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A Staub

Publications and source records attributed to A Staub.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.

Amino Acid Sequence↗
PubMed1992

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.

Actin Cytoskeleton↗
PubMed1991

NF-X, a transcription factor implicated in MHC class II gene regulation.

The X box has been shown in several assay systems to be a critical element of MHC class II gene promoters. Several X box-binding activities have been discovered in nuclear extracts from a variety of cell lines. The critical question is: which of these are responsible for mediating X box function? This report provides a further characterization of NF-X, a highly specific X box-binding activity we described previously. The cell-type distribution, structural features, and binding site characteristics of NF-X are analyzed in detail, to facilitate comparison with other reported activities. Most importantly, the functional relevance of NF-X is assessed by scanning mutagenesis, and the results indicate that this complex is indeed involved in regulating MHC class II gene expression. With these data in mind, the relationship between NF-X and RF-X, an X box-binding activity reported to be absent in patients with severe combined immunodeficiency, is discussed.

Animals↗
PubMed1990

Murine isoforms of retinoic acid receptor gamma with specific patterns of expression.

We have characterized seven murine retinoic acid receptor gamma cDNA isoforms (mRAR-gamma 1 to -gamma 7) generated by alternative splicing of at least seven exons. These isoforms differ from one another in their 5' untranslated region and in two cases (mRAR-gamma 1 and -gamma 2) differ in their N-terminal A region, which is known to be important for differential transactivation by other nuclear receptors. mRAR-gamma 1 and -gamma 2, the predominant isoforms, are differentially expressed in adult tissues and during embryogenesis. Most notably, skin contains almost exclusively mRAR-gamma 1 transcripts. The conservation of the RAR-gamma isoforms from mouse to human together with their patterns of expression suggests that they perform specific functions, which may account for the pleiotropic effect of retinoic acid in embryogenesis and development.

Animals↗
PubMed1989

The SV40 TC-II(kappa B) and the related H-2Kb enhansons exhibit different cell type specific and inducible proto-enhancer activities, but the SV40 core sequence and the AP-2 binding site have no enhanson properties.

The enhancer activity of the oligomerized SV40 TC-I and TC-II sequences has been investigated in lymphoid and non-lymphoid cell lines. While the TC-I sequence had no demonstrable enhanson activity, a class C enhanson (proto-enhancer), 5'-GGAAAGTCCCC-3', overlapping the TC-II sequence and the GT-I enhanson was identified. This TC-II enhanson, which is identical to the kappa B motif from the kappa chain enhancer, was active in both lymphoid and non-lymphoid cells, which contrasts with the previously reported lymphoid cell specificity of the kappa B motif. However, its activity in non-lymphoid cells is in agreement with our previous reports describing the effect of mutations in the 'TC region' within the total SV40 enhancer in lymphoid and non-lymphoid cells. The activity of the TC-II enhanson could be moderately increased in HeLa by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and cycloheximide treatment, indicating that the protein(s) mediating its activity may be partially repressed by the previously described inhibitor protein I kappa B. The TC-II related, H-2Kb element, 5'-TGGGGATTCCCCA-3', of the histocompatibility class I H-2Kb gene promoter is also a class C enhanson which is active in both lymphoid and non-lymphoid cells. However, in contrast to the TC-II enhanson, the H-2Kb enhanson exhibits a very low activity in HeLa cells, but can be strongly induced by TPA and/or cycloheximide treatments which suggests that its cognate factor is inactivated (repressed) by an inhibitor protein. Interestingly, cycloheximide, but not TPA treatment, could induce the activity of both the TC-II and H-2Kb enhansons in F9 embryonal carcinoma cells, suggesting that these cells lack some component(s) of the protein kinase C signal transduction pathway. We also show that oligomers of the SV40 'core' sequence, which overlaps the TC-II enhanson, had no enhanson activity in any of the cell types studied, which questions the possible role of the AP-3 protein in SV40 enhancer activity in these cell types. In addition, oligomers of the AP-2 binding sites which are present in the SV40 TC region and in the human metallothionein IIA promoter show no enhanson activity, irrespective of whether the cells are treated with TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence.

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spaced repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-I and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.

Allosteric Regulation↗
PubMed1987

A multiplicity of CCAAT box-binding proteins.

NF-Y is a sequence-specific DNA-binding protein that recognizes the Y box, a promoter element common to all major histocompatibility complex class II genes. Since the 14-base Y element harbors a CCAAT box in reverse, we were prompted to ask whether NF-Y is actually a CCAAT box-binding protein and whether it is related to the previously described CCAAT-binding factors CBP and CTF/NF-I. Data from gel retardation, methylation interference, saturation mutagenesis, and cross-competition experiments establish definitively that NF-Y is an entirely distinct CCAAT box-binding entity. Moreover, these experiments have uncovered a fourth CCAAT-binding protein, NF-Y(star) that interacts with the thymidine kinase promoter. Clearly, then, there exists a multiplicity of factors that recognize CCAAT sequences; it now becomes imperative to understand the functional significance of this multiplicity.

Animals↗
PubMed1987

Sequences coding for part of oncogene-induced transin are highly conserved in a related rat gene.

The transin gene is induced by oncogenes and epidermal growth factor (EGF). We report here the isolation of a related gene (transin-2 gene). The structures of these genes are very similar. Indeed, a stretch of 428 nucleotides of the transin gene containing both exon and intron sequences is 98% conserved in the transin-2 gene. However, the putative promoter regions of the genes show little sequence homology, apart from a short element related to a sequence involved in control of transcription by cyclic AMP or a tumour promoter. Expression of the transin-2 gene, unlike that of the transin gene, is not induced by EGF, dibutyryl cyclic AMP or cytochalasin D. Nevertheless, transin-2 RNA is expressed in several transformed rat embryo fibroblast cell lines, and can be induced by a tumour promoter. The proteins transin and transin-2 are approximately 71% homologous in sequence. Both proteins show significant sequence homology with two connective tissue degrading metalloproteases. These homologies raise the possibility that expression of transin and transin-2 in transformed cells might play a role in tumour invasion.

Amino Acid Sequence↗
PubMed1987

In vitro binding of several cell-specific and ubiquitous nuclear proteins to the GT-I motif of the SV40 enhancer.

We have investigated the specific in vitro binding of nuclear proteins from several cell lines to the GT-I motif of the SV40 enhancer which overlaps with the canonical enhancer "core" homology. The binding of three proteins (GT-IA, GT-IB, and GT-IC), one of which (GT-IC) exhibits cell specificity, was detected. Competition and direct binding experiments demonstrated that the two ubiquitous proteins also bind to the GC-rich motif III from the 21-bp repeat upstream element of the SV40 early promoter and that protein GT-IA is most probably the transcription factor Sp1. The third, cell-specific protein GT-IC exhibited a high affinity for both the GT-I motif and an upstream element in the promoter of the mouse beta-major-globin gene, suggesting that this protein can act both as an enhancer and an upstream element trans-acting factor. The good correlation between the known cell-specific in vivo activity of the wild-type and mutated GT-I motif and the cell-specific binding of protein GT-IC in vitro strongly supports the conclusion that this protein is an enhancer factor. Interestingly, its cognate recognition sequence does not coincide with the core homology.

Animals↗
PubMed1986

Demethylation of bacterial chemoreceptors is inhibited by attractant stimuli in the complete absence of the regulatory domain of the demethylating enzyme.

The CheB methylesterase catalyzes the demethylation of membrane receptors during chemotaxis in Salmonella typhimurium. The kinetic properties of the full length product of the cheB gene are compared to those of the isolated C-terminal catalytic domain. The fragment has at least a 15-fold higher specific activity than the intact protein. In intact cells receptor demethylation is inhibited by attractants such as L-aspartate. We show here that both forms of the enzyme are similarly inhibited in vitro. Thus, the C-terminal catalytic domain of the CheB protein is sufficient for this aspect of esterase regulation. Inhibition by attractants appears to be caused by changes in receptor conformation rather than by changes in the activity of the demethylating enzyme.

Aspartic Acid↗
PubMed1986

Functional sites on Ia molecules: a molecular dissection of A alpha immunogenicity.

Ia antigens are polymorphic cell-surface molecules that control the immune response. We have begun to localize important functional sites on one of the Ia molecules, A alpha. Herein, we focus on the A alpha k and A alpha b alleles and ask what defines "b-ness" and "k-ness" for a panel of monoclonal antibodies. Two independent experimental strategies are employed: the ability of 12 monoclonal antibodies to recognize L cell transfectants bearing chimeric and mutant A alpha chains is assessed, and the amino acid sequences of A alpha chains expressed by immunoselected B lymphoma mutants are deduced. For each antibody, we identify a stretch of polymorphic residues critical for recognition; for several, we can pinpoint a single amino acid. Certain stretches of A alpha (depending on the allele) appear strikingly immunodominant.

Amino Acid Sequence↗
PubMed1986

Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor.

Site-directed mutagenesis was used to prepare a series of human oestrogen receptor (hER) deletion mutants. The ability of these mutant receptors to bind oestradiol, either after being transiently expressed in HeLa cells or produced synthetically in vitro using T7 polymerase coupled with a rabbit reticulocyte lysate translation system, was analysed. The results indicate that a region which is highly conserved (94% amino acid identity) between the human and chicken ERs (region E) contains all of the sequence necessary to bind oestradiol with high affinity. When tight nuclear association of the oestradiol-receptor complex was investigated using the oestradiol-binding mutants of the same series, two regions of the hER sequence were found to be important. One of these regions is completely conserved (100% amino acid identity) between the human and chicken ERs (region C). This region is rich in cysteine and basic amino acids and contains motifs similar to those which have been proposed to be important for DNA binding in other eukaryotic transcriptional regulatory proteins. The other region (region D), which is comparatively poorly conserved (38% amino acid identity), is located between the putative DNA-binding domain (region C) and the oestradiol-binding domain (region E).

Animals↗
PubMed1985

Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis.

We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis. Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length. The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector. The oligonucleotides are hybridized and ligated to the M13 vector without any purification of the synthetic DNA segment. After cloning, about half of the progeny from such shot-gun ligations contained the predicted sequence demonstrating the efficacy of this method for gene synthesis and its potential for the extensive mutational analysis of genes.

Cloning, Molecular↗
PubMed1985

Cloning of the human estrogen receptor cDNA.

Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.

Animals↗