Yves Malpiece

Insertions in the hepatitis B surface antigen: Effect on assembly and secretion of 22-nm particles from mammalian cells

The envelope protein of hepatitis B virus carrying the surface antigen, HBsAg, has the unique property of mobilizing cellular lipids into spherical or elongated particles, about 22 nm in diameter, which are secreted from mammalian cells. We have created mutant envelope proteins by insertion of various sequences of different lengths into two regions of the S gene encoding the major envelope protein. S genes carrying inserts in phase with HBsAg were expressed in mouse L cells from the simian virus 40 early promoter. Various single or double inserts in the two major hydrophilic domains of HBsAg were compatible with secretion of 22-nm particles. In all mutant envelope proteins studied, the HBsAg domains required for intracellular aggregation appeared to be intact. However, assembly into particles was not sufficient to assure transport into the extracellular space. The 22-nm HBsAg particle may be a useful vehicle for the export and presentation of foreign peptide sequences.

Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors

This paper describes a new method of lasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultra-centrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or 5uperose 6B. It permits recovery oI plasmids : (I) in preparative quantities (from 300 gg to 4 mg), (II) exempt from RNA, DNA and protein contamination, and (III) suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purilication, the plasmids were used to construct thermoampliIiable vectors, carrying the tacUV5 promoter and the 5′ end of the β -gallactosidase gone with a single EcoRl site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins inEscherichia coli.

A transformed Vero cell line stably producing the hepatitis B virus surface antigen

We have constructed plasmids in which transcription of the surface antigen gene of the human hepatitis B virus (HBsAg) is under the control of the SV40 early promoter. These plasmids have been used to analyze the expression of the HBsAg gene, both in mouse cells and in African green monkey kidney (Vero) cells. We have established a Vero cell line continuously expressing HBsAg that is indistinguishable from authentic 22 nm HBsAg particles. Post-transcriptional modification of HBsAg mRNA also appears to occur normally in the monkey cells. These cells might be useful as a source of antigen for the preparation of an antihepatitis vaccine.

The gene S promoter of hepatitis B virus confers constitutive gene expression

The properties of the promoter of the hepatitis B surface antigen (HBsAg) were studied using recombinants containing either this promoter or the SV40 early promoter. Mouse L cells were transfected with these recombinants and the levels of gene expression obtained with the two promoters were compared. The level of expression of a cellular gene, the human fibroblast interferon gene, obtained with the HBsAg promoter was comparable to that obtained with the SV40 early promoter. Similarly when the HBsAg gene was controlled by the SV40 early promoter the level of HBsAg synthesis is in the same range as that observed with its own promoter. Together these results suggest that although the HBsAg gene codes for a structural viral protein, its expression is constitutive as for an early gene. The implications of these observations on the synthesis of HBV particles in vivo are discussed.

Identification of vaccinia promoters by heterologous expression of hepatitis B surface antigen in mouse cells infected by recombinant vaccinia viruses

DNA fragments preceding open reading frames in a conserved segment of the vaccinia virus genome (Plucienniczak A., et al. (1985) Nucleic Acids Res. 13, 985-998) were cloned into plasmids upstream of the S gene of the hepatitis B virus encoding the surface antigen (HBsAg). Recombinant vaccinia virus obtained after insertion of these constructs into the thymidine kinase gene were used to infect mouse 1D cells. HBsAg was assayed in cellular supernatants. A strong promoter was thus identified in a 295 bp fragment preceding the coding region of the 147 kDa subunit of the vaccinia RNA polymerase.

Colony hybridization method for screening in situ of eukaryotic amplified genes

A new method for autoradiographic screening of amplified genes in cellular clones is described. The main feature of the device is to keep viable cells from each clone, which can subsequently be regrown. The availability of this biochemical screening method allows screening for recombinants harboring unselectable markers as well.

Constitutive expression of the human fibroblast interferon gene is elicited in cultured mouse L cells by the long terminal repeat of moloney sarcoma virus

SummaryWe have used a chimerical plasmid containing the long terminal repeat of Moloney sarcoma virus to direct the expression of the human fibroblast interferon gene in mouse L cells. Constitutive secretion in cell culture supernatants was achieved at a level higher than 2.103 U/ml/72 h. The antiviral activity was indistinguishable from that of authentic human fibroblast interfereon by both immunological and physical criteria. Northern blotting analysis showed unambiguously that the expression was under the control of the putative transcriptional regulatory sequences previously described in the long terminal repeat of Moloney murine sarcoma virus (Dhar et al. 1980).