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Biomedical subjects

R Pictet

Publications and source records attributed to R Pictet.

At least 37 records · Page 2Linked to original sources
PubMed1991

In vivo footprinting of rat TAT gene: dynamic interplay between the glucocorticoid receptor and a liver-specific factor.

HNF5, a liver-specific DNA-binding protein, interacts with DNA in a manner that allows DNAase I cleavage in the middle of its recognition sequence. Using this property we have identified in vivo HNF5 bound to its sites within two glucocorticoid-responsive units of the rat tyrosine aminotransferase (TAT) gene. One HNF5-binding site is also a glucocorticoid receptor-binding site; glucocorticoid-dependent HNF5 binding could be detected at this site even though it is incompatible with glucocorticoid receptor binding. HNF5 binds within 10 min of hormone addition, indicating that it participates in transcriptional activation. In the TAT gene glucocorticoid-dependent HNF5 binding occurs where there is glucocorticoid-dependent disruption of nucleosomal structure; constitutive binding occurs in constitutively disrupted regions. These results suggest a hit-and-run mechanism of transcriptional activation by glucocorticoid receptor: the activated receptor binds its target sequence, modifies local chromatin structure, then leaves its site accessible to another factor.

Animals↗
PubMed1991

Trimethoprim binds in a bacterial mode to the wild-type and E30D mutant of mouse dihydrofolate reductase.

Previous crystallographic studies of the antibacterial trimethoprim in complexes with bacterial and avian dihydrofolate reductases have shown substantial differences in the mode of binding, providing plausible explanations for the origin of the remarkable species selectivity of this inhibitor (Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., Kaufman, B. T., Beddell, C. R., Champness, J. N., Stammers, D. K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391; Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Volz, K. W., and Kraut, J. (1985) J. Biol. Chem. 260, 392-399). A major species difference between the active sites is that the only carboxylate present is always Glu in vertebrates and Asp in bacteria. Crystallographic studies of the wild-type and E30D mutant of the enzyme from mouse now reveal that in both cases trimethoprim is bound in an identical fashion to that observed with the bacterial enzyme, and there is no obvious single explanation for the origin of the 10(5)-fold selectivity of trimethoprim binding. In an earlier study of a mouse wild-type enzyme using more limited data it was proposed that trimethoprim bound in the avian mode (Stammers, D. K., Champness, J. N., Beddell, C. R., Dann, J. G., Eliopoulos, E. E., Geddes, A. J., Ogg, D., and North, A. C. T. (1987) FEBS Lett. 218, 178-184), but a re-examination indicates that the occupancy of the active site by trimethoprim is less than had been thought, and we are currently unable to make an unambiguous interpretation of the electron density maps and cannot confirm the avian mode of binding in those crystals.

Animals↗
PubMed1991

Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor.

The structures of two remote glucocorticoid responsive units (GRUs) that cooperatively interact to promote cell-type specific glucocorticoid induction of rat tyrosine aminotransferase gene expression have been analyzed. DNAase I footprinting and gel mobility shift analyses reveal a complex array of contiguous and overlapping sites for cell type-specific DNA binding proteins. Apart from the glucocorticoid receptor, two liver-specific nuclear factors possess multiple binding sites in each of these GRUs: C/EBP and a newly identified liver-specific factor: HNF5. C/EBP possesses four binding sites in each GRU; a DNA-binding protein with similar binding specificity has been identified in fibroblasts; this protein could be related to AP-3. HNF5 possesses two binding sites in one GRU and four in the other. There are also HNF5 binding sites in numerous regulatory regions of other liver-specific genes. The interaction of HNF5 with DNA gives a characteristic DNAase I footprint with hypersensitive sites in the middle of the recognition sequence. Some of the C/EBP and HNF5 binding sites overlap in a conserved arrangement.

Animals↗
PubMed1990

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase.

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.

Animals↗
PubMed1990

Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice.

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.

Animals↗
PubMed1989

Two remote glucocorticoid responsive units interact cooperatively to promote glucocorticoid induction of rat tyrosine aminotransferase gene expression.

Tyrosine aminotransferase (TAT) gene transcription is specifically activated by glucocorticoid hormones in liver cells. This regulation involves a glucocorticoid responsive region located 2,500 bases upstream from the transcription start site of the rate gene. By transient transfection of TAT-CAT fusion genes into a rat hepatoma cell line expressing the TAT gene we found that this region promotes only 30% of the glucocorticoid stimulation. We have identified a new cis-acting region far upstream (-5,400) from the transcription start site that is essential to achieve the physiological level of glucocorticoid stimulation of endogenous TAT gene expression. This region corresponds to a tissue-specific DNAse I hypersensitive site which is constitutive despite the fact it possesses a glucocorticoid receptor binding site. It is by itself almost inactive on a promoter but it cooperatively enhances the action of the proximal glucocorticoid responsive region. Its activity requires both the glucocorticoid receptor binding site and its flanking sequences.

Animals↗
PubMed1989

Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and Tn10.

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.

Base Sequence↗
PubMed1989

In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis.

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.

Animals↗
PubMed1989

B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice.

Transgenic mouse lines carrying the human insulin gene were previously shown to express it in pancreas but not in other tissues. The present study reports evidence that the expression of the transgene is restricted to a single category of cells. Immunofluorescence staining of frozen pancreas sections showed that the human C-peptide was present in pancreatic islets only, and more precisely in the B cells of the islets. Human insulin transcripts were initiated correctly in mouse pancreas at the same site as in human pancreas. Three different transgenic lines with different insertion sites and various copy numbers of the human insulin transgene had the same high levels of the transgene transcripts corresponding to a well-balanced contribution in insulin gene expression.

Animals↗
PubMed1989

Positive and negative regulation of a transfected chimeric tyrosine aminotransferase gene: effect of copy number.

To define a selective system for the study of rat tyrosine aminotransferase (TAT; EC 2.6.1.5) gene expression, we have introduced into cultured cells the selectable bacterial gene gpt linked to TAT gene flanking sequences. After integration in host cell DNA, the chimeric gene exhibits the same pattern of regulation as the TAT gene. In hepatoma cells, its expression is induced after glucocorticoid hormone treatment and repressed after fusion with fibroblasts. In fibroblasts, the chimeric gene is not expressed. The correct pattern of regulation is lost when the number of integrated copies is high. At copy number above 10, the transfected gene responds poorly to glucocorticoids in hepatoma cells. At copy number above 50, the gene is expressed in fibroblasts. Another gene present in the same construction and controlled by the SV40 early promoter and enhancer is positively regulated by glucocorticoids in hepatoma cells but not after fusion with fibroblasts. These data indicate that in hybrid cells, both TAT promoter and glucocorticoid-responsive elements are negatively regulated.

Animals↗
PubMed1988

Site-directed mutagenesis of mouse dihydrofolate reductase. Mutants with increased resistance to methotrexate and trimethoprim.

Site-directed mutagenesis was used to generate mutants of recombinant mouse dihydrofolate reductase to test the role of some amino acids in the binding of two inhibitors, methotrexate and trimethoprim. Eleven mutations changing eight amino acids at positions all involved in hydrogen bonding or hydrophobic interactions with dihydrofolate or one of the two inhibitors were tested. Nine mutants were obtained by site-directed mutagenesis and two were spontaneous mutants previously obtained by in vivo selection (Grange, T., Kunst, F., Thillet, J., Ribadeau-Dumas, B., Mousseron, S., Hung, A., Jami, J., and Pictet, R. (1984) Nucleic Acids Res. 12, 3585-3601). The choice of the mutated positions was based on the knowledge of the active site of chicken dihydrofolate reductase established by x-ray crystallographic studies since the sequences of all known eucaryotic dihydrofolate reductases are greatly conserved. Enzymes were produced in great amounts and purified using a plasmid expressing the mouse cDNA into a dihydrofolate reductase-deficient Escherichia coli strain. The functional properties of recombinant mouse dihydrofolate reductase purified from bacterial extracts were identical to those of dihydrofolate reductase isolated from eucaryotic cells. The Km(NADPH) values for all the mutants except one (Leu-22----Arg) were only slightly modified, suggesting that the mutations had only minor effects on the ternary conformation of the enzyme. In contrast, all Km(H2folate) values were increased, since the mutations were located in the dihydrofolate binding site. The catalytic activity was also modified for five mutants with, respectively, a 6-, 10-, 36-, and 60-fold decrease of Vmax for Phe-31----Arg, Ile-7----Ser, Trp 24----Arg and Leu-22----Arg mutants and a 2-fold increase for Val-115----Pro. All the mutations affected the binding of methotrexate and six, the binding of trimethoprim: Ile-7----Ser, Leu-22----Arg, Trp-24----Arg, Phe-31----Arg, Gln-35----Pro and Phe-34----Leu. The relative variation of Ki for methotrexate and trimethoprim were not comparable from one mutant to the next, reflecting the different binding modes of the two inhibitors. The mutations which yielded the greatest increases in Ki are those which involved amino acids making hydrophobic contacts with the inhibitor.

Amino Acid Sequence↗
PubMed1988

Chromosome localization of the human insulin gene in transgenic mouse lines.

Three transgenic mouse lines, Tg 74, Tg 174, and Tg 171, were obtained by microinjection of an 11-kb human DNA fragment carrying the insulin gene into pronuclei of fertilized mouse eggs. The human insulin gene was expressed in all three transgenic mouse lines as shown by the presence of human C peptide in serum and urine and of human insulin transcripts in RNA prepared from pancreas. Several copies of the human DNA fragment arranged in head-to-tail arrays were present in each line. The human DNA insert was transmitted to the progeny as a single genetic locus. The chromosomal integration of the human insulin transgene was directly demonstrated by in situ hybridization to metaphase chromosomes of mitotic cells prepared from spleen and bone marrow. The insert appeared unique and located on a different chromosome in each line, namely 7 for Tg 74, 13 for Tg 174, and 18 for TG 171. Separation of DNA fragments larger than 20 kb by pulse-field electrophoresis showed that several insertion sites were present in each chromosome locus. This is the first direct evidence in transgenic mice that a gene located at various chromosome loci can be correctly expressed.

Animals↗
PubMed1988

Human insulin gene in transgenic mouse lines.

Transgenic mouse lines carrying the human insulin gene flanked by 4 kilobases (kb) in 5' and 5.5 kb in 3' were obtained. The presence of the human C-peptide in serum and urine, and of specific transcripts of the human gene in pancreas RNA indicate that the human DNA fragment contains the sequences necessary for correct phenotypic expression of the gene.

Animals↗
PubMed1987

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.

Amino Acid Sequence↗