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R Pictet

Publications and source records attributed to R Pictet.

At least 55 records · Page 3Linked to original sources
PubMed1986

Hormonal and developmental specificities of transcription lie within a 1.5 kb region 5' to the ecdysone inducible Drosophila P1 gene.

P-element-mediated transformation was used to investigate 5' cis-acting regulatory sequences flanking the P1 gene of Drosophila melanogaster, which is selectively expressed in the fat body of late third instar larvae under the positive control of ecdysone. A hybrid gene was constructed by fusing a 1.5 kb DNA fragment directly adjacent to and including the first 25 transcribed bases of the P1 gene to the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene itself linked at its 3' end to the SV40 t antigen splicing and polyadenylation sequences. Five transformed lines of D. melanogaster containing only one copy of the hybrid gene were established. In each of these lines the gpt sequence is transcribed with the same spatial, temporal and hormonal specificities as those of the P1 gene. This provides evidence that control elements essential for the ecdysone and developmentally regulated expression of P1 are located within a 1.5 kb region 5' to this gene.

Journal Article↗
PubMed1986

Pancreatic expression of human insulin gene in transgenic mice.

We have investigated the possibility of obtaining integration and expression of a native human gene in transgenic mice. An 11-kilobase (kb) human chromosomal DNA fragment including the insulin gene (1430 base pairs) was microinjected into fertilized mouse eggs. This fragment was present in the genomic DNA of several developing animals. One transgenic mouse and its progeny were analyzed for expression of the foreign gene. Synthesis and release of human insulin was revealed by detection of the human C-peptide in the plasma and urine. Human insulin mRNA was found in pancreas but not in other tissues. These findings indicate that the 11-kb human DNA fragment carries the sequences necessary for tissue-specific expression of the insulin gene and the human regulatory sequences react to homologous signals in the mouse.

Animals↗
PubMed1985

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.

Amino Acid Sequence↗
PubMed1985

Differential expression of two linked selection genes (HSVI-tk and Eco.gpt) in transformed teratocarcinoma and in L cells.

Upon transfection of (TK-)F9 teratocarcinoma stem-cells and (TK-)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI-tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV-gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk-regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low.

Animals↗
PubMed1984

Expression of the mouse dihydrofolate reductase cDNA in B. subtilis: a system to select mutant cDNAs coding for methotrexate resistant enzymes.

With the aim to obtain a cDNA coding for a mammalian methotrexate resistant dihydrofolate reductase (Dhfr) a plasmid ( pQS1 ) harboring the mouse wild type Dhfr cDNA was constructed and used to transform a methotrexate sensitive bacteria: B. subtilis. A plasmid, pQS4 , expressing large amount of Dhfr in both E. coli and B. subtilis was isolated through a two steps selection with two substrate analogues, trimethoprim followed by methotrexate. This new plasmid has a 54 bp duplication including the beta-lactamase promoter and a deletion of 564 bp removing the 5' end of the beta-lactamase coding region. These changes create a new -35 region TTGAAA and a potentially stronger binding site for both E. coli and B. subtilis 16S ribosomal RNA. pQS4 transformed B. subtilis were then grown in the presence of high level of methotrexate and resistant mutants isolated. One of them, pQS6 , which codes for an enzyme about 50 times more resistant to methotrexate than the wild type Dhfr was sequenced. It shows that a point mutation replaces the glutamine residue at position 35 by a proline.

Animals↗
PubMed1984

Isolation and characterization of the human prolactin gene.

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900-bp sequenced in the 5'-flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Animals↗
PubMed1984

Transformation of teratocarcinoma stem cells and fibroblasts with various vectors containing the Eco.gpt gene as a selection marker.

Eco.gpt, which codes for xanthine guanine phosphoribosyltransferase (XGPRT), when placed under the control of SV40 early genes regulating sequences (pSV2gpt) selects transformed teratocarcinoma cells with a low efficiency. The SV40 promoter may not function efficiently in teratocarcinoma stem cells, as suggested by the fact that such cells do not support SV40 T antigen expression. We have tested whether one could change the efficiency of gpt as a dominant selective marker in transformation by several operations. (1) Deletion of 121 base pairs (bp) upstream the bacterial coding sequence gpt (pQS14) did not make any difference. (2) Replacement of the SV40 regulating sequences by the HSV tk regulating sequences (pQS15) resulted in ten times fewer transformants with PCC4 teratocarcinoma cells as well as with L cells. No XGPRT activity was detectable in cultures 48 h after transfection. (3) Reintroduction of the PvuII-HindIII SV40 fragment (which contains an enhancing sequence together with the origin of replication and the early promoter of the virus) into the pQS15 vector, either in 5' or 3' from tk-gpt composite gene (pQS20 and pQS22) allows selection of ten to twenty times more transfected PCC4 or L cells colonies and restores transient XGPRT activity upon transfection. Whatever the vector used, the transformation frequency of PCC4 teratocarcinoma cells remains ten times lower than that of L cells. It appears that the presence of the SV40 PvuII-HindIII fragment in the vector increases cell transformation even with PCC4 cells and that the low frequency obtained with pSV2pgt is likely not due to the use of the SV40 early promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Persistence of transcription of the human beta-globin gene in hybrids between transfected F9 embryonal carcinoma and L cells.

Extinction of the phenotypic properties of the embryonal carcinoma (EC) cell parent is always observed in hybrids between EC cells and L fibroblasts. On the other hand, human beta-globin genes can be expressed when recombined to EC cell genome. We wanted to examine whether this ectopic expression would be extinguished when phenotypic changes are induced by cell hybridization. F9 transformants carrying the human beta-globin gene were hybridized with L fibroblasts. Human beta-globin transcripts were found in the two hybrid cell lines examined. Thus, the human beta-globin genes escaped extinction and behaved as genes involved in housekeeping functions.

Animals↗
PubMed1983

Stable transformation of mouse teratocarcinoma stem cells with the dominant selective marker Eco.gpt and retention of their developmental potentialities.

Transformation of PCC4 mouse teratocarcinoma stem cells was obtained using a dominant selective marker, the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT), coded by the bacterial Eco.gpt gene placed under the control of the early SV40 genes in the vector pSV2gpt. An average of 20 colonies of transformed cells was obtained, using the calcium phosphate technique, 10 microg DNA vector, no carrier DNA and 1 x 10(6) recipient cells. Five independent Eco.gpt-transformed PCC4 cell lines were propagated in selective medium and assayed for XGPRT activity. All of them had the ability to convert [14C]xanthine to xanthine monophosphate. pSV2gpt sequences were present and associated with high mol. wt. cellular DNA. pSV2gpt sequences and XGPRT activity were both conserved in the three clones that were propagated in non-selective medium for 30 generations. The transformed PCC4 cells retained their ability to produce, in host mice, teratocarcinoma tumors composed of embryonal carcinoma and various differentiated tissues. Thus, pSV2gpt can be used as a dominant marker to select teratocarcinoma stem cells co-transformed with genes that are not selectable by themselves.

Animals↗
PubMed1980

Cloning and sequence analysis of cDNAs encoding two distinct somatostatin precursors found in the endocrine pancreas of anglerfish.

Complementary DNAs for two distinct anglerfish somatostatin peptides (termed I and II) have been cloned in bacterial plasmids and sequenced. The nucleotide sequence for somatostatin I encodes a large percursor peptide (molecular weight 13,300) in which the somatostatin hormones is at the carboxyl terminus. The predicted 14-amino acid sequence for anglerfish somatostatin I is the same as mammalian somatostatin. Somatostatin II is also synthesized as part of a larger precursor (molecular weight 14,100) with the presumptive somatostatin hormone also at the carboxyl terminus. The 14-amino acid sequence of somatostatin II differs from somatostatin I at two internal residues (Tyr in place of Phe 7 and Gly in place of Thr 10). The two different somatostatins may have distinct biological activities. Homologies in the amino acid sequences of the two peptides outside the somatostatin moiety suggest other regions of the molecules have biological functions.

Amino Acid Sequence↗
PubMed1980

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.

Animals↗
PubMed1979

Isolation and characterization of a cloned rat insulin gene.

The two nonallelic genes, insulin I and II of the rat, are separated by at least 7 kb of DNA. There is no obvious similarity in the sequence organization surrounding each gene, although the coding regions of the genes themselves share extensive homology. In three strains of rat, the insulin II gene lies predominantly on a 4.0 kb Eco RI restriction fragment, whereas the insulin I gene is located on a 9.4 kb Eco RI fragment in the Hooded strain, on a 7.2 kb Eco RI fragment in the Osborne-Mendel strain, and on both a 9.4 and a 7.2 kb Eco RI fragment in Sprague-Dawley rats. The 9.4 kb Eco RI fragment from Hooded rat DNA was isolated using the lambda cloning system, and the nucleotide sequence of this isolated rat insulin I gene and adjacent regions was determined. A translation in one frame of the sequence of the cloned gene confirms the protein sequence determined for rat pre-proinsulin I. The coding region of this gene lacks intervening sequences, although a presumptive intervening sequence of 119 bp is located in the 5 untranslated region preceding the prehormone sequence. The junctions around the 119 bp segment are identical to those which flank intervening sequences of other eucaryotic genes, AGGT. The site of polyadenylation was determined by direct sequence comparison with rat insulin cDNA clones, and a potential 5 "capping" site is proposed. A DNA sequence preceding this 5 "capping" site in the rat insulin I gene, TATAAAGC, is homologous to corresponding regions in other eucaryotic genes that have been proposed as putative promoter sites for the initiation of transcription.

Animals↗
PubMed1977

Rat insulin genes: construction of plasmids containing the coding sequences.

Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

Animals↗
PubMed1977

Glucocorticoids modulate the in vitro development of the embryonic rat pancreas.

The effect of the glucocorticoid analogue, dexamethasone, on the development of the embryonic pancreas was studied in tissue culture. It specifically enhances the accumulation of exocrine enzymes without altering the level of general cell proteins. The enhancement, however, is not symmetrical: the cellular levels of the two major exocrine products, amylase and chymotrypsinogen, are increased about 10- and 2-fold, respectively. Two other zymogens that are present in minor quantities, procarboxypeptidases A and B, are also increased, whereas no effect is seen on lipase A. Coordinate with these effects on synthesis, there is a dramatic change in the morphology of dexamethasone-stimulated acinar cells. Their number of zymogen granules is higher and crystalline arrays are found in the rough endoplasmic reticulum. Dexamethasone also inhibits cell replication, perhaps by selectively inhibiting the last cell divisions of the culture period. At the same time, there is a disproportionate reduction in the insulin content of cultured rudiments. We find that pancreatic development is normal in the absence of dexamethasone and that this glucocorticoid does not precociously induce the appearance of the specific secretory products, but rather enhances by a constant degree their synthesis and accumulation. Therefore, we conclude that glucocorticoids may play a modulatory but not an inductive role in pancreatic development.

Animals↗
PubMed1976

5-bromodeoxyuridine may alter the differentiative program of the embryonic pancreas.

The thymidine analog, 5-bromodeoxyuridine (BrdU), inhibits the differentiation of the acinar cells of the embryonic rat pancreas, while having little effect on the growth of the tissue. The BrdU-treated pancreas contains elevated alkaline phosphatase and carbonic anhydrase activities, and, unlike the normal pancreas, contains numerous extracellular fluid-filled vacuoles, surrounded by ductlike cells. Both alkaline phosphatase and carbonic anhydrase activities are located preferentially in the ductlike cells lining the vacuoles. The biochemical, morphological, and functional features of these epithelial cells are therefore characteristic of the normal pancreatic duct cell. Thus, in the exocrine pancreas, BrdU seems to alter the normal program of differentiation by favoring the functional duct cells while inhibiting the differentiation of acinar cells.

Adenosine Triphosphatases↗