Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-08-20
2004-03-02
Shukla, Ram R. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023100, C536S023400, C536S023510
Reexamination Certificate
active
06699686
ABSTRACT:
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 &lgr; 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).
Brocard Jacques Bertrand
Chambon Pierre Henri
Gronemeyer Hinrich
Metzger Daniel
Nicolas Jean-Claude
Association pour le Development de la Recherche en Genetique Mol
Burns Doane Swecker & Mathis L.L.P.
Shukla Ram R.
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