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Elisabeth Sheer

Bio: Elisabeth Sheer is an academic researcher from French Institute of Health and Medical Research. The author has contributed to research in topics: Protein engineering & In vivo. The author has an hindex of 1, co-authored 1 publications receiving 624 citations.

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TL;DR: The pSG5 vector as mentioned in this paper was constructed by combining the eukaryotic expression vector, pKCR2, and the high copy plasmid vector Bluescribe Ml3+ (Stratagene).
Abstract: The ability to engineer specific modifications within the coding region of a structural gene is a powerful tool with which to study the relationship between protein structure and function. The process of site-directed mutagenesis involves a) mutation of the target gene, b) verification of the mutation by sequencing and c) expression of the mutated gene. We describe here a vector, pSG5 (Fig.lA), in which each of these steps may be performed. pSG5 was constructed essentially by combining the eukaryotic expression vector, pKCR2 (1), and the high copy plasmid vector Bluescribe Ml3+ (Stratagene). The principle features of pSG5 are a) unique EcoRI and BamHI restriction enzyme sites for insertion of cDNAs, b) production of single stranded DNA containing the antisense strand of the structural gene for mutagenesis and sequencing, c) high yields of double stranded DNA, d) in vivo expression from the SV40 early promoter and e) in vitro expression from the T7 promoter situated just upstream of the cDNA insertion site. Expression from pSG5 was tested after insertion of a cDNA encoding the human oestrogen receptor (2) (ER) into the EcoRI site to produce pSG5-ER. Fig.IB shows an SDS polyacrylamide gel of the 66Kd ER protein synthesised in a rabbit reticulocyte cell-free translation cocktail programmed with mRNA transcribed from the T7 promoter of either Bluescribe-ER (BSM-ER (3)) or pSG5-ER. The ER when expressed in HeLa cells stimulates transcription from oestrogen-responsive 'reporter' genes (4) (vitellogenin-TK-globin (VTG) in Fig. 1C). Using quantitative SI nuclease analysis with an internal reference gene (RXF in Fig.lC), the ER expressed from either pSG5-ER or pKCR2-ER stimulates gene transcription equally well indicating that equivalent levels of ER are expressed in vivo using either of these two vectors.

628 citations


Cited by
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Journal ArticleDOI
18 Oct 1990-Nature
TL;DR: A member of the steroid hormone receptor superfamily of ligand-activated transcription factors is cloned that is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes.
Abstract: We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.

3,370 citations

Journal ArticleDOI
17 Aug 1995-Nature
TL;DR: A role for PKB in PI(3)K-mediated signal transduction is suggested and a constructed Gag–PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity.
Abstract: A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.

2,137 citations

Journal ArticleDOI
16 Aug 1996-Science
TL;DR: A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs.
Abstract: Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

2,064 citations

Journal ArticleDOI
05 Apr 1991-Cell
TL;DR: Using a monoclonal antibody raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule.

1,703 citations

Journal ArticleDOI
23 Jan 1992-Nature
TL;DR: The identification of a stereoisomer of retinoic acid, 9-cis retinol, which directly binds and activates RXRα, which suggests a new role for isomerization in the physiology of natural retinoids.
Abstract: Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

1,230 citations