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Biomedical subjects

F Galibert

Publications and source records attributed to F Galibert.

At least 91 records · Page 5Linked to original sources
PubMed1994

Woodchuck hepatitis virus surface antigen produced in vitro fails to bind polymerized woodchuck serum albumin.

Using a plasmid (pSWS) similar to one that has been successfully used for large-scale production of hepatitis B virus (HBV) envelope protein particles (pSVS) but containing the corresponding woodchuck hepatitis virus (WHV) envelope gene sequences, we have stably transformed the rodent dihydrofolate reductase-deficient cell line CHO dhfr-. Although production of WHV envelope particles in CHO/pSWS cell lines was low, it was sufficient to test whether these particles could bind to polymerized serum albumin. Whereas binding of HBV particles produced in CHO/pSVS cells to polymerized human serum albumin could readily be detected, we found no evidence that the WHV envelope protein particles produced in vitro bind to either human or woodchuck polymerized serum albumin.

Animals↗
PubMed1994

Construction of a cosmid contig and of an EcoRI restriction map of yeast chromosome X.

We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.

Chromosomes, Fungal↗
PubMed1994

Assessing genetic stability at the nucleic acid level.

Due to the natural genetic instability of any living organisms as well as faulty amino acid incorporation by the translational machinery or by post-processing alteration, a recombinant protein is prone to contamination at various levels by mutated or altered molecules. However, it should be borne in mind that alteration at the nucleic acid level will be amplified and may affect a substantial percentage of the whole population, while alterations introduced at the protein level are not further amplified and will at the most constitute a harmless background. While it is the protein and not the gene that is used as a medicine, it is important to recognize that analytical results obtained at the protein level are better interpreted when they are backed by good knowledge of the genetic system. Direct sequencing of a gene borne by a plasmid or of a reverse transcriptase-polymerase chain reaction (RT PCR) product can provide the sequence of the majority of the molecules. However, it fails to detect mutated molecules corresponding to a low level of contaminant. Therefore, a statistical treatment of the polymorphism of the population or the use of alternative methods such as those based on single-strand conformational polymorphism (SSCP) or chemical cleavage modification (CCM) are recommended.

Biotechnology↗
PubMed1993

Implication of cysteine residues in the activity of single-chain urokinase-plasminogen activator.

Single-chain urokinase-plasminogen activator contains 24 cysteine residues involved in 12 disulfide bonds and distributed all along the three domains of the protein. In order to investigate the role of these disulfide bridges in the catalytic activities of scu-PA, we used site-specific mutagenesis to construct 10 mutants in which some cysteine residues were changed to serine residues. Each mutated DNA fragment was cloned into a procaryotic expression vector and the protein expressed in E. coli. Mutant proteins of the expected size were produced and analyzed for amidolytic and fibrinolytic activities. From this, it is shown that: i) the disulfide bonds in the epidermal growth factor (EGF)-like and in the kringle domains are not necessary. Moreover, disulfide bond deletion in the kringle domain improves those catalytic activities; ii) on the contrary, the disulfide bridges in the catalytic domain are essential for maintaining both activities.

Amino Acid Sequence↗
PubMed1993

The expression of cytokine and cytokine receptor genes in long-term bone marrow culture in congenital and acquired bone marrow hypoplasias.

A number of cytokines have been shown to have stimulatory activity on multipotent haematopoietic precursors. These include kit ligand (KL), interleukins (IL) 1, 3 and 6 and granulocyte macrophage-colony stimulating factor (GM-CSF). Using reverse transcriptase/polymerase chain reaction method (RT/PCR) we have examined the expression of these cytokines, the c-kit and IL-6 receptors, in long-term bone marrow culture (LTC) adherent layer cells in human bone marrow hypoplasia syndromes. Disorders studied include Fanconi's anaemia (FA, n = 16), idiopathic aplastic anaemia (AA, n = 11), Seckel's syndrome (n = 2), dyskeratosis congenita (n = 2), Shwachman-Diamond syndrome (n = 1), thrombocytopenia with absent radii syndrome (n = 1), acquired amegakaryocytosis (n = 1), paroxysmal nocturnal haemoglobinuria (n = 1) and acquired agranulocytosis (n = 1). IL-6 and GM-CSF expression appeared reduced in most patients with FA, suggesting that impaired production of these cytokines may contribute to the bone marrow failure seen in most patients with FA. In contrast, abundant IL-6 and GM-CSF expression were seen in most patients with AA when compared with the FA group and controls; these may be mediators of a stromal response in this disorder. No obvious differences were seen between the different patients' groups and controls in expression of the other cytokines or cytokine receptors studied.

Anemia, Aplastic↗
PubMed1993

The molecular cloning and sequence of the common marmoset interferon-gamma (MaIFN-gamma) gene.

A DNA fragment covering marmoset interferon gamma (MaIFN-gamma) was cloned from the DNA of peripheral blood leucocytes, sequenced and compared to its human IFN-gamma counterpart. The two nucleotide sequences were found to be highly homologous (90.3%). The position of the exons are directly comparable with those of the human IFN-gamma gene and follows the pattern of structural conservation found elsewhere. The present work makes available recombinant MaIFN-gamma for animal experiments.

Amino Acid Sequence↗
PubMed1992

Gibbon and marmoset c-myc nucleotide sequences.

The nucleotide sequences of the gibbon and marmoset myc loci have been determined by the dideoxy ribomethod. The number of mutations which occurred during evolution and the branches affected were deduced according to the principle of maximum parsimony, from a comparison with known mammal sequences. As previously observed for the human and chimpanzee myc genes, an Alu repeat belonging to subclass III was observed in the second intron of the gibbon myc gene. In contrast, no such element was found in the marmoset gene. Alignment of the Myc amino acid (aa) sequences provided clues for detecting which aa or which protein regions have been more heavily mutated. Conversely, some regions remained free of mutations and remained unchanged from mouse to human, most probably in connection with some important embedded property(ies). An intriguing feature of the human Myc protein is duplication of 50 aa out of 439. Strikingly, most of these aa remain unchanged in mouse, rat, cat, marmoset, gibbon, chimpanzee and human.

Amino Acid Sequence↗
PubMed1992

Sequence analysis of the hepatitis B virus pre-C region in hepatocellular carcinoma [HCC] and nontumoral liver tissues from HCC patients.

We investigated whether replication-competent pre-C/C defective mutants of hepatitis B virus (HBV) are detectable in primary human hepatocellular carcinoma (HCC) tissues from patients of a geographic area endemic for such mutants. DNAs extracted from formalin-fixed paraffin-embedded HCC samples were checked for the presence of specific HBV DNA sequences using the polymerase chain reaction (PCR). Amplified pre-C regions from nine HCC samples were directly sequenced as were samples of nontumoral liver tissues from five of these patients. The data show that hypervariable distal pre-C sequences were present in all nine HCC samples; this high variability was dependent on point mutations, which led to amino acid substitutions in nearly all cases. Interestingly, seven of the nine HBV DNA-positive samples from HCC tissues (but not samples from peritumoral liver tissue) showed mutations leading to amino acid substitution at the level of a distal cysteine residue. No mutation generating a translationally defective pre-C/C region was detectable in the tumor samples. Otherwise, in four of the six nontumoral liver tissues available from the same patients, a pre-C sequence with an in-frame TAG stop codon was detectable, although in three cases as a component of mixed population.

Amino Acid Sequence↗
PubMed1992

Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties.

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.

Amino Acid Sequence↗
PubMed1992

[Genes of the lipase family: comparison of nucleic and proteinic sequences].

Vertebrates' plasmatic apolipoproteins and a few number of lipases in their metabolism present sequence homologies. They are grouped in genes families. The four exons apolipoproteins gene family includes nine human genes: the divergence rate of their sequences allows to place the first ancestral gene very high in the phylogenetic tree of the evolution. However, a more recent duplication of apolipoprotein C-I gene dating from 40 millions years, may be a phylogenetic marker for the radiation of Monkeys. Pancreatic lipase and isoforms, lipoprotein-lipase and hepatic triacylglycerol-lipase form by their homologies a "superfamily" of genes, which also includes yolk proteins of Dipterians eggs. Sequence homologies of PL, LPL and HL are analysed and compared with multiple alignments of amino-acids and nucleotides on spreadsheets. From these comparisons we may characterize four classes of phylogenetic markers: 1) repetitive DNA sequence (Alu, B1, PRE-1) appeared during Mammals evolution, 2) short insertions or deletions (within N-terminal domain) and a gene conversion in guinea-pig lineage, 3) a progressive reduction of intron number during the lipases evolution, 4) several duplications of genes which have produced the five genes of this superfamily currently known in the human genome.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the human c-kit gene: evolution of the receptor tyrosine kinase subclass III.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor. It belongs to receptor tyrosine kinase subclass III, which also includes the colony-stimulating factor I receptor (c-fms), platelet-derived growth factor receptors A and B (PDGFRA and PDGFRB), as well as FLT1 and FLT3/FLK2. c-kit and PDGFRA, c-fms and PDGFRB, FLT1 and FLT3/FLK2 are grouped by pair in three clusters in man on chromosome 4 band q11-q13, chromosome 5 band q31-q33 and chromosome 13 band q12 respectively. Here, we report the genomic organization of the human c-kit gene, which is composed of 21 small coding exons, distributed over 80 kb. Comparison of the c-kit and c-fms oncogenes shows that they share identified exon/intron boundaries in their two kinase domains, as well as a similar exon/intron organization in the extracytoplasmic domain. Comparison with the kinase domains of tyrosine kinase genes not belonging to subclass III suggests that the exon/intron organization of c-kit and c-fms is a characteristic feature of subclass III. The genomic similarities between c-kit and c-fms, in conjunction with the location in pairs on different chromosomes of the subclass III genes, has led us to hypothesize that cis and trans duplications gave rise to this group of genes.

Amino Acid Sequence↗
PubMed1991

Sequence analysis of the simian foamy virus type 1 genome.

We have cloned the simian foamy virus type 1 genome (SFV1) and determined its nucleotide sequence. Analysis of this genome reveals, in addition to the usual genes encoding retroviral capsid, reverse transcriptase, and envelope protein (respectively, gag, pol, and env), two open reading frames (ORFs) between env and the long terminal repeat with partial homology to the human foamy virus (HFV) bel1 and bel2 genes. The first ORF could code for a polypeptide of 312 amino acids (aa) showing 40% homology with the HFV bel1 putative gene product. A more detailed analysis showed that the protein encoded by this ORF would have features characteristic of known trans-activating proteins. The second ORF could code for a polypeptide of 403 aa showing 38% homology with the putative HFV bel2 gene product. Moreover, the 5' extremity of the RNA genome can be folded into a secondary structure identical to the Tat-response element of human immunodeficiency viruses. A phylogenetic tree of retroviruses, including SFV1 and HFV, was constructed. It showed at the molecular level that Spumavirinae, previously classified on the basis of their morphology and their biological properties, constitute a separate group. The homology between SFV1 and HFV reaches 89% in the reverse transcriptase domain of the pol gene. but is much smaller in other parts of the genome.

Amino Acid Sequence↗
PubMed1991

Cloning and nucleotide sequence of the chimpanzee c-myc gene.

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.

Animals↗
PubMed1991

Recognition of the N-terminal, C-terminal, and interior portions of HBx by sera from patients with hepatitis B.

We have cloned and expressed in Escherichia coli three different parts of the HBx open reading frame, the N- and C-termini and the interior or central portion, using two vector systems. The sera of 43 hepatitis B virus patients representing three clinical categories--asymptomatic carriers, chronic active hepatitis, and hepatitis B patients with cirrhosis--known to be anti-HBx positive, were tested for reactivity against these constructs by Western blotting. The great majority of sera, regardless of the clinical categories, clearly recognise all three parts of HBx, strongly suggesting that the normal mechanism of biosynthesis of the HBx gene product is a straight-forward translation of the open reading frame starting from the first ATG. However, asymptomatic carriers show a marked, often almost exclusive, preference for recognition of the central portion of HBx, while patients with chronic hepatitis and patients with cirrhosis generally recognise all three parts of HBx to a similar extent.

Amino Acid Sequence↗