(en)A DNA fragment coding for a modified nuclear glucocorticoid receptor, particularly one mutated in the region coding for the ligand binding domain, so that receptor activity is more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand, is disclosed. A recombination system inducible in mammals by means of a fusion protein produced between a recombinase and the binding domain of the ligand derived from the modified glucocorticoid receptor of which the activity is more strongly inducible by synthetic glucocorticoids than by natural glucocorticoids, is also disclosed.

1.ApplicationNumber: US-12549198-A
1.PublishNumber: US-6699686-B1
2.Date Publish: 20040302
3.Inventor: BROCARD JACQUES BERTRAND
CHAMBON PIERRE HENRI
GRONEMEYER HINRICH
METZGER DANIEL
NICOLAS JEAN-CLAUDE
ROUX SYLVIE

4.Inventor Harmonized: BROCARD JACQUES BERTRAND(FR)
CHAMBON PIERRE HENRI(FR)
GRONEMEYER HINRICH(DE)
METZGER DANIEL(FR)
NICOLAS JEAN-CLAUDE(FR)
ROUX SYLVIE(FR)

5.Country: US
6.Claims:
(en)A DNA fragment coding for a modified nuclear glucocorticoid receptor, particularly one mutated in the region coding for the ligand binding domain, so that receptor activity is more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand, is disclosed. A recombination system inducible in mammals by means of a fusion protein produced between a recombinase and the binding domain of the ligand derived from the modified glucocorticoid receptor of which the activity is more strongly inducible by synthetic glucocorticoids than by natural glucocorticoids, is also disclosed.

7.Description:
(en)FIELD OF THE INVENTION
The present invention relates to a DNA fragment coding for a modified nuclear glucocorticoid receptor (GR), and a DNA fragment coding for a ligand binding domain (LBD) of said receptor, as well as a DNA fragment coding for a fusion protein comprising said receptor of said ligand binding domain of said receptor fused to a protein whose activity is induced in the presence of glucocorticoid ligands.
The present invention also relates to a modified nuclear glucocorticoid receptor, in particular the human receptor and its modified ligand binding domain, as well as a fusion protein comprising said receptor or said ligand binding domain.
The present invention also relates to a vector for conditionally expressing a protein, in particular a foreign protein, in human or animal host cells, in particular mammalian cells. The present invention also relates to a method of expressing a protein in said cells. Moreover, the present invention relates to a method for the conditional excision, insertion, inversion or translocation of a DNA fragment in human or animal, in particular mammalian, host cells.
The present invention relates in addition, to a vector for transferring a DNA fragment into said human or animal, in particular mammalian, host cells and to its use as a medicament, as a tool for analyzing and studying the function of a gene as well as to a method of treating cells ex vivo or in vitro.
Finally, the present invention relates to human or animal cells transfected with an expression and/or transfer vector according to the invention as well as to transgenic animals derived therefrom.
BACKGROUND OF THE INVENTION
The glucocorticoid receptor (GR) is a protein for regulating transcription of genes which mediate the action of glucocorticoids in target cells. The GR and other members of the super family of nuclear receptors (ligand-activated transcription factors) exhibit a common modular structure. The variable N-terminal region contains the constitutive transactivation activity AF-1. The core DNA binding domain (DBD) is highly conserved between various species and allows the binding of the receptor to its related DNA response element (GRE in the case of GR). The ligand binding domain (LBD), located in the C-terminal region of GR, not only binds the ligand, but also comprises multiple distinct activities: a surface for interaction with hsp90 and a distinct surface for homodimerization, a nuclear localization signal and a ligand-dependent transactivation function AF-2 (for review articles and references, see (1-5)). The LBD domain is highly conserved between various species. A transactivation function AF-2 has been identified in the C-terminal part of various LBDs (6-10). The integrity of the core part of the region containing AF-2 is required for the ligand-dependent transcription activation by the corresponding nuclear receptors, and for the interaction between this region and related transcriptional intermediate factors (TIF, also called coactivators or mediators) (11-19). A general model has been proposed (19) in which the binding of the ligand induces a conformational change in the ligand binding domain by calling into play in particular the C-terminal region harboring the core part of AF-2, thus generating a surface for an effective interaction with the TIFs. The LBDs of the nuclear receptors contain conserved regions possessing a canonic structure In the form of a helix. These structural data have made it possible to carry out a common alignment or the LBDs of all nuclear receptors. The H12 helix contains the transactivation function AF-2 (19). The m no acids of helices H11 and H12, in particular of the glucocorticoid receptors from various species such as humans, rats, mice and xenopus, are conserved.
Several mutations in the LBD of GR have been previously described. Two major types of mutation can be distinguished:
the first type consists in mutations which positively or negatively affect the affinity for binding to the ligand. For example, the replacements of the cysteine residue 656 by glycine in rat GR (20) and of the methionine residue 565 by arginine or of the alanine residue 573 by glutamine in the human GR (hGR) (21) increase the affinity for binding to the ligand and lead to a shift in the dose-response curve for the transactivation in the direction of the lowest ligand concentrations. These mutants are consequently designated “super GR”. Likewise, mutants have been reported which have a lower affinity for binding to the ligand (21-24). LBD mutations which shift the ligand dose-response curve in the direction of the highest ligand concentrations result from an altered hormone binding affinity (25-26);
the second group of LBD mutants comprises those which affect the transcriptional activation function (AF-2) without altering the binding to the ligand. As examples, there may be mentioned mutations in the region containing AF-2 which are linked to a loss or a decrease in the transactivation potential, but do not affect the affinity for binding to the ligand (6-10).
The fusion of the ligand binding domain (LBD) of nuclear receptors to heterologous proteins has made it possible to control their activity in numerous cases.
The activities of Myc, c-Abl, Src, erbB1, Raf, E1A Cre and FLP may be modulated by producing fusion proteins with the LBD of nuclear receptors (27-29). However, the ligands for these receptors are present in numerous biological systems and are thus capable of inducing a basal activity level. To avoid such problems, a mutated LBD of the estrogen receptor (ER) has been fused to the c-Myc protein (30), or to the Cre and FLP recombinases (28 and 29).
The recombinases of the family of λ integrases catalyze the excision, insertion, inversion or translocation of DNA fragments at the level of specific sites of recognition of said recombinases (31-36). The recombinases are active in animal cells (35).
The Cre recombinase, an integrase of 38 KDa from bacteriophage P1, catalyzes recombination between two DNA sequences of 34 base pairs called loxP in the absence of cofactors (32, FIG. 1 ).
The position on one or more DNA molecules and the orientation of loxP sites relative to each other determine the type of function of the recombinase, excision, insertion, inversion or translocation, in particular the Cre recombinase (FIG. 1 ). Thus, the recombinase activity of Cre is an inversion when two LoxP sites are head-to-tail on the same DNA fragment and an excision when the LoxP sites are a direct repeat on the same DNA fragment. The recombinase activity is an insertion when a loxP site is present on a DNA fragment, it being possible for a DNA molecule such as a plasmid containing a loxP site to be inserted at the level of said loxP site (FIG. 1 ). The Cre recombinase can also induce a translocation between two chromosomes provided that a loxP site is present on each of them (37) (FIG. 1 ). More generally, the Cre recombinase is therefore capable of inducing recombination between one or more different DNA molecules provided that they carry loxP sites.
Likewise, the FLP recombinase, a recombinase of 43 KDa from Saccharomyces cerevisiae , is capable of the same type of action on DNA fragments containing recognition sites FRT (34).
By producing a fusion protein (chimera) between the Cre recombinase and the C-terminal region of the human receptor for estrogens containing a Valine at position 400, a molecule is obtained which is capable of excising, in the presence of the receptor ligand, estradiol, the DNA sequences located between two loxP sites, as well as one of the loxP sites, when the latter are a direct repeat, whereas in the absence of ligand, the excision does not take place (28).
The activity of the FLP recombinase can also be regulated by producing chimeras between the FLP and the binding domain of nuclear receptors. By fusing the FLP with the LBD of the estrogen receptor or of the glucocorticoid receptor in the absence of ligand, the recombinase activity is very low, whereas it is rapidly induced by the respective ligands for the LBDs (29).
However, the use of fusion proteins between recombinases and LBDs of nuclear receptors to excise, in a controlled manner, DNA fragments situated between sites of recognition of said recombinases can pose a problem, given that the ligand concentrations present in animals or in cell culture media may be sufficient to induce, at least partially, the recombinase activity.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a recombination system inducible in animals, in particular mammals, by virtue of the use of a fusion protein produced between a recombinase and the ligand binding domain derived from the glucocorticoid receptor whose activity is inducible by synthetic glucocorticoids but not by natural glucocorticoids in particular at concentrations of less than 10 −6 M, that is to say physiological concentrations.
To avoid induction of the recombinase activity of the chimeric protein by the endogenous ligands, the present invention indeed proposes to provide a mutation in the LBD of the glucocorticoid receptor allowing It to be more strongly activated in the presence of synthetic glucocorticoid ligands than in the presence of natural glucocorticoid ligands at physlologlcal concentrations.
The subject of the present invention is indeed a modified nuclear glucocorticoid receptor, in particular mutated in the region of the ligand binding domain, so that the activity of said receptor is more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand.
According to the present invention, “(mutated) glucocorticoid receptor more strongly inducible by a synthetic glucocorticoid ligand than by a natural glucocorticoid ligand” is understood to mean that the activity of the mutated receptor according to the invention is induced more strongly by synthetic glucocorticoid ligands than by natural glucocorticoid ligands at a given concentration and that the mutated glucocorticoid receptor according to the invention is a lot less sensitive to the natural glucocorticoids than the wild-type receptor.
“Activity of said receptor” is understood to mean here the transcriptional activity of said receptor and/or, where appropriate, the activity of regulating the activity of a protein fused to said receptor or to said ligand binding domain, induced in the presence of glucocorticoids. There may be mentioned more particularly the activity of a recombinase protein fused to said receptor or to said ligand binding domain of said receptor.
“Natural glucocorticoids” are understood to mean here steroid hormones secreted by the cortex of the adrenal glands such as cortisol, corticosterone or aldosterone. “Synthetic glucocorticoids” are understood to mean chemically synthesized agonists of said receptor for the relevant activity. There may be mentioned, among the letter, dexamethasone, triamcimolone acetonide RU 486, RU 28362, bimedrazol, fluocinolone acetonide.
Some synthetic ligands will be agonists for inducing said particular activity of the receptor and antagonists in relation to another activity of the receptor. Thus, the compound RU 486 induces the activity of a recombinase protein fused to said receptor or to said ligand binding domain of the receptor but does not induce the transcriptional activity of the receptor.
The synthetic glucocorticoid ligand may bind to the (nonmutated) wild-type nuclear receptor, but it is possible that said synthetic ligand binds to the mutated receptor and not to the natural receptor. It is also possible that if said synthetic ligand binds to the wild-type receptor, the activity of the latter is nevertheless not induced by binding to the synthetic ligand.
A mutation has in particular been identified in the LBD domain of the nuclear glucocorticoid receptors (isoleucine→threonine) situated between helices H11 and H12 (19). This mutation causes a loss of activity for the receptor in the presence of natural glucocorticoids. More precisely, this glucocorticoid receptor mutated between H11 and H12 has no or very little transcriptional activity in the presence of natural glucocorticoids at their natural physiological concentrations, in particular concentrations of less than 10 −6 M. Its activity may, however, be induced by synthetic glucocorticoid ligands, in particular dexamethasone (dex).
The subject of the present invention is more particularly a mutated human receptor called hGR (I747/T) characterized in that it has, as sequence, the amino acid sequence of the human wild-type nuclear glucocorticoid receptor with an isoleucine→threonine mutation at position 747, and still more particularly a modified human nuclear receptor which has, as amino acid sequence, a sequence substantially as represented in the SEQ ID NO: 1.
The isoleucine 747 to threonine mutation in the C-terminal part of the ligand binding domain (LBD) of the glucocorticoid receptor alters the capacity of the natural ligands to induce the transactivation activity thereof. Natural glucocorticoids such as cortisol, aldosterone or corticosterone are very weakly active or completely inactive with the GR mutant (I747/T), whereas synthetic glucocorticoids such as dexamethasone (Dex) efficiently stimulate the GR-mediated transactivation (I747/T). However, the corresponding ligand dose-response curve, for the transactivation induced by Dex, is shifted toward the highest concentrations, compared with that obtained with the wild-type (WT) GR. Neither the shift nor the inability of cortisol to efficiently activate GR (I747/T) are due to an altered affinity for binding to the ligand in vitro.
Indeed, the mutation in the LBD of GR (I747/T) does not substantially alter the affinity for binding to the ligand in vitro. However, this mutant exhibits a substantial shift compared with the WT GR for the activity curve toward the high ligand concentrations.
Moreover, so-called physiological concentrations of natural glucocorticoid ligands which are sufficient to activate the WT GR cause only a residual or zero activity in the mutant GR.
This mutant differs from the other GR LBD mutants previously described (22, 38) whose altered activation function is closely correlated with a modification in their properties for binding to the ligand in vitro.
The subject of the present invention is also a ligand binding domain of the nuclear glucocorticoid receptor according to the invention and particularly the LBD domain [GR(I747/T)], characterized in that it has an isoleucine→threonine mutation between helices H11 and H12. More particularly, it has, as sequence, the ainmo acid sequence of the ligand binding domain of the human glucocorticoid receptor substantially as represented in SID No. 1 from amino acid 532 to amino acid 777 with an isoleucine→threonine mutation at position 747.
The present invention also provides a DNA fragment encoding a modified nuclear glucocorticoid receptor, in particular mutated in the region of the ligand binding domain (LBD) according to the invention, as well as a DNA fragment encoding a ligand binding domain (LBD) of the modified receptor according to the invention.
The subject of the present invention is therefore more precisely a DNA fragment encoding a wild-type nuclear glucocorticoid receptor with an isoleucine to threonine mutation situated between helices H11 and H12 of the amino acid sequence of said receptor, and still more particularly a DNA fragment, characterized in that it has, as sequence, a coding sequence of the human nuclear glucocorticoid receptor whose amino acid sequence is substantially that represented in SEQ ID NO: 1.
In one embodiment, the subject of the present invention is a DNA fragment, characterized in that it has, as sequence, the cDNA sequence of the human nuclear glucocorticoid receptor substantially as represented in SEQ ID NO: 1 comprising the ACC codon coding for threonine at position 747 of the amino acid sequence of said receptor.
Likewise, the subject of the present invention is a DNA fragment encoding the ligand binding domain (LBD) of the modified nuclear glucocorticoid receptor with an isoleucine→threonine mutation situated between helices H11 and H12 and in particular at a position corresponding to position 747 of the amino acid sequence of the human nuclear glucocorticoid receptor.
More particularly, the present invention provides a DNA fragment encoding the ligand binding domain of the modified human nuclear glucocorticoid receptor, characterized in that it has, as sequence, a sequence coding for the amino acids of the ligand binding domain of the human nuclear glucocorticoid receptor whose amino acid sequence is substantially as represented in SEQ ID NO: 1 from amino acid 532 to amino acid 777.
Still more particularly, the present invention provides a DNA fragment, characterized in that it has, as sequence, the cDNA sequence encoding the ligand binding domain (LBD) of the human nuclear glucocorticoid receptor substantially as represented in SEQ ID NO: 1 from codon 532 to codon 777 with the ACC codon at position 747.
The subject of the present invention is, in addition, a vector system for conditionally expressing a protein, in particular a foreign protein, in host cells comprising an element for control of transcription inducible by a complex formed by a nuclear glucocorticoid receptor and a ligand, characterized in that it comprises:
a first DNA fragment consisting of a DNA fragment coding for said protein under the control of elements ensuring its expression in said host cells, said elements ensuring its expression comprising a sequence for control of transcription (RE) inducible by the receptor according to the invention complexed with a synthetic glucocorticoid ligand, and
a second DNA fragment consisting of a functional DNA fragment coding for said receptor according to the invention or only part of said fragment comprising the region which recognizes the ligand (LBD) according to the invention, and the DNA binding region (DBD) which attaches to said sequence for control of transcription (RE),
it being possible for said first and second DNA fragments to be carried by the same vector or two vectors separately.
Some cells have endogenous GR which may be activated by natural ligands and synthetic ligands. If it is desired to activate a given gene at will in such cells, without it being possible for it to be activated by endogenous ligands or by endogenous receptors, it is necessary for this gene to contain an RE different from GRE and to use a chimeric activator comprising the corresponding DBD and a mutated LBD according to the invention. This gene can therefore be activated by synthetic ligands, but not by natural ligands.
Accordingly, in one embodiment, the DNA binding domain (DBD) of the glucocorticoid receptor is replaced by that of another transactivator, in particular that of the yeast protein Gal 4 and the sequence for control of transcription (RE) of the glucocorticoid receptor is replaced by that of another transactivator, in particular the 17 mer (17m) sequence recognized by the Gal4 protein.
The subject of the present invention is therefore also a method of expressing a foreign protein in human or animal, in particular mammalian, cells, characterized in that cells are cultured which contain an expression vector system according to the invention, and in that said synthetic ligand is added to the culture medium and then the protein synthesized is recovered.
The synthetic glucocorticoid ligands diffuse freely in the cells when they added to the culture medium or injected into an animal.
In this method, a synthetic ligand chosen from dexamethasone, triamcinolone acetonide, RU2836, bimetrazole, deacylcortivazol and fluocinolone acetonide will be used in particular.
The subject of the present invention is, in addition, a fusion protein comprising a receptor according to the invention or a ligand binding domain according to the invention and a protein whose activity is more strongly inducible by binding of said receptor or of said ligand binding domain (LBD) of said receptor with a said synthetic glucocorticoid ligand than with a natural glucocorticoid ligand.
In particular, the subject of the present invention is a fusion protein which comprises a recombinase protein and more particularly of the family of λ integrases and still more particularly the Cre protein of the bacteriophage P 1 .
In one embodiment, the fusion protein comprises the C-terminal part of the hinge region D of the human nuclear glucocorticoid receptor, intercalated between the Cre protein and said ligand binding domain of the modified receptor according to the invention.
The actual recombinase activity of the fusion protein is similar to that of Cre. It therefore makes it possible, like Cre, to excise or reverse the DNA sequences located between the loxP sites, to insert a plasmid containing a loxP site at the level of a loxP site located in the genome, or to carry out a translocation between two chromosomes each containing a loxP site.
The subject of the present invention is also a fusion gene coding for the fusion protein according to the invention comprising a DNA fragment coding for a modified nuclear glucocorticoid receptor or a ligand binding domain of said receptor according to the invention, as well as a DNA fragment coding for a protein of which it is desired to regulate the activity, said activity being inducible by binding of said receptor or of said ligand binding domain with a said synthetic glucocorticoid ligand, but not by binding with a natural glucocorticoid ligand when said protein is fused to said receptor or to said ligand binding domain of said receptor.
More particularly, the subject of the present invention is a fusion gene comprising, in the 5′→3′ direction:
a DNA fragment coding for the Cre recobinase of bacteriophage P 1 :
a DNA fragment coding for all or part of the hinge region D of the nuclear glucocorticoid receptor, a region situated between the DBD domain and the LBD domain, and
the DNA fragment coding for the modified, in particular mutated, ligand binding domain (LBD) of the nuclear glucocorticoid receptor according to the invention.
And still more particularly, the subject of the present invention is a fusion gene, characterized in that it has, as sequence, a sequence coding for the amino acid sequence of SEQ ID NO: 2 comprising:
amino acids 1 to 343 which correspond to the Cre recombination;
amino acids 346 to 377 which correspond to the C-terminal region of the D region of the human glucocorticoid receptor;
amino acids 378 to 623 which correspond to the LBD [GR(I747/T)].
In a specific embodiment, the fusion gene has substantially, as sequence, the Cre-LBD[GR(I747/T)] sequence as represented in SEQ ID NO: 2 in which amino acids 626 to 667 correspond to the F region of the human estrogen receptor.
The subject of the present invention is also a vector for expressing the fusion protein encoded by the fusion gene according to the invention, in animal, in particular mammalian, host cells, characterized in that it comprises the fusion gene according to the invention, placed under the control of elements for expression ensuring its expression in said host cells. The vector for expressing the fusion protein may also be a vector or integrating into the genome of the host cells.
In the vectors according to the invention, to express the receptor, the LBD or the fusion protein according to the invention, it is necessary for the DNA fragments coding for these molecules to be placed under the control of a regulatory sequence, in particular of the promoter/enhancer sequences. Promoter/enhancer sequences of the SV 40 virus may be used in particular.
The subject of the present invention is, in addition, a method of recombination, in particular of conditional excision, insertion, inversion or translocation at the level of a DNA fragment containing one or two sites for specific recognition of a recombinase protein in human or animal, in particular mammalian, host cells, characterized in that it comprises the steps in which:
1) a fusion protein according to the invention or a vector for expressing said fusion protein according to the invention is introduced into said host cells under conditions allowing the expression of the fusion protein according to the invention and,
2) said fusion protein is complexed with a said synthetic glucocorticoid ligand by exposing said ligand to said host cells.
The present invention therefore provides a method for the conditional deletion of a DNA fragment in which a method of excision according to the invention is used, and in which said DNA fragment to be excised is integrated between two recombinase protein recognition sites oriented as a direct repeat. In particular, said DNA fragment may be chosen so that the excision of said DNA fragment has, as effect, the inactivation of said gene. The excision of said DNA fragment can also allow the synthesis of a functional protein if said fragment contains, for example, a stop codon or a polyadenylation signal.
In the preferred embodiment according to the invention, said recombinase protein specific recognition sites are the loxP sites and said recombinase protein is the Cre protein of bacteriophage P 1 .
Said DNA fragment which it is desired to recombine, in particular to excise and said recognition site(s) specific for a recombinase protein may be carried by a plasmid or viral vector, or may be integrated into the chromosome of the host cells.
The integration of a gene carrying the loxP sites in a genome may be random or targeted, In particular, the integration of the recombinase protein specific recognition sites, in particular of the loxP site(s) for the Cre recombinase, may take place by homologous recombination of the gene comprising said DNA fragment to be excised or inverted (2 loxP sites) or respectively inserted or translocated (1 loxP site) with a said modified gene comprising said DNA fragment to be excised flanked in 5′ and/or 3′ by said recombinase recognition site(s) depending on the desired application, in particular the loxP sites.
A chimeric protein, Cre-LBD[GR(747/T)] of SID No. 2, composed of the Cre recombinase fused to the LBD of GR (I747/T) was constructed. The fusion protein Cre-LBD[GR(I747/T)] makes it possible to carry out a recombination between the loxP sites, in a mammalian cell, following a treatment with synthetic glucocorticoids. In the absence of treatment, or in the presence of concentrations of natural glucocorticoids of up to 10 −6 M, no excision is observed. This chimera is capable of excising the DNA sequences situated between the loxP sites previously integrated into the genome of F9 cells (mouse embryo carcinoma) or present on a plasmid after transfection of these cells with a vector expressing Cre-LBD[GR(I747/T)] following a treatment with Dex at 10 −6 M or 10 −7 M, whereas at such concentrations, cortisol does not induce recombinase activity. As already stated, the Cre-LBD[GR(I747/T)] expression vector may also be a vector for integration into the aenome of the host cells.
This system therefore makes it possible to release the recombinase activity of a chimeric protein at given and chosen time. The fusion protein Cre-LTD[GR(I747/T)] may be expressed in cells containing loxP sites without modifying the locus containing the loxP sites. Recombination at the level of the loxP sites takes place only after treatment with synthetic glucocorticoids. Furthermore, by expressing the fusion protein Cre-LBD[GR(I747/T)] in an animal under the control of a promoter exhibiting cellular specificity, it is possible to obtain recombination between loxP sites, specifically in these cells.
The subject of the present invention is also a vector for transferring a DNA fragment into human or animal, in particular mammalian, host cells, characterized in that it comprises a said DNA fragment to be transferred comprising recognition sites specific for the recombinase, in particular two loxP sites, oriented as direct repeats and a cassette for expression of a fusion gene according to the invention. Appropriately, the two sites are placed at each end of said DNA fragment.
Preferably, the fusion gene is placed under the control of expression elements specific for the host cells; in particular, it comprises the sequence of the plasmid pCre-LBD[GR(I747/T)] of SEQ ID NO: 3.
In the excision method according to the invention, said DNA fragment to be excised may be transferred into the host cells before, at the same time, or after the step of introducing the fusion protein or a transfer vector.
The transfer vector according to the invention may be a plasmid or a viral vector. The mode of transfer varies according to the type of target cell and the desired efficiency.
When the material is transferred into the germ cells, microinjection of the male pronucleus of a fertilized oocyte is preferably used. To introduce the DNA into pluripotent embryonic cells (ES cells), the appropriate technique will instead be electroporation or the use of retroviral vectors.
For experiments on somatic cells, it is sought to obtain a maximum efficiency, hence the preferential use of viral vectors: mainly retroviruses and adenoviruses. These viruses should be defective, in particular for application in human health to prevent any multiplication or any risk of revertion.
After integration or otherwise of this type of vectors into the genome, the Cre-lox system makes it possible to excise certain viral sequences which might possibly present a risk in relation to the subsequent propagation of the virus. This is very advantageous for the use of such vectors in gene therapy. Vectors containing a cassette for expression of the Cre-LBD[GR(I747/T)] gene and lox sites delimiting a potentially “dangerous” gene and this cassette make it possible, where appropriate after integration into the genome of the recombinant virus, to eliminate these elements. Conversely, it is also possible to activate a gene which has been integrated in an inactive form. This activation may result from the deletion of a fragment from said gene, for example a stop codon or a polyadenylation signal delimited by loxP sites.
When it is desired to transfer genetic material into a cell, constructs are generally used which comprise the gene to be transferred, to which a helper gene has optionally been added, conferring a selective advantage (for example the neo gene conferring resistance to the antibiotic G418).
Selectable markers may also be excised by the excision method according to the invention.
By using conventional techniques, it is possible to modify, by homologous recombination, mammalian, in particular mouse, genes. A loxP site may be introduced into a gene which it is desired to modify, or two loxP sites may delimit sequences which it is desired to modify. In particular, the selectable marker used to make it possible to identify the homologous recombination events may be problematic, and may be removed in a first instance, if necessary, if it is itself delimited by recombinase recognition sites such as loxP or FRT sites, by microinjecting the corresponding recombinase protein, by transfecting the cells, in particular ES cells, with an expression vector for a recombinase protein, or by crossing mice carrying the selectable marker with mice expressing the desired recombinase, in particular the Cre recombinase. This makes it possible to obtain mice whose sole modification in the modified locus is the insertion of recognition sites such as loxP. These mice may subsequently be crossed with mice expressing Cre-LBD[GR(I747/T)] and the modification of the locus obtained by treatment with the ligand such as Dex. This makes it possible to inactivate or modify a gene at a given time, and therefore to study the function of these genes at various times of development. This is particularly advantageous for studying the genes which are essential for the good progress of embryonic development. Indeed, in some cases, conventional homologous recombination techniques cause the death of the embryo and do not allow the function of the gene to be studied at later stages.
The transfer of genes into a given cell forms the basis of gene therapy. The most efficient vectors in this regard are viral vectors, in particular retroviral or adenoviral vectors (39). The subject of the present invention is therefore also a transfer vector according to the invention comprising said DNA fragment to be transferred comprising two loxP sites oriented as a direct repeat, for use as a medicament in gene therapy, when it is administered in combination with a said synthetic glucocorticoid ligand such as dexamethasone.
This type of vector makes it possible to transfer the desired DNA fragment comprising two recombinase specific recognition sites into a gene so that it will be possible for the DNA sequences situated between the two loxP sites to be conditionally excised after administration of said synthetic glucocorticoid
The present invention provides, if addition, human or animal, in particular mammalian, cells transfected with a vector for expression and transfer of a fusion protein according to the invention, or human or animal, in particular mammalian, cells into which a DNA fragment has been transferred with the aid of a transfer vector according to the invention, or alternatively human or animal, in particular mammalian, cells constitutively expressing the fusion protein according to the invention.
A further subject of the present invention is finally a transgenic animal, in particular a mouse, comprising a functional gene for the fusion protein according to the invention, said gene being in particular integrated into one of its chromosomes.
Alternatively, a transgenic animal comprising a functional gene for the fusion protein according to the invention, in which a gene of interest is modified by insertion of loxP site(s), in particular integrated into one or more chromosomes.
The subject of the present invention is finally the use of an expression or transfer vector of cells or of an animal according to the invention as a tool for analyzing or studying the function of a given gene of a host cell and a method of treating cells ex vivo or in vitro involving the use of a method of excision, insertion, inversion or translocation according to the invention and the use of an expression or transfer vector according to the invention.
Other characteristics and advantages of the present invention will emerge in the light of the detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
SEQ ID NO: 1 represents the nucleotide and peptide sequences of GR (I747/T) with the DBD corresponding to aa 421-487 and the LBD corresponding to aa 532-777;
SEQ ID NO: 2 represents the nucleotide and peptide sequences of Cre-LBD[GR(I747/T)].
Nucleotides 1-1029 code for the Cre recombinase (aa 1-343).
Nucleotides 1036-1131 code for the C-terminal region of the D region or the human glucocorticoid receptor [aa 500-531 of GR (I747/T); aa 346-377 of Cre-LBD[GR(I747/T)].
Nucleotides 1132-1869 code for the LBD of GR (I747/T) [aa 532-777 of GR (I747/T); aa 378-623 of Cre-LBD[GR(I747/T)].
Nucleotides 1876-2001 code for the F region of the human estrogen receptor [aa 554-595 of HEGO; aa 626-667 of Cre-LBD[GR(I747/T)].
Nucleotides 1777-1779 correspond to the mutation which replaces isoleucine with a threonine.
SEQ ID NO: 3 represents the nucleotide sequence of the plasmid pCre-LBD[GR(I747/T)].
SEQ ID NOS: 4 to 17 represent the nucleotide sequences of the various oligonucleotides used according to the invention.
FIG. 1 [SEQ ID NOS.: 20-21] is a schematic representation of Cre recombinase activities.
FIG. 2 represents the transcriptional activity of the mutant and wild-type GR receptor in response to dexamethasone and the capacity for attachment of dexamethasone to the two receptors.
FIG. 3 represents a Scatchard analysis of the binding of [ 3 H]-dexamethasone to the wild-type and I747/T mutant receptors.
FIG. 4 represents competition by RU 486 for the Dex binding and the transactivation activities of the wild-type (WT) and GR (I747/T) receptor.
FIG. 5 is a schematic representation of the Cre, hER, Cre-ER, hGR and Cre-LBD[GR(I747/T)] proteins.
FIG. 6 is a schematic representation of the plasmid pCre-LBD[GR(!747/T)].
FIGS. 7A-D represents the nucleotide sequence of pCre-LBD[GF(I747/T)](SEQ ID NO: 5) and the peptide sequence of Cre-LBD[GF(I747/T)] (SEQ ID NO. 5.
FIG. 8 represents the PCR strategy used to detect the wild-type, mutated and recombinant alleles in the test for recombinase activity of Cre-LBD[GR(I747/T)].
FIG. 9 represents the PCR analysis of the excision of the sequences situated between the loxP sites after transient transfection of pCre-LBD[GR(I747/T)] in the murine cells RXRα +/−(LNL) without addition of ligand, or treated with cortisol (10 −6 M) or with dexamethasone (10 −6 M).
FIG. 10 represents the PCR analysis of the recombinase activity of Cre-GR(I747/T) in the cells RXRα +/−(LNL) :Cre-GR(I747/T) not treated or treated with cortisol or dexamethasone at 10 −6 M.
FIG. 11 summarizes the recombinase activity of Cre-LBD[GR(I747/T)] in the RXRα +/−(LNL) line constitutively expressing the recombinase, in the presence of increasing concentrations of various ligands.
FIG. 12 represents the PCR analysis of the recombinase activity of Cre-LBD[GR(I747/T)] induced by synthetic ligands in spite of the presence of natural ligands.
FIG. 13 represents the test for the recombinase activity of Cre-LBD[GR(I747/T)] in human HeLa cells.
FIG. 14 is a schematic representation of the trangene Cre-LBD[GR(I747/T)]. Part (a) represents the DNA fragment containing the sequence coding for the Cre-LBD[GR(I747/T) ] gene used to establish transgenic mice and part (b) represents the genomic structure of the wild-type allele RXRα, of the target allele RXRα ΔAF1 (LNL) and of the excised allele RXRα ΔAF1 (L) as well as the PCR strategy for analyzing the excision of the marker.
FIG. 15 represents the PCR analysls of the recombinase activity of Cre-LBD[GR(I747/T)] in mice treated with dexamethasone.


DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
Replacement of isoleucine 747 with threonine in the human nuclear glucocorticoid receptor.
A human GR mutant was obtained comprising a single amino acid replacement, that of the isoleucine 747 residue by a threonine residue, located between helices H11 and H12, in the C-terminal part of the ligand binding domain. This mutation appeared spontaneously in the vectors derived from HG1 (40). This mutant is different from other GR LBD mutants previously described (22, 38), in which the altered activation function is closely correlated with a modification in their ligand binding properties in vitro.
1. Mutation in the Human GR
The vector for expression of the wild-type human GR (pSG5HG0) was obtained by cloning the BamHI fragment of about 2.9 kb (containing the open reading frame of the wild-type hGR) isolated from the HG0 vector previously described (40), into the BamHI site of the expression vector pSG5 (41).
All the DNA constructions were performed by means of conventional techniques (42). The expression vector GR (I747/T)hGR, coding for a mutant carrying the I 747→T mutation, was obtained from the fragment of PstI-PflMI digestion of the mutated expression vector HG1 (40) which was ligated into the corresponding PstI and PflMI sites of pSG5HG0.
In addition to the isoleucine 747 residue to a threonine residue mutation present in HG1, this mutant also contains a mutation of the EcoRI site which does not result in any alteration of the peptide sequence.
The presence of the mutation was confirmed by sequencing (Sequenase 2.0 protocol, U.S. Biochemical Corp., Cleveland, Ohio).
2. Results
2.1. The Replacement of Isoleucine 747 by Threonine Leads to Shift in the Dexamethasone Dose-response Curve for Transactivation, Without Substantially Affecting the Ligand Binding Affinity.
The transcriptional characteristics of the wild-type (WT) hGR and of a mutant in which isoleucine 747 was replaced by threonine [designated hereinafter GR (I747/T)] were compared. The receptors were transiently expressed in GR-free CV-1 cells, conjointly with a luciferase reporter gene under the control of the tyrosine aminotransferase promoter (pTAT-tk-Luc). FIG. 2 shows the corresponding curves of response as a function of ligand dose used, allowing the EC 50 (ligand concentration giving 50% of the maximum transcriptional response) values to be determined. The dose-response curve for WT hGR exhibits a maximum response at 10 −8 M Dex and a response equal to half the maximum response at 4×10 −8 M, whereas the transcriptional response curve for the GR (I747/T) mutant is markedly shifted toward the highest ligand concentrations, resulting in 100-fold higher values of the EC 50 for Dex (4×10 −8 M) (see Table I below). The maximum stimulation mediated by GR (I747/T) was reached at about 10 −6 M Dex. However, it is important to note that the GR (I747/T) receptor is capable of activating the transcription of pTAT-tk-Luc to the same degree as the WT GR. Consequently, the replacement of the isoleucine residue by threonine at position 747 does not reduce the ability of GR to activate transcription but specifically modified the ligand sensitivity of the response.
The effect of this mutation is independent of the N-terminal A/B region and of its associated transactivation function AF-1. The deletion mutants lacking AF-1 but containing the I747/T[ΔA/B-GR (I747/T)] mutation require 3×10 −8 M Dex for a stimulation representing half the maximum stimulation, whereas only 6×10 −10 M are sufficient for the ΔA/B-GR derived from the WT (data not represented; see also Table II for the corresponding GAL chimeras). Thus, the I747/T mutation exerts a similar effect on the complete GCr and the CR with N-terminal deletion. It can be concluded therefrom that the shift observed in the response as a function of the ligand and dose is not the consequence of modified interaction between AF-1 land AF-2.
To determine the effect of the I747/T mutation on the binding of the ligand, the binding of Dex was measured in a cytosol of Cos-7 cells transiently expressing the mutant hGR and the wild-type hGR. The K d values obtained from a Scatchard analysis are very similar since the I747/T mutant only manifests an affinity for Dex which is twice as low as the WT GR (FIG. 3 ). Thus, the order of magnitude of the shift in the dose-response curves toward the high concentrations of hGR (I747/T), compared with the WT hGR (about 100 fold) is not very likely to result from a decrease in affinity for the ligand for the mutated hGR (factor of 2 only; compare FIGS. 2 and 3 ).
2.2. hGR (I747/T) Does Not Respond to the Natural Ligands
To further characterize the binding characteristics of the I747/T mutant, the activation by various known synthetic agonists for the WT GR (triamcinolone acetonide, RU28362, bimedrazol), a partial agonist (fluocinolone acetonide) and natural glucocorticoids (cortisol, corticosterone), was compared. With the mutant receptor, triamcinolone acetonide and RU28362 give the same transactivation as with the wild-type receptor, whereas bimedrazol and fluocinolone acetonide manifested a higher activity (Table I). For all these analogs, the I747/T mutant manifests a shift in the dose-response curve in the direction of the higher ligand concentrations, although this shift is less pronounced for high affinity ligands (Table 1).
Natural ligands, such as cortisol or corticosterone or aldosterone, the natural ligand for the mineralocorticoid receptor, bind to GR with a lower affinity than Dex. With the wild-type GR, these ligands are as effective as Dex for stimulating the luciferase reporter (Table I). However, in the presence of these natural ligands, and in notable contrast with the effects observed with Dex, transactivation by the I747/T mutant is greatly reduced (cotisol) or suppressed (corticosterone, aldosterone).
2.3. Similar Agonist and Antagonist Activities of RU486 With Wild-type hGR and Mutant hGR
RU486 is a potent inhibitor of the expression of genes which are dependent on glucocorticoids. In the presence of this antagonist, the AF-2 function of GR is blocked, but it has been possible to observe some AF-1-dependent agonist activity [(43) and references contained therein]. A mutation or a deletion of the C-terminal part of the LBD can alter the response to agonist/antagonist ligands, although the roles of AF-1 and AF-2 have not been clearly defined in the studies reported (44-46).
To test the relative binding affinity of RU486 for the I747/T mutant and the wild-type, the ability of RU486 to enter into competition with Dex for binding to the receptors was examined. Cells transfected with mutant or WT hGR were treated with 10 nM [ 3 H]-Dex, a concentration sufficient to generate essentially mutant and WT receptors complexed with Dex, in conformity with the in vitro ligand binding tests (see above), and with increasing concentrations of RU486 (FIG. 4 ). Very similar IC 50 (the antagonist concentration blocking 50% of the maximum transactivation) values were obtained for the two receptors (10 −8 M for the WT hGR and 6×10 −9 M for the mutant hGR). These results are in agreement with the tests for binding to Dex and show that the affinities for binding to Dex and RU486 are not modified by the I747/T mutation.
2.4. The Shift in the Transcriptional Activity of the Mutant Receptor is Independent of the Promoter
The results described above were obtained in transfected cells using a reporter gene containing a promoter fragment of the TAT gene which harbors two glucocorticoid response elements (GRE). To distinguish between a modification of the intrinsic transactivation properties of the receptor and modification of the cooperative activity resulting from the binding of several receptor molecules, the transcriptional activities of the mutant receptor GR (I747/T) and of the wild-type receptor were compared using a synthetic promoter containing a single GRE, which is palindromic and perfect. The corresponding dose-response curves have the same profiles as those obtained with the TAT promoter. The response corresponding to half the maximum response is obtained at 10 −10 M for the WT GR and at 2×10 −8 M for GR (I747/T) (Table II). This indicates that the shift observed with the mutant receptor reflects the intrinsic transactivation properties of the mutant GR (I747/T) and are not the result of an alteration in the cooperation between the receptors.
The replacement of the DNA binding domain of GR by the first 147 amino acids of the yeast Gal4 transactivator gives a hybrid activator protein having a new promoter specificity. The Gal4-GR hybrids consist of the DNA binding domain (DBD) of Gal4(1-147) fused with the LBD of WT or mutant hGR. Their capacities to activate transcription were tested in transient transfection tests in HeLa cells. The same shift was observed as with the complete GR (I747/T) mutant (Table II). These results show that the shift in the transcriptional response as a function of the ligand dose is independent of promoter and of response element (RE). In addition, this effect is apparently not specific for the strains of cells used, as has been observed both in human HeLa cells and in simian Cos cells. To further exclude a possible involvement of factors binding to the tk or β-globin promoter of the reporter gene (47), a reporter gene was used containing a Gal4 response element preceding a minimal promoter. Although transactivation by the Gal4 chimeric protein was reduced with this minimal reporter gene, it was still possible to observe a shift in the response curve toward the increasing doses for the mutant chimeric protein Gal4-GR (I747/T) (Table II).
2.5. Transcriptional Activity of Mutant and Wild-type GR Receptors in Response to Dexamethasone (FIG. 2 ).
CV-1 cells were transiently transfected with 0.5 μg of vectors for expression of wild-type GR (□) or of GR (I747/T) (o), 2.5 μg of pTAT-tk-Luc as reporter plasmid and 0.5 μg of pCMV-βGal as internal control. After transfection, the cells were incubated for 24 hours with increasing concentrations of Dex, and they were then subjected to an assay of the β-galactosidase and luciferase activities, as described in “Materials and Methods”. The luciferase activity was normalized with the β-galactosidase activity, and the value obtained in the absence of ligand was subtracted for each point. The results were expressed as a percentage of the maximum luciferase activity obtained for each receptor. Each point is the mean±standard deviation of the mean of four separate determinations carried out in duplicate. The analyses of binding at saturation were carried out on cytosols obtained from Cos-7 cells transfected with 40 μg of vector for expression of wild-type hGR (□) or of hGR (I747/T) (•), with 20 μg of pCMV-βGal incubated overnight at 4° C. in the presence of increasing concentrations of [ 3 H]-dexamethasone±a 100× excess of radio-inert Dex. The [ 3 H] steroid attached was measured after treatment with dextran-coated charcoal. The results presented are those for a single assay carried out in duplicate and representative of three separate assays. The saturation curves for the binding of [ 3 H]-dexamethasone are represented as a percentage of the maximum binding capacity obtained for each receptor. The binding capacity of the GR (I747/T) mutant is reduced to reach about 40-60% of that of the wild-type hGR (623±65 fmol/mg of protein, compared with 1340±285 fmol/mg of protein).
2.6. Scatchard Analysis of the Binding of [ 3 H-dexamethasone to the Wild-type and I747/T Mutant Receptors (FIG. 3 ).
The Scatchard analysis was applied to the binding performed as described in FIG. 2 . The apparent dissociation constant for the GR (T747/T) mutant (o) is only twice as high as that for the wild-type hGR (υ) (K o =5.1±1.9 compared with 2.7±1.3 nM).





TABLE I



2.7.

Transcriptional activities and EC 50 of various steroids

with wild-type hGR and hGR(I747/T)







Luciferase




activity %

EC50









Wild
hGR

hGR


Ligand
type
(I747/T)
Wild type
(I747/T)
% EC 50










Dexamethasone
100
100
4 × 10 −10 M
4 × 10 −8 M
100

TA
98
102
5 × 10 −10 M
2 × 10 −8 M
40

RU28362
90
88
3 × 10 −10 M
7 × 10 −9 M
23

Fluocinolone
75
100
1.5 × 10 −10 M
3 × 10 −9 M
20

Bimedrazol
100
180
10 −10 M
2 × 10 −9 M
20

Cortisol
110
15
7 × 10 −9 M
nm

Corticosterone
100
0
7 × 10 −8 M
nm

Aldosterone
90
0
5 × 10 −8 M
nm







































Co-transfections were carried out and dose-response curves were constructed as described in FIG. 2 . Six increasing concentrations were tested, in duplicate, triamcinolone acetonide (TA), RU28362, fluocinolone acetonide (fluocinolone), cortisol, corticosterone and aldosterone. The assays of luciferase and β-galactosidase activity were carried out as described in “Materials and Methods”. The mean values of three independent assays are given in this table. nm=not measurable.
2.8. Competition for the Dex Binding and for the Transactivation Activities by RU486 (FIG. 4 ).
Cos-7 cells transfected with the wild-type hGR (□) or I747T mutant (e) were incubated for 1 hour at 37° C. with 10 nM [3H]-dexamethasone alone or in the presence of the indicated concentration of nonlabeled RU486, as descibed in “Materials and Methods”. The data are expressed as a percentage of the specific binding observed the control not subjected to the competition. These data represent the mean of determinations carried out in duplicate for each antagonist concentration.
The antagonist activity was determined on CV-1 cells transfected as described in FIG. 4 and treated simultaneously with 10 −7 M Dex and the indicated concentration of RU486, 24 hours before harvesting for the assay of the luciferase and β-galactosidase activities. 100% activity is defined for each receptor as the luciferase activity of the cells treated with Dex alone. Each point represents the mean of at least two independent tests carried out in duplicate. (□) wild-type hGR; (o): I747T mutant.





TABLE II



2.9.

EC 50 obtained with various reporter genes







Receptor
Reporter gene
EC 50





Wild-type GR
PGRE-tk-Luc
10 −10 M


GR(I747/T)
″
2 × 10 −8 M


Gal4-GR
p(17M) 5 -βGlob-Luc
1.7 × 10 −9 M


Gal4-GR (I747/T)
″
1.5 × 10 −7 M


Gal4-GR
p(17M) 5 -tata-Luc
2 × 10 −9 M


Gal4-GR (I747/T)
″
2 × 10 −7 M




























HeLa cells (for the Gall chimeras) or CV-1 cells were transiently cotransfected with various expression vectors and reporter genes. The transfection efficiency was standardized by cotransfecting with 0.5 μg of vector pCMV-βgal. The cells were treated for 24 hours with increasing concentrations of dexamethasone. The luciferase and β-galactosidase assays were carried out as described in “Materials and Methods”. The luciferase activity is expressed as a percentage of the maximum activity obtained for each receptor and each reporter gene. The EC 50 values were determined graphically.
2.10. Outline
The isoleucine 747 mutation of the human glucocorticoid receptor to threonine results in a very high reduction, or even in an abolition, of the transactivation activity of the receptor, in the presence of natural glucocorticoids, whereas at similar concentrations, synthetic glucocorticoids efficiently stimulate the transactivation activity of this mutated receptor.
It is important to underline that hGR (I747/T) responds fully to a high concentration of Dex, which indicates that it can, in principle, transactivate as efficiently as its wild-type counterpart. Consequently, the transcriptional activation function of AF-2 itself is apparently not altered by the mutation, as is the case for receptors comprising mutations in the core part of AF-2 (see the introduction for the references).
3. Materials and Methods
3.1. Cell Cultures and Transfection
The CV-1 and Cos-7 cells were cultured in Dulbeco's modified Eagle's medium (DMEM) containing 10% (v/v) of fetal calf serum (FCS) (Gibco, Grand Island, N.Y.), in a humidified atmosphere containing 5% CO 2 . The HeLa cells were cultured under the same conditions. Transient transfections were carried out in 6-well multiplates according to the calcium phosphate procedure already described (42). A reference recombinant plasmid, pCMV-βgal, expressing bacterial β-galactosidase, was transfected conjointly with the receptor expression vector and the corresponding reporter gene so as to correct possible variations in he transfection efficiency. The precipitate was removed by washing after 24 hours, and the cells were kept in DMEX without FCS but containing hormone. After another 24 hours, the cells were rinsed and lysed for 15 minutes with 0.3 ml of lysis buffer [25 mM Tris.phosphate, pH 7.8, 8 mM MgCl 1 , 1% Triton X100, 10% glycerol (48)]. The corresponding luciferase activity as determined on an aliquot fraction (0.1 ml), by evaluation of the luminescence peak (for an integration time of 15 seconds), after injection of 0.1 ml of 1 mM luciferin in an LKB luminometer. The β-galactosidase was determined by the method modified by Bocquel et al. (49), for controlling the efficiency of each transfection.
3.2. Plasmids Used
The N-terminal deletion GRs, ΔA/B-GR or ΔA/B-GR5I747/T), which expressed only amino acids 368 to 777, were obtained from the fragment of PstI-Pfl MI digestion of the expression vector HG0 or pGR (I747/T), respectively, ligated into the corresponding sites of HG8 (49). The plasmid pGAL-GR (gift from T. Lerouge) consisted of the DNA binding domain of the yeast Gal4 protein (aa1-147) and of the hormone binding domain of GR (a500-777); pGal-GR (I747/T) was constructed by insertion of the mutation into pGal-GR.
The reporter gene pTAT-tk-Luc was provided by D. Gagne and was constructed as follows: an intermediate plasmid pTAT was first generated by insertion of the 966 bp EcoRI-BamHI segment obtained from pKT531 [offered freely by T. Grande (50)] into the corresponding sites of the vector pUC19 (Pharmacia). The fragment containing the sequence coding for the firefly luciferase and the thymidine kinase (tk) minimal promoter obtained from the Herpes simplex virus was isolated in the form of a BamHI fragment from pvit-tk-Luc (51) and inserted, downstream of the TAT promoter, into the corresponding site of pTAT. The reporter plasmid pGRE-tk-Luc was constructed by ligation of the fragment of HindIII-BglII digestion of pGRE-tk-CAT (52), which contains a synthetic GRE (AGAACAcagTGTTCT) upstream of the tk minimal promoter, into the HindIII-BglII sites of pLuc (53). The reporter gene p(17M) 5 -βGlob-Luc has been previously described (54). The reporter plasmid p(17M) 5 -tata-Luc was constructed from the plasmid p(17M) 5 -tata-CAT (55) by digestion with XhoI-BamHI and isolation of the fragment containing (17M 5 )-tata. This fragment was then inserted into the vector pGL 2 (Promega) digested with the same restriction enzymes.
The β-galactosidase expression vector pCMV-βgal contains the sequence coding for β-galactosidase, under the control of the strong constitutive cytomegalovirus (CMV) promoter.
3.3. Test of Binding to a Hormone
Cos-7 cells transfected with pCMV-βgal and wild-type hGR or I747/T mutant were harvested 48 hours after the transfection, in a phosphate-buffered salt solution (STP), by scraping with the aid of a rubber spatula. The cells were washed twice in ice-cold STP. All the steps for the preparation of cytosols were carried out at 4° C. The cells were resuspended in 3 volumes of binding buffer (20 mM Na-HEPES, pH 7.3, 20 mM sodium molybdate and 5 mM EDTA (22)] and with protease inhibitors [leucopeptin (5 μg/ml), aprotinin (5 μg/ml), PMSF (40 μg/ml) and pepstatin A (5 μg/ml)], and they were placed for 15 minutes on ice. The cells were then lysed by 30-40 piston strokes in a Dounce glass homogenizer, and the resulting homogenate was then centrifuged at 110,000 g for 30 minutes. The supernatant, called cytosol, was used immediately for the test of binding to a hormone. The protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, Calif., USA). For the test of binding to a hormone, a cytosol, containing 1-3 mg of protein/ml, was incubated overnight at 4° C. with [ 3 H]-Dex (49 CI/mmol; Radiochemical Center, Amersham, England) or cortisol (67 Ci/mmol; Radiochemical Center, Amersham, England) without (total binding) or with (non-specific binding) a 100-fold excess of radio-inert compound (10 −5 M, Sigma Chemical Co., St. Louis, Mo., USA). The samples were then treated with 1 volume of dextran-charcoal (5% charcoal, 0.5% dextran in the binding solution) for 15 minutes on ice, with constant stirring, and then they were subjected to centrifugation for 10 minutes at 12,000 g. The [ 3 H] ligand attached was measured in an aliquot portion of the supernatant, by scintillation counting. The equilibrium dissociation constant (Kd) of the receptor for dexamethasone or cortisol was determined by Scatchard analysis.
For the competitive binding analysis, the cytosols were incubated with 10 nM [ 3 H]-Dex and various concentrations of nonradioactive competitive steroids, under the same conditions as above. After treatment with dextran-charcoal and scintillation counting, the specific binding was expressed as a percentage of the control not subjected to competition, and plotted as a function of the concentration of steroid in competition. Competitive binding tests in whole cells were carried out on 10 6 cells incubated for 1 hour at 37° C. with 10 nM [ 3 H]-Dex and various concentrations of steroids in competition. After sonication in 1 ml of KCl buffer (1.5 mM EDTA, 20 mM Tris.HCl, pH 7.4, 0.5 M KCl), the unbound steroid was removed by addition of two volumes of dextran-coated charcoal, in KCl buffer with gelatin (0.05% dextran, 0.5% charcoal, 0.1% gelatin). The mixture was incubated for 30 minutes at 4° C. and it was centrifuged at 3000 g for 5 minutes. The radioactivity was determined by liquid scintillation counting and the specific binding was plotted on a curve as above.
EXAMPLE 2
Chimeric Cre Recombinase Whose Activity is Inducible by Synthetic Glucocorticoids, but not by Natural Glucocorticoids
1. Materials and Methods
1.1. Construction of the Vector pCre-LBD[GR(I747/T)]
A ClaI-XbaI DNA fragment of about 1300 base pairs (bp), containing the sequence coding for the ligand binding domain of the human glucocorticoid receptor was isolated from the plasmid HG1 (see Example 1 (40)) and its ends were filled by T4 polymerase. This fragment was mutated so that the nucleotides ATT coding for an isoleucine at position 747 of the wild-type receptor (56) are replaced by the nucleotides ACC, coding for a threonine. This mutated fragment was cloned into the vector pCre-ER (28) digested with XhoI and PstI whose ends were also filled by T4 polymerase [plasmid pCr⊕GR(I747/T)+F; coding sequence of the LBD of hGR (I747/T) in the same orientation as that of Cre]. pCre-LBD[GR(I747/T)] was obtained by site-directed mutagenesis carried out on the single-stranded DNA prepared from the plasmid pCre+GR (I747/T)+F, with the synthetic oligonucleotides:
SG52 (5′-CTGGAAGATGGCGATCTCGAGATTCAGCAGGCCACT-3′) (SEQ ID NO: 6)
SG53 (5′-CTGTTTCATCAAAAGGGTACCAGCCGTGGAGGGGCAT-3′) (SEQ ID NO. 7)
making it possible to fuse, on the one hand, the DNA sequence coding for Cre [amino acids (aa) 1-343 (orf2(cre); (31)], and that which codes for the region containing the mutated LBD of HG1 [aa 500-777], on the other hand, the previously modified DNA sequence and that coding for the F region of the human estrogen receptor (ER) [aa 553-595 (57)] (FIG. 5 ). This operation restores the XhoI restriction site situated at the end of the Cre sequences in pCre-ER and introduces a KpnI restriction site between the sequences coding for GR and the F region of ER (FIGS. 6 and 7 ).
1.2. Mutation of the RXRα Gene
The vector (pRXRα (LNL) ), used to mutate the RXRαgene (58) by homologous recombination in F9 cells was constructed in the following manner:
A λ phage containing a genomic fragment of 11.8 kb, comprising exons 2, 3 and 4 of RXRα, was isolated from a genomic library prepared with the P19 murine embryo carcinoma genomic DNA, cloned into the EMBL3λ vector. A SalI fragment isolated from this phage (whose ends were filled with T4 polymerase) was subcloned into the BamHI site (after treatment with T4 polymerase) of a derivative of the vector pBluescriptIIsK+ (Stratagene). In this derivative, the XhoI site has been eliminated. An XhoI site was introduced into exon 4 of RXRα at the level of the AccI site corresponding to nucleotide 535 of the cDNA for RXRα (the cDNA sequence for RXR∝ is given in Genbank reference: M 84817), by cloning the following synthetic oligonucleotides into the AccI site:
5′-ATTATTATTACTCGAGTGATGTG-3′ (SEQ ID NO: 8) and,
5′-ATCATCATCATCCGAGTAATAATA-3′ (SEQ ID NO: 9).
Finally, the XhoI fragment, containing the cassette for expression of the neomycin resistance gene, surrounded by loxP sites, isolated from the vector pHR56 (28) was cloned into the XhoI site of the preceding vector.
The mutation of an allele of the RXRα gene in F9 wells was carried out by homologous recombination with the aid of the vector pRXRα (LNL) (28). This line was called RXRα +/−(LNL) .
1.3. Expression of Cre-LBD[GR(I747/T)] in Cells Comprising a Modified DNA Fragment: RXRα (LNL)
Transient transfection: 5×10 6 RXRα +/−(LNL) cells or HeLa cells resuspended in 500 μl of PBS were electroporated with 5 μg of plasmid pCre-LBD[GR(I747/T)] [optionally cotransfected with 5 μg of the vector pRXRα (LNL) for the HeLa cells] with the aid of an electroporator (Bio-Rad gene pulser), set at 200 V and 960 μF. The cells were then inoculated at 10 6 cells per 100 mm dish and treated, if necessary, with ligands.
Stable transfection: 5×10 6 RXRα +/−(LNL) cells, resuspended in 500 μl of PBS were electroporated with 5 μg of pCre-LBD[GR(I747/T)] and 1 μg of pPGK-Hyg (59), digested respectively with SalI and PvuII, with the aid of an electroporator (Bio-Rad gene pulser), set at 200 V and 960 μF. The resistant clones were obtained according to the procedure described by Metzger et al. (28).
1.4. “PCR” Amplification of a Genomic DNA Region Located in Exon 4 of RXRα
The DNA amplifications were carried out using the “Polymerase Chain Reaction” (PCR) technique according to the protocol described by Chen and Evans (60). The primers used are: SB211 and PZ105, having respectively as nucleotide sequence 5′-GGCAAACACTATGG-3′ (SEQ ID NO: 10) and 5′-TTGCGTACTGTCCTCTT-3′ (SEQ ID NO: 11). After denaturation for 8 min at 94° C., 2.5 units of Taq polymerase are added. The amplification is carried out by performing 35 cycles (30 sec. at 94° C., 30 sec. at 50° C.) followed by one cycle (30 sec. at 94° C., 30 sec. at 50° C., 5 min at 72° C.), the product of which is stored at 4° C.
The products of the reactions were separated on polyacrylamide (10%) or agarose (2.5%) gel. The DNA was visualized by UV followed by ethidium bromide staining, or by Southern blotting (42).
1.5. Test of Excision of the Sequences Situated Between the LoxP Sites
The DNA of RXRα +/−(LNL) cells, transfected with pCre-LBD[GR(I747/T)] or the parental vector pSG5, not treated or treated with cortisol (10 −6 M) or dexamethasone (10 −6 M) for 72 hours, was subjected to a PCR reaction according to the strategy described in FIG. 8 . The products of the reaction were separated on acrylamide gel and stained with ethidium bromide. Lane M in FIG. 9 corresponds to the loading of the pBR322 size marker digested with HinfI. The position of the wild-type and recombinant alleles is indicated.
1.6. PCR Analysis of the Recombinase Activity of Cre-LBD[GR(I747/T)] in the RXRα +/−(LNL) Line Constitutively Expressing the Chimeric Protein
For FIG. 10, the DNA prepared from RXRα +/−(LNL) :Cre-GR(I747/T) cells not treated or treated with cortisol (10 −6 M) or with dexamethasone (10 −6 M) served as a template for carrying out a PCR amplification, according to the procedure described in FIG. 8 . The amplified DNA was separated on acrylamide gel and stained with ethidium bromide. Lanes 1, 2, 3 and 4 contain respectively the PCR products produced with DNA extracted from wild-type (WT) F9 cells, RXRα +/−(LNL) , RXRα +/−(L) cells and C2RXRα +(L)/−(L) cells (73). Lane 8 contains a size marker (1 kb ladder; GibcoBRL).
In FIG. 11, the DNA prepared from RXRα +/−(LNL) :Cre-GR(I747/T) cells not treated or treated with aldosterone, cortisol, corticosterone, dexamethasone, triamcinolone acetonide or RU486, at concentrations ranging from 10 −9 to 10 −6 M, served as a template for carrying out a PCR amplification, according to the procedure described in FIG. 8 . The amplified DNA was separated on agarose gel and analyzed according to the Southern technique. The amplified fragments corresponding to the wild-type and recombinant alleles were detected by hybridization with the aid of the synthetic oligonucleotide (5′-AAGAAGCCCTTGCAGCCCTC-3′) (SEQ ID NO: 12), radiolabeled at the 5′ end with T4 polynucleotide kinase. The signal was visualited and quantified with the aid of a “phosphoimager” (Fujix bas2000).
For FIG. 12, RXR∝ +/−(LNL) :Cre-GR(I747/T) cells were cultured for 72 h in culture medium to which three natural ligands (aldosterone, cortisol and corticosterone) at the concentration of 10 −9 M or 10 −8 M and dexamethasone (10 −7 or 10 −6 M) have been added as indicated. The recombinant allele was detected according to the procedure described in FIG. 8 . The amplified DNA was separated on agarose gel and analyzed according to the Southern technique. The amplified fragments corresponding to the recombinant allele were detected by autoradiography following hybridization with the synthetic oligonucleotide 5′-AATTATAACTTCGTATAATGTATGCTATACGAAGTTATTC-3′ (SEQ ID NO: 13 ), (containing a loxP site) radiolabeled with Phosphorus 32 at the 5′ end by T4 polynucleotide kinase.
For FIG. 13, HeLa cells were cotransfected with the plasmids pRXRα (LNL) and pCre-LBD[GR(I747/T)] and then cultured for 72 hours with or without addition of the appropriate ligands, at the concentration of 10 −7 M. The DNA of these cells was then subjected to PCR amplification according to the strategy described. The products of the reaction were separated on acrylamide gel and stained with ethidium bromide. Lane 1 corresponds to the loading of the 1 kb ladder size marker (GibcoBRL).
2. Results
In order to express a Cre recombinase whose activity is inducible by synthetic ligands for the glucocorticoid receptor, but not by natural glucocorticoid ligands at “physiological” concentrations, an expression vector, pCre-LBD[GR(I747/T)], derived from the vector pCre-ER (28) by replacing the DNA sequence coding for amino acids 344-618 containing the LBD of the estrogen receptor (ER), by that coding for amino acids 500-777 of a mutated glucocorticoid receptor (GR) containing the mutated binding domain (LBD) of GR (I747/T), was constructed. The construction of this plasmid led to the insertion of the sequence GGTACC, coding for the aa Gly-Thr at position 624-625 of Cre-LBD[GR(I747/T)] (FIG. 7 ).
The recombinase activity of Cre-LBD[GR(I747/T)] was tested in a murine cell line RXRα +/−(LNL) . This line was obtained by integrating, by homologous recombination, the TK-neo gene, delimited by loxP sites (as a direct repeat) (LNL), into exon 4 of an allele of the RXRα gene (28). The TK-neo gene is a chimeric gene allowing the expression of a thymidine kinase/neomycin resistance gene fusion protein. Electroporation of this line with the vector pCre-LBD[GR(I747/T)] made it possible to evaluate its recombinase activity without addition of ligand, or after addition of cortisol (natural ligand) or of dexamethasone (FIG. 9) (Dex; synthetic ligand). The DNA of the cells thus treated is indeed subjected to a PCR with synthetic oligonucleotides located in exon 4, one upstream of the AccI site, the other downstream of this site. The size of the PCR fragments obtained with this pair of oligonucleotides is 65 bp for the wild-type allele, 3345 bp for the mutated allele (after integration of LNL) and 191 bp for the recombinant allele (after excision of the sequences situated between the two lox sites and of one of the lox sites, see FIG. 8 ). Following treatment of the cells transfected with dexamethasone (10 −6 M), a 191 bp fragment is observed, reflecting the presence of sequences corresponding to the recombinant allele, whereas in the absence of treatment or following a treatment with cortisol (10 −6 M), no fragment of this size is observed. These results therefore show that the recombinase activity of the fusion protein studied may be induced by treating cells expressing this protein with a synthetic GR ligand such as dexamethasone but not with a natural ligand such as cortisol, at the concentration of 10 −6 M.
An RXRα +/−(LNL) line constitutively expressing Cre-LBD[GR(I747/T)] was also established by cointegrating the cassette for expression of the Cre-LBD[GR(I747/T)] gene with a vector expressing the hygromycin resistance gene (RXRα +/−(LNL) :Cre-GR(I747/T); see “Materials and Methods”; data not presented). The recombinase activity was tested following a treatment of these cells with Dex (10 −6 M) for 72 hours. Analysis of the products of the PCRs carried out on the DNA of these cells reveals a fragment of 191 bp corresponding to the recombinant allele alone after treatment with Dex (FIG. 10 ).
Thus, it is shown that Cre-LBD[GR(I747/T)] is inactive without addition of ligand or in the presence of cortisol at 10 −6 M, but active in the presence of Dex, when its expression cassette is integrated into the genome.
The kinetics of recombinase action was also evaluated: its activity is detectable from 12 h of treatment with dexamethasone at 10 −6 M, but it is considerably stronger after 72 h of treatment (results not presented).
The recombinase activity of Cre-LBD[GR(I747/T)] was also tested following the addition of various concentrations of ligands. No recombinase activity could be observed in the RXRα +/−(LNL) :Cre-GR(I747/T) cells treated with aldosterone, with cortisol or with corticosterone (ligand concentration ranging from 10 −9 to 10 −6 M), whereas in the presence of dex, of triamcinolone acetonide or of RU486, a recombinase activity is observed from 10 −9 M (FIG. 11 ).
It was also shown, with the aid of this cell line, that the recombinase activity of Cre-LBD[GR(I747/T)] may be induced by synthetic ligands even when the cells are cultured in the presence of natural ligands (aldosterone, cortisol and corticosterone) at 10 −9 or 10 −8 M. The excision of sequences located between the loxP sites is obtained following a treatment of 72 h with dexamethasone at 10 −7 M or 10 −6 M (FIG. 12 ).
The recombinase activity of Cre-LBD[GR(I747/T)] was finally tested in human cells: HeLa cells were transiently cotransfected with the vectors pCre-LBD[GR(I747/T)] and pRXRα (LNL) , and then cultured either without addition of ligand, or in the presence of the ligands indicated in FIG. 13 . As in the F9 cells, a 191 bp band corresponding to the recombinant fragment is amplified from DNA isolated from the cells treated with synthetic ligands (Dexamethasone, Triamcinolone acetonide or RU486 at 10 −7 M), whereas without addition of ligand, or in the presence of natural ligands such as aldosterone, corticosterone or cortisol at the same concentrations, only the fragment corresponding to the wild-type allele can be detected.
These results therefore show that the recombinase activity of the fusion protein Cre-LBD[GR(I747/T)] is induced by synthetic ligands for the glucocorticoid receptor, whereas it is insensitive to the natural ligands such as cortisol, corticosterone or aldosterone, even at concentrations as high as 10 −6 M. Furthermore, the presence of natural ligands does not prevent the induction of the recombinase activity of Cre-LBD[GR(I747/T)] by the synthetic ligands.
EXAMPLE 3
Induction of the Activity of a Chimeric Cre Recombinase in Mice
1. Materials and Methods
1.1. Construction of Plasmids and Establishment of Transgenic Mice
The vector pCMVCre-LBD[GR(I747/T)] was constructed by cloning the 2 kb EcoRI fragment isolated from pCre-LBD[GR(I747/T)] into the EcoRI site of pMGSV1 (60). The 4.64 kb PvuII fragment isolated from pCMVCre-LBD[GR(I747/T)] was injected into F1 zygotes (C57BL/6×SJL) at a concentration of 4 ng/μl so as to produce transgenic mice according to the conventional procedure (61).
1.2. PCR Conditions
The PCR amplifications were carried out in a buffer solution containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM of each primer, 2 units of Taq polymerase and 1 μg of genomic DNA as template. After 30 cycles (30 sec at 94° C., 30 sec at 550° C., 1 min at 72° C.) and then 1 cycle (30 sec at 90° C., 30 sec at 550° C., 5 min at 72° C.), the amplification products were separated on a 2.2% agarose gel stained with ethidium bromide.
1.3. Analysis of the Genotype of the Mice.
The transgene Cre-LBD[GR(I747/T)] and the wild-type allele and the excised allele RXR∝ ΔAF2(LNL) were detected by PCR. The detection of the transgene Cre-LBD[GR(I747/T)] was carried out with the aid of the nucleotide primers 5′-ATCCGAAAAGAAAACGTTGA-3′ (SEQ ID NO: 14) and 5′-ATCCAGGTTACGGATATAGT-3′ (SEQ ID NO: 15), that of the alleles of the RXR∝ gene using the primers 5′-GGTTCTCCGGCCGCTTGGGT-3′ (SEQ ID NO: 16) and 5′-GAAGGCGATGCGCTGCGAAT-3′ (SEQ IN NO: 17). In the same matter, the excision of the tk-neo marker delimited by loxP sites was followed by PCR with the aid of the primers A (5′-CAAGGAGCCTCCTTTCTCTA-3′) (SEQ ID NO: 18) and B (5′-CCTGCTCTACCTGGTGACTT-3′) (SEQ ID NO 19. These primers amplify a DNA fragment of 156 base pairs from the wild-type allele and a fragment of 190 base pairs from the excised allele RXR∝ ΔAF2(L) .
2. Results
The recombinase activity of Cre-LBD[GR(I747/T)] was also tested in mice. Thus, transgenic mice expressing the fusion protein Cre-LBD[GR(I747/T)] under the control of the promoter of the human cytomegalovirus “major IE” gene were established. The structure of the transgene is represented in FIG. 14 a . It contains the activating/promoter sequences of the human cytomegalovirus “major IE” gene, an intron of the rabbit β-globin gene, coding sequences of Cre-LBD[GR(I747/T)] and the simian virus SV40 polyadenylation signal. The site of initiation of transcription is indicated by an arrow. These mice were then crossed with “reporter” mice so as to demonstrate therein the existence of an induced recombinase activity. This “reporter” line contains an insertion of the tk-neo selectable marker, delimited by loxP sites, in the intron situated between exons 8 and 9 of the RXRα gene, on one of its two alleles [RXRα ΔAF2(LNL) ]. After recombination of the tkneo marker, a loxP site remains in place and constitutes the excised allele RXRα ΔAF2 (L) . The wild-type RXRα allele and the excised allele may be detected simultaneously by PCR amplification using an appropriate primer pair A and B. FIG. 14 b schematically represents this PCR strategy by indicating the A and B primers as well as certain restriction sites.
The progeny derived from crossing Cre-LBD[GR(I747/T)] mice and “reporter” mice, described above, containing both the transgene Cre-LBD[GR(I747/T)] and the allele RXRα ΔAF2 (LNL) was identified by establishing their genotype from a tail biopsy. One of these mice was subjected, at the age of four weeks, to a daily incraperitoneal injection of 0.3 mg of dexamethasone diluted in 100 μl of vegetable oil, for five days. A tail biopsy was taken the day before the first injection, as well as the day after the fifth injection. The DNA isolated from these samples was analyzed by PCR with the aid of the oligonucleotides A and B (FIG. 15, lanes 2 and 3) to determine if the treatment induces the deletion of the tk-neo marker. Lane 1 corresponds to a control reaction, carried out without DNA. The position of the PCR products amplified using the wild-type RXRα allele and the excised allele RXRα ΔAF2 (L) are indicated. The size marker (lane 4) corresponds to the 1 kb ladder (GibcoBRL). The size of the fragments is given as base pairs. According to FIG. 15, the induced excision indeed took place after treatment of the mouse tested with dexamethasone. Indeed, the analysis of the DNA fragments amplified from the tail before treatment reveals only the presence of the wild-type RXRα allele whereas the analysis of the tail of the same mouse, after treatment, indicates the presence of the excised allele RXRα ΔAF2 (LNL) (FIG. 15 ). Thus, it appears that the chimeric recombinase Cre-LBD[GR(I747/T)] exhibits no basic constitutive activity in the presence of the endogenous ligands. It is, however, induced and active in mice by treatment with dexamethasone.
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19



2334 base pairs
nucleic acid
single
linear


cDNA


CDS
1..2331



-
1..2331
/note= “GR(I747/T) (Human
glucocorticoid receptor mutated at position 747).”


1
ATG GAC TCC AAA GAA TCA TTA ACT CCT GGT AGA GAA GAA AAC CCC AGC 48
Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser
1 5 10 15
AGT GTG CTT GCT CAG GAG AGG GGA GAT GTG ATG GAC TTC TAT AAA ACC 96
Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr
20 25 30
CTA AGA GGA GGA GCT ACT GTG AAG GTT TCT GCG TCT TCA CCC TCA CTG 144
Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu
35 40 45
GCT GTC GCT TCT CAA TCA GAC TCC AAG CAG CGA AGA CTT TTG GTT GAT 192
Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp
50 55 60
TTT CCA AAA GGC TCA GTA AGC AAT GCG CAG CAG CCA GAT CTG TCC AAA 240
Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys
65 70 75 80
GCA GTT TCA CTC TCA ATG GGA CTG TAT ATG GGA GAG ACA GAA ACA AAA 288
Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys
85 90 95
GTG ATG GGA AAT GAC CTG GGA TTC CCA CAG CAG GGC CAA ATC AGC CTT 336
Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu
100 105 110
TCC TCG GGG GAA ACA GAC TTA AAG CTT TTG GAA GAA AGC ATT GCA AAC 384
Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn
115 120 125
CTC AAT AGG TCG ACC AGT GTT CCA GAG AAC CCC AAG AGT TCA GCA TCC 432
Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser
130 135 140
ACT GCT GTG TCT GCT GCC CCC ACA GAG AAG GAG TTT CCA AAA ACT CAC 480
Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His
145 150 155 160
TCT GAT GTA TCT TCA GAA CAG CAA CAT TTG AAG GGC CAG ACT GGC ACC 528
Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr
165 170 175
AAC GGT GGC AAT GTG AAA TTG TAT ACC ACA GAC CAA AGC ACC TTT GAC 576
Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp
180 185 190
ATT TTG CAG GAT TTG GAG TTT TCT TCT GGG TCC CCA GGT AAA GAG ACG 624
Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr
195 200 205
AAT GAG AGT CCT TGG AGA TCA GAC CTG TTG ATA GAT GAA AAC TGT TTG 672
Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu
210 215 220
CTT TCT CCT CTG GCG GGA GAA GAC GAT TCA TTC CTT TTG GAA GGA AAC 720
Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn
225 230 235 240
TCG AAT GAG GAC TGC AAG CCT CTC ATT TTA CCG GAC ACT AAA CCC AAA 768
Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys
245 250 255
ATT AAG GAT AAT GGA GAT CTG GTT TTG TCA AGC CCC AGT AAT GTA ACA 816
Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr
260 265 270
CTG CCC CAA GTG AAA ACA GAA AAA GAA GAT TTC ATC GAA CTC TGC ACC 864
Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr
275 280 285
CCT GGG GTA ATT AAG CAA GAG AAA CTG GGC ACA GTT TAC TGT CAG GCA 912
Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala
290 295 300
AGC TTT CCT GGA GCA AAT ATA ATT GGT AAT AAA ATG TCT GCC ATT TCT 960
Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser
305 310 315 320
GTT CAT GGT GTG AGT ACC TCT GGA GGA CAG ATG TAC CAC TAT GAC ATG 1008
Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met
325 330 335
AAT ACA GCA TCC CTT TCT CAA CAG CAG GAT CAG AAG CCT ATT TTT AAT 1056
Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn
340 345 350
GTC ATT CCA CCA ATT CCC GTT GGT TCC GAA AAT TGG AAT AGG TGC CAA 1104
Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln
355 360 365
GGA TCT GGA GAT GAC AAC TTG ACT TCT CTG GGG ACT CTG AAC TTC CCT 1152
Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro
370 375 380
GGT CGA ACA GTT TTT TCT AAT GGC TAT TCA AGC CCC AGC ATG AGA CCA 1200
Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro
385 390 395 400
GAT GTA AGC TCT CCT CCA TCC AGC TCC TCA ACA GCA ACA ACA GGA CCA 1248
Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro
405 410 415
CCT CCC AAA CTC TGC CTG GTG TGC TCT GAT GAA GCT TCA GGA TGT CAT 1296
Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His
420 425 430
TAT GGA GTC TTA ACT TGT GGA AGC TGT AAA GTT TTC TTC AAA AGA GCA 1344
Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala
435 440 445
GTG GAA GGA CAG CAC AAT TAC CTA TGT GCT GGA AGG AAT GAT TGC ATC 1392
Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile
450 455 460
ATC GAT AAA ATT CGA AGA AAA AAC TGC CCA GCA TGC CGC TAT CGA AAA 1440
Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys
465 470 475 480
TGT CTT CAG GCT GGA ATG AAC CTG GAA GCT CGA AAA ACA AAG AAA AAA 1488
Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys
485 490 495
ATA AAA GGA ATT CAG CAG GCC ACT ACA GGA GTC TCA CAA GAA ACC TCT 1536
Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser
500 505 510
GAA AAT CCT GGT AAC AAA ACA ATA GTT CCT GCA ACG TTA CCA CAA CTC 1584
Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu
515 520 525
ACC CCT ACC CTG GTG TCA CTG TTG GAG GTT ATT GAA CCT GAA GTG TTA 1632
Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu
530 535 540
TAT GCA GGA TAT GAT AGC TCT GTT CCA GAC TCA ACT TGG AGG ATC ATG 1680
Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met
545 550 555 560
ACT ACG CTC AAC ATG TTA GGA GGG CGG CAA GTG ATT GCA GCA GTG AAA 1728
Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys
565 570 575
TGG GCA AAG GCA ATA CCA GGT TTC AGG AAC TTA CAC CTG GAT GAC CAA 1776
Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln
580 585 590
ATG ACC CTA CTG CAG TAC TCC TGG ATG TTT CTT ATG GCA TTT GCT CTG 1824
Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu
595 600 605
GGG TGG AGA TCA TAT AGA CAA TCA AGT GCA AAC CTG CTG TGT TTT GCT 1872
Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala
610 615 620
CCT GAT CTG ATT ATT AAT GAG CAG AGA ATG ACT CTA CCC TGC ATG TAC 1920
Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr
625 630 635 640
GAC CAA TGT AAA CAC ATG CTG TAT GTT TCC TCT GAG TTA CAC AGG CTT 1968
Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu
645 650 655
CAG GTA TCT TAT GAA GAG TAT CTC TGT ATG AAA ACC TTA CTG CTT CTC 2016
Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu
660 665 670
TCT TCA GTT CCT AAG GAC GGT CTG AAG AGC CAA GAG CTA TTT GAT GAA 2064
Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu
675 680 685
ATT AGA ATG ACC TAC ATC AAA GAG CTA GGA AAA GCC ATT GTC AAG AGG 2112
Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg
690 695 700
GAA GGA AAC TCC AGC CAG AAC TGG CAG CGG TTT TAT CAA CTG ACA AAA 2160
Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys
705 710 715 720
CTC TTG GAT TCT ATG CAT GAA GTG GTT GAA AAT CTC CTT AAC TAT TGC 2208
Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys
725 730 735
TTC CAA ACA TTT TTG GAT AAG ACC ATG TCC ACC GAG TTC CCC GAG ATG 2256
Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Thr Glu Phe Pro Glu Met
740 745 750
TTA GCT GAA ATC ATC ACC AAT CAG ATA CCA AAA TAT TCA AAT GGA AAT 2304
Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn
755 760 765
ATC AAA AAA CTT CTG TTT CAT CAA AAG TGA 2334
Ile Lys Lys Leu Leu Phe His Gln Lys
770 775




777 amino acids
amino acid
linear


protein

2
Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser
1 5 10 15
Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr
20 25 30
Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu
35 40 45
Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp
50 55 60
Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys
65 70 75 80
Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys
85 90 95
Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu
100 105 110
Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn
115 120 125
Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser
130 135 140
Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His
145 150 155 160
Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr
165 170 175
Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp
180 185 190
Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr
195 200 205
Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu
210 215 220
Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn
225 230 235 240
Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys
245 250 255
Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr
260 265 270
Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr
275 280 285
Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala
290 295 300
Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser
305 310 315 320
Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met
325 330 335
Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn
340 345 350
Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln
355 360 365
Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro
370 375 380
Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro
385 390 395 400
Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro
405 410 415
Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His
420 425 430
Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala
435 440 445
Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile
450 455 460
Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys
465 470 475 480
Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys
485 490 495
Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser
500 505 510
Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu
515 520 525
Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu
530 535 540
Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met
545 550 555 560
Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys
565 570 575
Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln
580 585 590
Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu
595 600 605
Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala
610 615 620
Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr
625 630 635 640
Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu
645 650 655
Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu
660 665 670
Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu
675 680 685
Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg
690 695 700
Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys
705 710 715 720
Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys
725 730 735
Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Thr Glu Phe Pro Glu Met
740 745 750
Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn
755 760 765
Ile Lys Lys Leu Leu Phe His Gln Lys
770 775




2004 base pairs
nucleic acid
single
linear


cDNA


CDS
1..2001



-
1..2001
/note= “CRE-LBD (GR(I747/T) (Fusion
protein for the LBD domain of the human glucocorticoid
receptor mutated at position 747 and the Cre recombinase
protein).”


3
ATG TCC AAT TTA CTG ACC GTA CAC CAA AAT TTG CCT GCA TTA CCG GTC 48
Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val
780 785 790
GAT GCA ACG AGT GAT GAG GTT CGC AAG AAC CTG ATG GAC ATG TTC AGG 96
Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg
795 800 805
GAT CGC CAG GCG TTT TCT GAG CAT ACC TGG AAA ATG CTT CTG TCC GTT 144
Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val
810 815 820 825
TGC CGG TCG TGG GCG GCA TGG TGC AAG TTG AAT AAC CGG AAA TGG TTT 192
Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe
830 835 840
CCC GCA GAA CCT GAA GAT GTT CGC GAT TAT CTT CTA TAT CTT CAG GCG 240
Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala
845 850 855
CGC GGT CTG GCA GTA AAA ACT ATC CAG CAA CAT TTG GGC CAG CTA AAC 288
Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn
860 865 870
ATG CTT CAT CGT CGG TCC GGG CTG CCA CGA CCA AGT GAC AGC AAT GCT 336
Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala
875 880 885
GTT TCA CTG GTT ATG CGG CGG ATC CGA AAA GAA AAC GTT GAT GCC GGT 384
Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly
890 895 900 905
GAA CGT GCA AAA CAG GCT CTA GCG TTC GAA CGC ACT GAT TTC GAC CAG 432
Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln
910 915 920
GTT CGT TCA CTC ATG GAA AAT AGC GAT CGC TGC CAG GAT ATA CGT AAT 480
Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn
925 930 935
CTG GCA TTT CTG GGG ATT GCT TAT AAC ACC CTG TTA CGT ATA GCC GAA 528
Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu
940 945 950
ATT GCC AGG ATC AGG GTT AAA GAT ATC TCA CGT ACT GAC GGT GGG AGA 576
Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg
955 960 965
ATG TTA ATC CAT ATT GGC AGA ACG AAA ACG CTG GTT AGC ACC GCA GGT 624
Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly
970 975 980 985
GTA GAG AAG GCA CTT AGC CTG GGG GTA ACT AAA CTG GTC GAG CGA TGG 672
Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp
990 995 1000
ATT TCC GTC TCT GGT GTA GCT GAT GAT CCG AAT AAC TAC CTG TTT TGC 720
Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys
1005 1010 1015
CGG GTC AGA AAA AAT GGT GTT GCC GCG CCA TCT GCC ACC AGC CAG CTA 768
Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu
1020 1025 1030
TCA ACT CGC GCC CTG GAA GGG ATT TTT GAA GCA ACT CAT CGA TTG ATT 816
Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile
1035 1040 1045
TAC GGC GCT AAG GAT GAC TCT GGT CAG AGA TAC CTG GCC TGG TCT GGA 864
Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly
1050 1055 1060 1065
CAC AGT GCC CGT GTC GGA GCC GCG CGA GAT ATG GCC CGC GCT GGA GTT 912
His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val
1070 1075 1080
TCA ATA CCG GAG ATC ATG CAA GCT GGT GGC TGG ACC AAT GTA AAT ATT 960
Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile
1085 1090 1095
GTC ATG AAC TAT ATC CGT AAC CTG GAT AGT GAA ACA GGG GCA ATG GTG 1008
Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val
1100 1105 1110
CGC CTG CTG GAA GAT GGC GAT CTC GAG ATT CAG CAG GCC ACT ACA GGA 1056
Arg Leu Leu Glu Asp Gly Asp Leu Glu Ile Gln Gln Ala Thr Thr Gly
1115 1120 1125
GTC TCA CAA GAA ACC TCT GAA AAT CCT GGT AAC AAA ACA ATA GTT CCT 1104
Val Ser Gln Glu Thr Ser Glu Asn Pro Gly Asn Lys Thr Ile Val Pro
1130 1135 1140 1145
GCA ACG TTA CCA CAA CTC ACC CCT ACC CTG GTG TCA CTG TTG GAG GTT 1152
Ala Thr Leu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val
1150 1155 1160
ATT GAA CCT GAA GTG TTA TAT GCA GGA TAT GAT AGC TCT GTT CCA GAC 1200
Ile Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp
1165 1170 1175
TCA ACT TGG AGG ATC ATG ACT ACG CTC AAC ATG TTA GGA GGG CGG CAA 1248
Ser Thr Trp Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gln
1180 1185 1190
GTG ATT GCA GCA GTG AAA TGG GCA AAG GCA ATA CCA GGT TTC AGG AAC 1296
Val Ile Ala Ala Val Lys Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn
1195 1200 1205
TTA CAC CTG GAT GAC CAA ATG ACC CTA CTG CAG TAC TCC TGG ATG TTT 1344
Leu His Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr Ser Trp Met Phe
1210 1215 1220 1225
CTT ATG GCA TTT GCT CTG GGG TGG AGA TCA TAT AGA CAA TCA AGT GCA 1392
Leu Met Ala Phe Ala Leu Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala
1230 1235 1240
AAC CTG CTG TGT TTT GCT CCT GAT CTG ATT ATT AAT GAG CAG AGA ATG 1440
Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile Ile Asn Glu Gln Arg Met
1245 1250 1255
ACT CTA CCC TGC ATG TAC GAC CAA TGT AAA CAC ATG CTG TAT GTT TCC 1488
Thr Leu Pro Cys Met Tyr Asp Gln Cys Lys His Met Leu Tyr Val Ser
1260 1265 1270
TCT GAG TTA CAC AGG CTT CAG GTA TCT TAT GAA GAG TAT CTC TGT ATG 1536
Ser Glu Leu His Arg Leu Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met
1275 1280 1285
AAA ACC TTA CTG CTT CTC TCT TCA GTT CCT AAG GAC GGT CTG AAG AGC 1584
Lys Thr Leu Leu Leu Leu Ser Ser Val Pro Lys Asp Gly Leu Lys Ser
1290 1295 1300 1305
CAA GAG CTA TTT GAT GAA ATT AGA ATG ACC TAC ATC AAA GAG CTA GGA 1632
Gln Glu Leu Phe Asp Glu Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly
1310 1315 1320
AAA GCC ATT GTC AAG AGG GAA GGA AAC TCC AGC CAG AAC TGG CAG CGG 1680
Lys Ala Ile Val Lys Arg Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg
1325 1330 1335
TTT TAT CAA CTG ACA AAA CTC TTG GAT TCT ATG CAT GAA GTG GTT GAA 1728
Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Met His Glu Val Val Glu
1340 1345 1350
AAT CTC CTT AAC TAT TGC TTC CAA ACA TTT TTG GAT AAG ACC ATG TCC 1776
Asn Leu Leu Asn Tyr Cys Phe Gln Thr Phe Leu Asp Lys Thr Met Ser
1355 1360 1365
ACC GAG TTC CCC GAG ATG TTA GCT GAA ATC ATC ACC AAT CAG ATA CCA 1824
Thr Glu Phe Pro Glu Met Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro
1370 1375 1380 1385
AAA TAT TCA AAT GGA AAT ATC AAA AAA CTT CTG TTT CAT CAA AAG GGT 1872
Lys Tyr Ser Asn Gly Asn Ile Lys Lys Leu Leu Phe His Gln Lys Gly
1390 1395 1400
ACC AGC CGT GGA GGG GCA TCC GTG GAG GAG ACG GAC CAA AGC CAC TTG 1920
Thr Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp Gln Ser His Leu
1405 1410 1415
GCC ACT GCG GGC TCT ACT TCA TCG CAT TCC TTG CAA AAG TAT TAC ATC 1968
Ala Thr Ala Gly Ser Thr Ser Ser His Ser Leu Gln Lys Tyr Tyr Ile
1420 1425 1430
ACG GGG GAG GCA GAG GGT TTC CCT GCC ACA GTC TGA 2004
Thr Gly Glu Ala Glu Gly Phe Pro Ala Thr Val
1435 1440




667 amino acids
amino acid
linear


protein

4
Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Va
1 5 10 15
Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg
20 25 30
Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val
35 40 45
Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe
50 55 60
Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala
65 70 75 80
Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn
85 90 95
Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala
100 105 110
Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly
115 120 125
Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln
130 135 140
Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn
145 150 155 160
Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu
165 170 175
Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg
180 185 190
Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly
195 200 205
Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp
210 215 220
Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys
225 230 235 240
Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu
245 250 255
Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile
260 265 270
Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly
275 280 285
His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val
290 295 300
Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile
305 310 315 320
Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val
325 330 335
Arg Leu Leu Glu Asp Gly Asp Leu Glu Ile Gln Gln Ala Thr Thr Gly
340 345 350
Val Ser Gln Glu Thr Ser Glu Asn Pro Gly Asn Lys Thr Ile Val Pro
355 360 365
Ala Thr Leu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val
370 375 380
Ile Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp
385 390 395 400
Ser Thr Trp Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gln
405 410 415
Val Ile Ala Ala Val Lys Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn
420 425 430
Leu His Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr Ser Trp Met Phe
435 440 445
Leu Met Ala Phe Ala Leu Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala
450 455 460
Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile Ile Asn Glu Gln Arg Met
465 470 475 480
Thr Leu Pro Cys Met Tyr Asp Gln Cys Lys His Met Leu Tyr Val Ser
485 490 495
Ser Glu Leu His Arg Leu Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met
500 505 510
Lys Thr Leu Leu Leu Leu Ser Ser Val Pro Lys Asp Gly Leu Lys Ser
515 520 525
Gln Glu Leu Phe Asp Glu Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly
530 535 540
Lys Ala Ile Val Lys Arg Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg
545 550 555 560
Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Met His Glu Val Val Glu
565 570 575
Asn Leu Leu Asn Tyr Cys Phe Gln Thr Phe Leu Asp Lys Thr Met Ser
580 585 590
Thr Glu Phe Pro Glu Met Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro
595 600 605
Lys Tyr Ser Asn Gly Asn Ile Lys Lys Leu Leu Phe His Gln Lys Gly
610 615 620
Thr Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp Gln Ser His Leu
625 630 635 640
Ala Thr Ala Gly Ser Thr Ser Ser His Ser Leu Gln Lys Tyr Tyr Ile
645 650 655
Thr Gly Glu Ala Glu Gly Phe Pro Ala Thr Val
660 665




6094 base pairs
nucleic acid
single
circular


cDNA


-
1..6091
/note= “pCRE-LBD[GR(I747/T]
(Plasmid for expressing the fusion protein Cre-LBD(GR(I747/
T)].”


5
GTCGACTTCT GAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG 60
TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAAC 120
AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAA 180
TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAG 240
TCCGCCCATT CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGC 300
GCCTCGGCCT CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTT 360
TGCAAAAAGC TGGATCGATC CTGAGAACTT CAGGGTGAGT TTGGGGACCC TTGATTGTT 420
TTTCTTTTTC GCTATTGTAA AATTCATGTT ATATGGAGGG GGCAAAGTTT TCAGGGTGT 480
GTTTAGAATG GGAAGATGTC CCTTGTATCA CCATGGACCC TCATGATAAT TTTGTTTCT 540
TCACTTTCTA CTCTGTTGAC AACCATTGTC TCCTCTTATT TTCTTTTCAT TTTCTGTAA 600
TTTTTCGTTA AACTTTAGCT TGCATTTGTA ACGAATTTTT AAATTCACTT TTGTTTATT 660
GTCAGATTGT AAGTACTTTC TCTAATCACT TTTTTTTCAA GGCAATCAGG GTATATTAT 720
TTGTACTTCA GCACAGTTTT AGAGAACAAT TGTTATAATT AAATGATAAG GTAGAATAT 780
TCTGCATATA AATTCTGGCT GGCGTGGAAA TATTCTTATT GGTAGAAACA ACTACATCC 840
GGTCATCATC CTGCCTTTCT CTTTATGGTT ACAATGATAT ACACTGTTTG AGATGAGGA 900
AAAATACTCT GAGTCCAAAC CGGGCCCCTC TGCTAACCAT GTTCATGCCT TCTTCTTTT 960
CCTACAGCTC CTGGGCAACG TGCTGGTTAT TGTGCTGTCT CATCATTTTG GCAAAGAA 1020
GTAATACGAC TCACTATAGG GCGAATTCCA CCATGTCCAA TTTACTGACC GTACACCA 1080
ATTTGCCTGC ATTACCGGTC GATGCAACGA GTGATGAGGT TCGCAAGAAC CTGATGGA 1140
TGTTCAGGGA TCGCCAGGCG TTTTCTGAGC ATACCTGGAA AATGCTTCTG TCCGTTTG 1200
GGTCGTGGGC GGCATGGTGC AAGTTGAATA ACCGGAAATG GTTTCCCGCA GAACCTGA 1260
ATGTTCGCGA TTATCTTCTA TATCTTCAGG CGCGCGGTCT GGCAGTAAAA ACTATCCA 1320
AACATTTGGG CCAGCTAAAC ATGCTTCATC GTCGGTCCGG GCTGCCACGA CCAAGTGA 1380
GCAATGCTGT TTCACTGGTT ATGCGGCGGA TCCGAAAAGA AAACGTTGAT GCCGGTGA 1440
GTGCAAAACA GGCTCTAGCG TTCGAACGCA CTGATTTCGA CCAGGTTCGT TCACTCAT 1500
AAAATAGCGA TCGCTGCCAG GATATACGTA ATCTGGCATT TCTGGGGATT GCTTATAA 1560
CCCTGTTACG TATAGCCGAA ATTGCCAGGA TCAGGGTTAA AGATATCTCA CGTACTGA 1620
GTGGGAGAAT GTTAATCCAT ATTGGCAGAA CGAAAACGCT GGTTAGCACC GCAGGTGT 1680
AGAAGGCACT TAGCCTGGGG GTAACTAAAC TGGTCGAGCG ATGGATTTCC GTCTCTGG 1740
TAGCTGATGA TCCGAATAAC TACCTGTTTT GCCGGGTCAG AAAAAATGGT GTTGCCGC 1800
CATCTGCCAC CAGCCAGCTA TCAACTCGCG CCCTGGAAGG GATTTTTGAA GCAACTCA 1860
GATTGATTTA CGGCGCTAAG GATGACTCTG GTCAGAGATA CCTGGCCTGG TCTGGACA 1920
GTGCCCGTGT CGGAGCCGCG CGAGATATGG CCCGCGCTGG AGTTTCAATA CCGGAGAT 1980
TGCAAGCTGG TGGCTGGACC AATGTAAATA TTGTCATGAA CTATATCCGT AACCTGGA 2040
GTGAAACAGG GGCAATGGTG CGCCTGCTGG AAGATGGCGA TCTCGAGATT CAGCAGGC 2100
CTACAGGAGT CTCACAAGAA ACCTCTGAAA ATCCTGGTAA CAAAACAATA GTTCCTGC 2160
CGTTACCACA ACTCACCCCT ACCCTGGTGT CACTGTTGGA GGTTATTGAA CCTGAAGT 2220
TATATGCAGG ATATGATAGC TCTGTTCCAG ACTCAACTTG CAGGATCATG ACTACGCT 2280
ACATGTTAGG AGGGCGGCAA GTGATTGCAG CAGTGAAATG GGCAAAGGCA ATACCAGG 2340
TCAGGAACTT ACACCTGGAT GACCAAATGA CCCTACTGCA GTACTCCTGG ATGTTTCT 2400
TGGCATTTGC TCTGGGGTGG AGATCATATA GACAATCAAG TGCAAACCTG CTGTGTTT 2460
CTCCTGATCT GATTATTAAT GAGCAGAGAA TGACTCTACC CTGCATGTAC GACCAATG 2520
AACACATGCT GTATGTTTCC TCTGAGTTAC ACAGGCTTCA GGTATCTTAT GAAGAGTA 2580
TCTGTATGAA AACCTTACTG CTTCTCTCTT CAGTTCCTAA GGACGGTCTG AAGAGCCA 2640
AGCTATTTGA TGAAATTAGA ATGACCTACA TCAAAGAGCT AGGAAAAGCC ATTGTCAA 2700
GGGAAGGAAA CTCCAGCCAG AACTGGCAGC GGTTTTATCA ACTGACAAAA CTCTTGGA 2760
CTATGCATGA AGTGGTTGAA AATCTCCTTA ACTATTGCTT CCAAACATTT TTGGATAA 2820
CCATGTCCAC CGAGTTCCCC GAGATGTTAG CTGAAATCAT CACCAATCAG ATACCAAA 2880
ATTCAAATGG AAATATCAAA AAACTTCTGT TTCATCAAAA GGGTACCAGC CGTGGAGG 2940
CATCCGTGGA GGAGACGGAC CAAAGCCACT TGGCCACTGC GGGCTCTACT TCATCGCA 3000
CCTTGCAAAA GTATTACATC ACGGGGGAGG CAGAGGGTTT CCCTGCCACA GTCTGAGA 3060
TCCCTGGAAT TCGGATCTTA TTAAAGCAGA ACTTGTTTAT TGCAGCTTAT AATGGTTA 3120
AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAG 3180
GTGGTTTGTC CAAACTCATC AATGTATCTT ATCATGTCTG GTCGACTCTA GACTCTTC 3240
CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGC 3300
ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACAT 3360
CAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTT 3420
ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCG 3480
ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTC 3540
CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGT 3600
CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAA 3660
TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTA 3720
GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAA 3780
GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAA 3840
ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTT 3900
GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTT 3960
TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGAT 4020
TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCAT 4080
GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATC 4140
TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGC 4200
CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTA 4260
TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGA 4320
CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCG 4380
GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGC 4440
GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCAT 4500
TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAG 4560
GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGAT 4620
TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAA 4680
CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAA 4740
CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGA 4800
ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGG 4860
GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGC 4920
CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGG 4980
GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACT 5040
TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACAT 5100
TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGT 5160
CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTAT 5220
CGAGGCCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGC 5280
CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTC 5340
GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGC 5400
ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAA 5460
TACCGCATCA GGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTG 5520
AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAA 5580
AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAA 5640
ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACG 5700
AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAA 5760
CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAA 5820
AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCT 5880
GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCG CGCCATTC 5940
CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG CTATTACG 6000
AGCTGGCGAA AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTC 6060
AGTCACGACG TTGTAAAACG ACGGCCAGTG AATT 6094




36 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..36
/note= “SG52”


6
CTGGAAGATG GCGATCTCGA GATTCAGCAG GCCACT 36




37 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..37
/note= “SG53”


7
CTGTTTCATC AAAAGGGTAC CAGCCGTGGA GGGGCAT 37




24 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..24
/note= “synthetic oligonucleotide”


8
ATTATTATTA CTCGAGTGAT GATG 24




24 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..24
/note= “synthetic oligonucleotide”


9
ATCATCATCA TCCGAGTAAT AATA 24




14 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..14
/note= “SB211”


10
GGCAAACACT ATGG 14




17 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..17
/note= “PZ105”


11
TTGCGTACTG TCCTCTT 17




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “synthetic oligonucleotide”


12
AAGAAGCCCT TGCAGCCCTC 20




40 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..40
/note= “synthetic oligonucleotide”


13
AATTATAACT TCGTATAATG TATGCTATAC GAAGTTATTC 40




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer”


14
ATCCGAAAAG AAAACGTTGA 20




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer”


15
ATCCAGGTTA CGGATATAGT 20




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer”


16
GGTTCTCCGG CCGCTTGGGT 20




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer”


17
GAAGGCGATG CGCTGCGAAT 20




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer A”


18
CAAGGAGCCT CCTTTCTCTA 20




20 base pairs
nucleic acid
single
linear


DNA (genomic)


-
1..20
/note= “primer B”


19
CCTGCTCTAC CTGGTGACTT 20