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.

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.

Evolution in the structure and distribution of 4F2-antigen from the oncofetal to the adult phenotype of human fibroblasts.

The monoclonal antibody (MAb) 4F2 defines an oncofetal antigen in human fibroblastic cells. Two-dimensional electrophoretic analysis reveals that tumor cell lines from mesenchymal tissues co-express two or more heavy-chain molecular variants of the antigen whereas the light subunit (41 kDa) is not affected. Among normal cells, only embryonic and newborn fibroblasts (from donors up to 20 days after birth) clearly co-express two distinct molecular forms of the heavy chain with MW of 85 and 75 kDa, respectively. Cells derived from 3-month-old donors express detectable amounts of the 85 kDA but only faint traces of the 75 kDa subunit, while fibroblastic cells derived from donors older than 3 months seem to express only the 85 kDa subunit. Immunofluorescence analysis performed on adherent living cells shows that, in the first months after birth, there is a gradual evolution from the oncofetal to the adult phenotype also in the cell distribution of the 4F2. This evolution is reflected by a progressive disappearance of the 4F2 antigen from the cell membrane becoming, in adult normal cells, inaccessible to anti-4F2 MAb. The existence of different molecular forms and different membrane positions of the 4F2 antigen could facilitate surveillance of morphological and structural changes in the evolution of human fibroblastic cells during the developmental process and neoplastic transformation.

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles.

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.

[Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells]. / Etude de l'expression du gène S du virus de l'hépatite B dans les cellules hybrides hépatocytes de rat X hépatome de souris.

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.

Analysis of the expression of the 4F2 surface antigen in normal and neoplastic fibroblastic human cells of embryonic and adult origin.

4F2 monoclonal antibody recognizes a 120-kD glycoprotein on the surface of human spread fibroblastic cells of embryonic and neoplastic origin, but it does not bind to normal spread adult fibroblasts. Flow cytometric analysis reveals that human adult fibroblasts become 4F2-positive when they are analyzed as round-shaped cells; this means that, in normal adult cells, 4F2 antigen behaves as a cryptic molecule. Thus, the basic difference between embryonic, neoplastic and normal adult cells consists in a different organization in the architecture of the cell membrane, since in embryonic and neoplastic cells there is a continuous expression of the 4F2 antigen independently of the cell shape and cell cycle phase. Quantitative flow cytometry shows that the mean surface density (MSD) of the 4F2 antigen 1, does not vary as a function of the cell cycle; 2, is inversely related to cell size and "metabolic time". This suggests that at the plateau phase the surface organization of G1 resting cells changes as a function of the number of days spent in culture; and 3, sarcoma and SV40-transformed cells show significantly increased MSD levels of the 4F2 antigen in comparison with normal cells of similar size. Electrophoretic analysis under reducing conditions confirms the quantitative differences in the expression of the 4F2 antigen described with the cell sorter. It also reveals, in a way different from that previously found with lymphoid cells, the coexistence of two molecules (85 and 73 kD) in the heavy chain regions. The 73 kD is, however, much more strongly expressed in the fibrosarcoma than in the embryonic cells. Finally, it shows that 4F2 antigen is a very useful tool for studying the organization and the structure of the cell membrane of human fibroblasts and can provide new insights to understand better the developmental and transformation processes.

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 plasmid 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 ultracentrifugation 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 Superose 6B. It permits recovery of plasmids: in preparative quantities (from 300 micrograms to 4 mg), exempt from RNA, DNA and protein contamination, and suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purification, the plasmids were used to construct thermoamplifiable vectors, carrying the lacUV5 promoter and the 5' end of the beta-galactosidase gene with a single EcoR1 site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.

Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin.

A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for polymerized human serum albumin (pHSA) and elicit in animals the synthesis of antireceptor antibodies. This property is ascribed to a 34,000-dalton polypeptide in the particles, which is most likely encoded by the S gene and part of the pre-S region. Especially because the pHSA receptor is most abundantly present on the virion and because, in hepatitis B infection, the appearance of anti-pHSA receptor antibodies seems to be a highly reliable criterion for viral clearance, the HBsAg particles obtained may constitute a particularly efficient vaccine.

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.

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.

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.

Synthesis of human interferon beta 1 in Escherichia coli infected by a lambda phage recombinant containing a human genomic fragment.

DNA from a human adult was fragmented by partial digestion with restriction endonuclease EcoRI and cloned in lambda Charon 4A. Clone C15, with a human DNA insert of 17 X 10(3) bases, was identified as containing a gene for the fibroblast interferon, interferon beta 1. Restriction mapping shows that this gene, located on a 1840-base EcoRI fragment, is not interrupted by introns. Moreover, we show that this human genomic DNA fragment is able to direct the synthesis of active human interferon beta 1 in Escherichia coli. Interferon activity of up to 7 X 10(6) U/l was recovered from phage lysates by chromatography on Cibacron blue--Sepharose, and had the same immunological properties and species specificity as interferon produced by human fibroblasts.

Kinetic, computer, and electron microscopic studies dealing with an artificial enzyme membrane: theoretical and experimental evaluation of the cytochemical demonstration of acetylcholinesterase.

The cytochemical demonstration of acetylcholinesterase is theoretically and experimentally evaluated with a model system. The model is an artificial proteic membrane in which acetylcholinesterase homogeneously is immobilized chemically by a bifunctional agent, glutaraldehyde. The copper-thiocholine histochemical method is studied kinetically and the system is simulated by computer calculations based on experimental kinetic parameters and numerical analysis methods. In addition, the corresponding electron micrographs are presented. These studies lead to the conclusions that the system is ruled by diffusional constraints and that enzyme distribution and repartition of the insoluble electron dense product are not circumscribed by any specific conditions.

A histochemical model dealing with an immobilized glucose oxidase-peroxidase system. The influence of diffusion limitations on histochemical results.

A histochemical model dealing with an immobilized bienzyme system (glucose oxidase-peroxidase) is presented. The model is an artificial proteic membrane obtained by a previously described co-cross-linking process. The kinetic properties of free and immobilized horseradish peroxidase were studied when 3,3'-diaminobenzidine is used as a hydrogen donor substrate. A new direct method was developed for immobilized enzyme activity measurements. Computer simulation based on experimental kinetic parameters was performed in order to discuss electron microscopy results. By changing diffusion limitations, various profiles of insoluble product were visualized inside the proteic film and no geometrical similarity was seen between enzyme distributions and insoluble osmiophilic product patterns.

Theoretical and experimental study of an immobilized bienzyme system. Computer calculations and electron microscopy visualization of local concentration profiles.

A cytochemical method was used in order to visualize the existence of local concentrations of metabolites within an immobilized bienzyme system. Glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) were immobilized together into an artificial proteic membrane obtained by a previously described co-cross-linking process using a bifunctional agent, glutaraldehyde. The cytochemical reagent 3,3'-diaminobenzidine was used as a hydrogen donor substrate and kinetic studies were performed with both free and immobilized forms of the enzymes; in the latter case, a new direct method was introduced which allowed kinetic studies on the visualization system itself. From experimental kinetic parameters, theoretical concentrations profiles were calculated by computer using numerical analysis methods. These results were discussed with special attention to the electron microscopic observations. Computer simulations were in good agreement with electron micrographs. No geometrical similarity was seen in our system between poly(3,3'-diaminobenzidine) precipitate distribution and peroxidase molecule homogeneous location.