ser Gln Pro Asp Leu Ser Ser Pro Leu Gly Ser Pro Ser Val Pro
660 665 670

Pro Lys Ala Pro Gly Gln Lys Phe Ser Cys Gln Val Glu Gly Cys Thr
675 680 685

Arg Thr Tyr Asn Ser Ser Gln Ser Ile Gly Lys His Met Lys Thr Ala
690 695 700

His Pro Asp Gln Tyr Ala Ala Phe Lys Leu Gln Arg Lys Thr Lys Lys
705 710 715 720

Gly Gln Lys Ser Asn Asn Leu Asn Thr Pro Asn His Gly Lys Cys Val
725 730 735

Tyr Phe Leu Pro Ser Gln Val Ser Ser Ser Asn His Ala Phe Phe Thr
740 745 750

Pro Gln Thr Lys Ala Asn Gly Asn Pro Ala Cys Ser Ala Gln Val Gln
755 760 765

His Val Ser Pro Ser Ile Phe Pro Ala His Leu Ala Ser Val Ser Ala
770 775 780

Pro Leu Leu Pro Ser Val Glu Ser Val Leu Ser Pro Asn Ile Pro Ser
785 790 795 800

Gln Asp Lys His Gly Gln Asp Gly Ile Leu Cys Ser Gln Met Glu Asn
805 810 815

Leu Ser Tyr Ala Pro Leu Pro Ala Gln Met Glu Asp Leu Thr Lys Thr
820 825 830

Val Leu Pro Leu Asn Ile Asp Ser Gly Ser Asp Pro Phe Leu Pro Leu
835 840 845

Pro Thr Glu Asn Ser Ser Leu Phe Ser Ser Pro Ala Asp Ser Glu Asn
850 855 860

Asn Ser Val Phe Ser Gln Leu Glu Asn Ser Thr Asn His Tyr Pro Ser
865 870 875 880

Gln Thr Asp Gly Asn Ile Asn Ser Ser Phe Leu Lys Gly Gly Ser Ser
885 890 895

Glu Asn Gly Val Phe Pro Ser Gln Val Ser Ser Ala Asp Asp Phe Ser
900 905 910

Ser Thr Ser Ala Gln Pro Ser Thr Pro Lys Lys Val Lys Lys Asp Arg
915 920 925

Gly Arg Gly Pro Asn Gly Lys Glu Arg Lys Pro Lys His Asn Lys Arg
930 935 940

Ala Lys Trp Pro Ala Ile Ile Arg Asp Gly Lys Phe Ile Cys Ser Arg
945 950 955 960

Cys Tyr Arg Ala Phe Thr Asn Pro Arg Ser Leu Gly Gly His Leu Ser
965 970 975

Lys Arg Ser Tyr Cys Lys Pro Leu Asp Gly Ala Glu Ile Ala Gln Glu
980 985 990

Leu Leu Gln Thr Asn Arg Gln Pro Ser Leu Leu Ala Ser Met Ile Leu
995 1000 1005

Ser Thr Ser Ala Val Asn Met Gln Gln Pro Gln Gln Ser Asn Phe
1010 1015 1020

Asn Pro Glu Thr Cys Phe Lys Asp Pro Ser Phe Leu Gln Leu Leu
1025 1030 1035

Asn Val Glu Asn Arg Pro Thr Phe Leu Pro Ser Thr Phe Pro Arg
1040 1045 1050

Cys Asp Val Ser Asn Phe Asn Ala Ser Val Ser Gln Glu Gly Ser
1055 1060 1065

Glu Ile Ile Lys Gln Ala Leu Glu Thr Ala Gly Ile Pro Ser Thr
1070 1075 1080

Phe Glu Ser Ala Glu Met Leu Ser Gln Val Val Pro Ile Gly Ser
1085 1090 1095

Val Ser Asp Ala Ala Gln Val Ser Ala Ala Gly Met Pro Gly Pro
1100 1105 1110

Pro Val Thr Pro Leu Leu Gln Thr Val Cys His Pro Asn Thr Ser
1115 1120 1125

Pro Ser Asn Gln Asn Gln Thr Pro Asn Ser Lys Thr Leu Lys Glu
1130 1135 1140

Cys Asn Ser Leu Pro Leu Phe Thr Thr Asn Asp Leu Leu Leu Lys
1145 1150 1155

Thr Ile Glu Asn Gly Leu Cys Ser Asn Ser Phe Ser Ser Ser Thr
1160 1165 1170

Glu Pro Pro Gln Asn Phe Thr Asn Asn Ser Ala His Val Ser Val
1175 1180 1185

Ile Ser Gly Pro Gln Asn Thr Arg Ser Ser His Leu Asn Lys Lys
1190 1195 1200

Gly Asn Ser Ala Ser Lys Lys Arg Lys Lys Val Ala Pro Ala Val
1205 1210 1215

Ser Val Ser Asn Thr Ser Gln Asn Val Leu Pro Thr Asp Leu Pro
1220 1225 1230

Val Gly Leu Pro Ser Lys Asn Leu Thr Val Pro Asp Thr Asn Thr
1235 1240 1245

Arg Ser Asp Met Thr Pro Asp Cys Glu Pro Arg Ala Leu Val Glu
1250 1255 1260

Asn Leu Thr Gln Lys Leu Asn Asn Ile Asp Asn His Leu Phe Ile
1265 1270 1275

Thr Asp Val Lys Glu Asn Cys Lys Ala Ser Leu Glu Pro His Thr
1280 1285 1290

Met Leu Thr Pro Leu Thr Leu Lys Thr Glu Asn Gly Asp Ser Arg
1295 1300 1305

Met Met Pro Leu Ser Ser Cys Thr Pro Val Asn Ser Asp Leu Gln
1310 1315 1320

Ile Ser Glu Asp Asn Val Ile Gln Asn Phe Glu Lys Thr Leu Glu
1325 1330 1335

Ile Ile Lys Thr Ala Met Asn Ser Gln Ile Leu Glu Val Lys Ser
1340 1345 1350

Gly Ser Gln Gly Thr Gly Glu Thr Thr Gln Asn Ala Gln Ile Asn
1355 1360 1365

Tyr Ser Met Gln Leu Pro Ser Val Asn Ser Ile Pro Asp Ser Lys
1370 1375 1380

Leu Pro Asp Ala Ser Gln Cys Ser Ser Phe Leu Thr Val Met Pro
1385 1390 1395

Thr Lys Ser Glu Ala Leu His Lys Glu Asp Gln Ile Gln Asp Ile
1400 1405 1410

Leu Glu Gly Leu Gln Asn Leu Lys Leu Glu Asn Asp Thr Ser Ala
1415 1420 1425

Pro Ala Ser Gln Ser Met Leu Met Asn Lys Ser Val Ala Leu Ser
1430 1435 1440

Pro Thr Pro Thr Lys Ser Thr Pro Asn Ile Val Val Gln Pro Val
1445 1450 1455

Pro Glu Val Ile His Val Gln Leu Asn Asp Arg Val Asn Lys Pro
1460 1465 1470

Phe Val Cys Gln Asn Gln Gly Cys Asn Tyr Ser Ala Met Thr Lys
1475 1480 1485

Asp Ala Leu Phe Lys His Tyr Gly Lys Ile His Gln Tyr Thr Pro
1490 1495 1500

Glu Met Ile Leu Glu Ile Lys Lys Asn Gln Leu Lys Phe Ala Pro
1505 1510 1515

Phe Lys Cys Val Val Pro Ser Cys Thr Lys Thr Phe Thr Arg Asn
1520 1525 1530

Ser Asn Leu Arg Ala His Cys Gln Leu Val His His Phe Thr Ile
1535 1540 1545

Glu Glu Met Val Lys Leu Lys Ile Lys Arg Pro Tyr Gly Arg Lys
1550 1555 1560

Ser Gln Ser Glu Asn Leu Ser Ser Pro Gln Asn Asn Gln Val Lys
1565 1570 1575

Lys Gln Pro Ser Met Ala Glu Glu Thr Lys Thr Glu Ser Gln Pro
1580 1585 1590

Ala Phe Lys Val Pro Ala Ala Thr Gly Asp Ala Ala Leu Ala Asn
1595 1600 1605

Ala Thr Val Ile Pro Glu Lys Gln Leu Ala Glu Lys Lys Ser Pro
1610 1615 1620

Glu Lys Pro Glu Ser Ser Ser Gln Pro Val Thr Ser Ser Ala Glu
1625 1630 1635

Gln Tyr Asn Ala Asn Leu Ala Asn Leu Lys Thr Lys Gly Arg Lys
1640 1645 1650

Asn Lys Arg His Arg Lys Glu Lys Glu Glu Lys Arg Glu Lys Asn
1655 1660 1665

Pro Val Ser Gln Ala Phe Glu Leu Pro Thr Lys Tyr Ser Ser Tyr
1670 1675 1680

Arg Pro Tyr Cys Cys Val His Gln Gly Cys Phe Ala Ala Phe Thr
1685 1690 1695

Ile Gln Gln Asn Leu Ile Leu His Tyr Gln Ala Val His Lys Ser
1700 1705 1710

Asn Leu Pro Thr Phe Ser Ala Glu Val Gln Glu Glu Ser Glu Ala
1715 1720 1725

Val Lys Glu Ser Glu Glu Thr Glu Pro Lys Gln Ser Met Lys Glu
1730 1735 1740

Phe Arg Cys Gln Val Ser Asp Cys Ser Arg Ile Phe Gln Ala Ile
1745 1750 1755

Thr Gly Leu Ile Gln His Tyr Met Lys Leu His Glu Met Thr Pro
1760 1765 1770

Glu Glu Ile Glu Ser Met Thr Ala Ala Val Asp Val Gly Lys Phe
1775 1780 1785

Pro Cys Asp Gln Leu Glu Cys Lys Leu Ser Phe Thr Thr Tyr Leu
1790 1795 1800

Ser Tyr Val Val His Leu Glu Val Asp His Gly Ile Gly Thr Arg
1805 1810 1815

Thr Ser Lys Ala Glu Glu Asp Gly Ile Tyr Lys Cys Asp Cys Glu
1820 1825 1830

Gly Cys Asp Arg Ile Tyr Ala Thr Arg Ser Asn Leu Leu Arg His
1835 1840 1845

Ile Phe Asn Lys His Asn Asp Lys His Lys Ala His Leu Ile Arg
1850 1855 1860

Pro Arg Lys Leu Thr Gly Gln Glu Asn Ile Ser Ser Lys Ala Asn
1865 1870 1875

Gln Glu Lys Ser Lys Ser Lys His Arg Thr Thr Lys Pro Asn Arg
1880 1885 1890

Ser Gly Lys Asp Gly Met Lys Met Pro Lys Thr Lys Arg Lys Lys
1895 1900 1905

Lys Ser Asn Leu Glu Asn Lys Ser Ala Lys Val Val Gln Ile Glu
1910 1915 1920

Glu Asn Lys Pro Tyr Ser Leu Lys Arg Gly Lys His Val Tyr Ser
1925 1930 1935

Ile Lys Ala Arg Asn Asp Ala Leu Ala Glu Cys Thr Ser Lys Phe
1940 1945 1950

Val Thr Gln Tyr Pro Cys Met Ile Lys Gly Cys Thr Ser Val Val
1955 1960 1965

Thr Ser Glu Ser Asn Ile Ile Arg His Tyr Lys Cys His Lys Leu
1970 1975 1980

Ser Arg Ala Phe Thr Ser Gln His Arg Asn Ile Leu Ile Val Phe
1985 1990 1995

Lys Arg Tyr Gly Asn Pro Gln Gly Arg Glu Ile Ser Glu Gln Glu
2000 2005 2010

Asp Glu Lys Asn Asp Lys Lys Gly Pro Asp Ser Ser Val Leu Glu
2015 2020 2025

Lys Asn Asp Asn Ser Glu Pro Ala Ala Ala Pro Gln Glu Glu Gly
2030 2035 2040

Arg Lys Gly Glu Lys Asp Glu Met Asp Glu Leu Thr Glu Leu Phe
2045 2050 2055

Ile Thr Lys Leu Ile Asn Glu Asp Ser Thr Asn Ala Glu Asn Gln
2060 2065 2070

Gly Asn Thr Thr Leu Lys Gly Asn Asn Glu Phe Gln Glu His Asp
2075 2080 2085

Ser Cys Thr Ser Glu Arg Gln Lys Pro Gly Asn Leu Lys Arg Val
2090 2095 2100

Tyr Lys Glu Lys Asn Thr Val Gln Ser Lys Lys Arg Lys Ile Asp
2105 2110 2115

Lys Thr Glu Pro Glu Val Ser Leu Val Val Asn Asn Thr Arg Lys
2120 2125 2130

Glu Glu Glu Pro Ala Val Ala Val Gln Thr Thr Glu Glu His Pro
2135 2140 2145

Ala Ser Phe Asp Trp Ser Ser Phe Lys Pro Met Gly Phe Glu Ala
2150 2155 2160

Ser Phe Leu Lys Phe Leu Glu Glu Ser Ala Val Lys Gln Lys Lys
2165 2170 2175

Asn Ser Asp Arg Asp His Ser Asn Ser Gly Ser Lys Arg Gly Ser
2180 2185 2190

His Ser Ser Ser Arg Arg His Val Asp Lys Ala Ala Val Ala Gly
2195 2200 2205

Ser Ser His Val Cys Ser Cys Lys Asp Ser Glu Ile Phe Val Gln
2210 2215 2220

Phe Ala Asn Pro Ser Lys Leu Gln Cys Ser Glu Asn Val Lys Ile
2225 2230 2235

Val Leu Asp Lys Thr Leu Lys Asp Arg Ser Glu Leu Val Leu Lys
2240 2245 2250

Gln Leu Gln Glu Met Lys Pro Thr Val Ser Leu Lys Lys Leu Glu
2255 2260 2265

Val Leu Ser Asn Ser Pro Asp Arg Thr Val Leu Lys Glu Ile Ser
2270 2275 2280

Ile Gly Lys Ala Thr Gly Arg Gly Gln Tyr
2285 2290