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Biomedical subjects

P Charnay

Publications and source records attributed to P Charnay.

87 records · Page 5Linked to original sources
PubMed1981

Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state.

The detection of hepatitis B virus (HBV) DNA in the liver and the serum permits direct study of the interaction between the virus and the liver cell. 40 HBV chronic carriers were studied by the blot technique of Southern to detect HBV DNA, and the results were compared with the serological status and histological status of the patients. It was possible to define two different chronic carrier states. The first is characterised by free viral DNA in the liver, with viral DNA and hepatitis B e antigen (HBeAG) in the serum; integrated HBV DNA is also present, at least in some patients. The second carrier state is characterised by the presence of only integrated HBV DNA sequences in the liver: viral DNA and HBeAg are not present in the serum. In one HBeAg-negative patient, however, free HBV DNA was detected in the liver and HBV DNA was present in the serum. The hybridisation technique appears to be a very sensitive test which could reflect viral multiplication better than HBeAg radioimmunoassay. Since a needle biopsy sample provides sufficient tissue for the Southern blot technique, it should be useful in understanding chronic hepatitis, the selection of patients for antiviral therapy, and the estimation of its efficiency.

Autoradiography↗
PubMed1981

Biology of hepatitis B virus.

Immunochemical investigations of the viral antigens and molecular characterization of the viral DNA have elucidated the nature of the hepatitis B virus infection underlying acute, chronic, and oncogenic disorders of the liver in man. Cloning and sequencing of viral DNA have made possible studies on the structure of the genome and on certain aspects of the biology of the virus, hitherto constrained for a lack of tissue culture systems and laboratory animal models useful in its propagation.

Amino Acid Sequence↗
PubMed1981

Structure and expression of the hepatitis B virus genome.

By fusion of the hepatitis B virus (HBV) surface antigen (HBsAg) gene to that of the E. coli lac Z gene carried by a phage lambda derivative, expression of HBsAg antigenic determinants was obtained and carried by a 138,000 dalton fusion polypeptide. Such a protein could be ultimately useful for second generation vaccine production. HBsAg gene expression was studied in eukaryotic cells using the mouse L cell (tk-) system. Cotransformation using a plasmid carrying two copies of the HBV genome in a tandem, head-to-tail arrangement (pCP10) and the cloned HSV-1 tk gene resulted in the excretion of 22 nm HBsAg particles in the supernatant. No other HBV markers were detected. These particles possess the same characteristics as the human serum particles (morphology, diameter, density, antigenicity). The purified HBsAg particles from L cells were found to be highly immunogenic in mice. HBV mRNA transcripts from these cells were analysed by Northern blotting. A major species of 2,300 bases was detected. This was mapped on the genome by hybridization with subgenomic fragments and in the L cell system using a series of plasmid derivatives carrying insertions at specific sites in the HBV genome and assaying for HBsAg expression. Thus the HBsAg gene promotor was localized between positions 2,400-2,800. Indeed there is only one TATA like sequence in this region, starting at position 2,776.

Animals↗
PubMed1980

Biosynthesis of hepatitis B virus surface antigen in Escherichia coli.

Hepatitis B is a widespread viral disease. In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers. The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg). Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins. Recently, the HBV genome has been cloned in E.coli. Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported. We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1). Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen.

DNA, Recombinant↗
PubMed1979

Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli.

The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the S chain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.

Base Sequence↗
PubMed1979

Localization on the viral genome and nucleotide sequence of the gene coding for the two major polypeptides of the hepatitis B surface antigen (HBs Ag).

The structural gene coding for both polypeptides I and II which are the two major polypeptides of the Hepatitis B surface antigen, is found to be localized on the viral genome. This gene, referred to as gene S, is located in the partially single stranded region. It maps between positions 73.6 and 95.1% of the genome length. It is composed of 678 nucleotides, which correspond to a theoretical polypeptide of 25,422 molecular weight.

Amino Acid Sequence↗
PubMed1979

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control. II. DNA fragment insertion at the vicinity of the lac UV5 promoter.

Bacteriophage vectors derived from lambda plac5 have been constructed. Their genomes have one EcoRI restriction site which is located at the very beginning of the lac Z gene. The major part of this gene was deleted by an in vivo intramolecular recombination. These vectors allow the fusion of a gene or an operon with the beginning of the lac Z gene, placing them under the control of the lac promoter, which carries the UV5 mutation. Some of these vectors (lambda Y) also include the lac Y gene and it too is under the control of the lac promoter. The lambda YEQS, which carries the Qam73 and Sam7 mutations, as safety mutations, has been certified as a B2 (EK2) vector by the French control commission "recombinaison génétique in vitro".

Coliphages↗
PubMed1979

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.

Chromosome Mapping↗
PubMed1978

Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.

We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).

Base Sequence↗
PubMed1978

[Cloning of the hepatitis B virus genome in Escherichia coli].

The whole genome of the hepatitis B virus (Dane particles) was inserted in vitro in the genome of the bacteriophage lambda gtWES . LAMBDA B. The recombinant DNA molecule was cloned in E. coli. Amplification of the hybrid bacteriophage enables the preparation of large amounts of hepatitis B virus DNA. The possibilities offered by the utilization of this recombinant bacteriophage are discussed.

Bacteriophage lambda↗