DNA of Rous sarcoma virus: its nature and significance.

Purified preparations of Rous sarcoma virus (an avian tumor virus with an RNA genome) contain small amounts of double-stranded DNA. This DNA cannot be hybridized to viral RNA, but will reanneal completely with the DNA of avian cells. Extensive substitution of bromodeoxyuridine for thymidine in "viral" DNA does not photosensitize the biological activity of the virus. These observations indicate that the DNA associated with Rous sarcoma virus is derived from the DNA of the avian host cell, and is probably devoid of any function in the life cycle of the virus.

Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin.

We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell line, 9-186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell lines, which resulted in a 4-5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration.

Gene transfer into mammalian cells.

The introduction of purified genes into animal cells allows the study of gene expression and regulation and the establishment of genetically modified cell lines. Several methods are available of transferring DNA into cells of higher eukaryotes: transfection with calcium phosphate, fusion of cells with liposomes or bacterial protoplasts, and micro-injection of DNA. Transient and/or permanent expression of foreign genes can be obtained in biochemically transformed cells.

DNA transfection into mammalian cells, selective markers and gene cointegration.

The dominant selective markers that we and others have constructed for the selection of cells which stably acquired a foreign DNA, are briefly described. The possibility of obtaining the cointegration of several genes, and of inducing their amplification in the recipient cell, is discussed.

Comparative expression of the hepatitis B surface antigen gene in biochemically transformed human, simian and murine cells.

In this paper, we show that the pattern of expression of the human hepatitis B surface antigen (HBs Ag) gene, transfected along with a dominant selectable marker into mammalian cells, is complex. In human hepatoma (HepG2) cells, late transient expression occurs and permanent expression takes place at high frequencies in the selected clones. In HeLa and human xeroderma pigmentosum (GM4312A)-derived cells, the late transient expression is barely seen or absent and permanent expression is only seen in a few selected clones. In monkey kidney Vero cells, late transient expression has been described and we show in this report that only 5% of the selected clones are capable of expressing HBs Ag in a permanent manner. In most of the Vero clones, the absence of HBs Ag expression is mainly due to HBs Ag gene rearrangements. We have selected and amplified more than 500 transfected Vero clones and have characterized in detail one clone (GAR1412) which is a permanent high-level HBs Ag expressor.

Cloning of the herpes simplex virus type 1 thymidine kinase gene in E. coli K 12: a selective marker for gene transfer into animal cells.

The Herpes Simplex Virus type 1 (HSV 1) thymidine kinase (TK) gene, carried by a 3.6 kb Bam H1 DNA fragment, was isolated and ligated to Bam H1 cleaved bacterial plasmid pBR 322. The TK gene, cloned in E. coli, is functional when it is transferred back into Eukaryotic cells, and can thus be used as a selective marker for gene transfer.

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

Addition of a foreign oligopeptide to the major capsid protein of poliovirus.

Insertion mutants of type 3 poliovirus (Sabin strain) were constructed that encode additional amino acid sequences at the level of residue 100 of the capsid polypeptide VP1 within the neutralization site 1, corresponding to a loop on the capsid surface. The addition of a tri- or hexapeptide did not hamper virus viability. The antigenic pattern of insertion mutants was only modified locally: all mutants lost reactivity of neutralization site 1 with the corresponding monoclonal antibodies, while the reactivity of sites 2 and 3 was unaffected by the insertion. We have shown for one of the mutants--vFG68--that the antigenic specificity of the neutralization site 1 was replaced by a new one. Although vFG68 differs from its parental Sabin strain only by the addition of three amino acids within VP1, neutralizing antibodies specific for vFG68 were induced by the native virion as well as by the heat-denatured mutated virions. Our results demonstrate that an oligopeptide of three or six amino acids can lengthen VP1 at the level of antigenic site 1 without affecting virus multiplication and that this foreign peptide is exposed on the virion surface.

Stable HBV surface antigen expression by Vero cell clones after transfection.

After transfection of Vero cells with the hepatitis B surface (HBs) antigen gene and the dominant selective marker amino-glycoside 3' phosphotransferase (APH3'), a late transient HBs expression is observed during the first weeks after transfection. We isolated 300 G418-resistant cell clones transformed by plasmids carrying the two genes. Only 6% of them were found to express the HBs antigen stably (versus 65% in murine cells). The HBs antigen expression is maximal after cells have reached confluency, and this level of antigen expression remains stable for months when cells are fed twice weekly without trypsinization. One clone - GAR 1412 - has been studied in greater detail. The cells (10(6] are not tumorigenic for nude mice, and the excreted HBs antigen, which is under the form 22 nm spherical particles, is immunogenic in Balb/C mice.

Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins.

Two genes of Mycobacterium tuberculosis, apa (Rv1860) and pro (Rv1796), coding for two glycosylated excreted proteins have been injected to mice and guinea pigs. They produce an extended immunological response of Th1 and Th2 types. Despite the fact that mycobacterial glycosylation is necessary for a high level of delayed-type hypersensitivity (DTH) reaction, plasmids bearing each of the two genes induced an elevated level of DTH sensitization. An inverse relation between the CpG-N hexamer cluster frequency and the protective effect of injected genes is described. A comparison of the strength of several eukaryotic promoters based on the diameter of the DTH reaction shows that CMVIE followed by the ubiquitin promoter are the most efficient among those tested. A significant protective effect (0.7 log unit CFU) in mice was found for the apa gene while the pro gene had no effect.

Deoxyribonucleic acid polymerase associated with avian tumor viruses: secondary structure of the deoxyribonucleic acid product.

The products of the deoxyribonucleic acid (DNA) polymerase associated with Rous sarcoma virus and avian myeloblastosis virus were characterized by correlative analyses with equilibrium centrifugation and stepwise elution from hydroxyapatite. The initial enzymatic product consists of nascent DNA chains which are hydrogen-bonded to 70S viral ribonucleic acid (RNA), whereas the final enzymatic product is double-stranded DNA. Appreciable amounts of free single-stranded DNA were not detected at any point during the course of the enzymatic reaction, but the data in this regard are not decisive. The time course of synthesis of DNA:RNA hybrids and double-stranded DNA has been analyzed. It is concluded that the synthesis of double-stranded DNA is a sequel to and is probably dependent upon the synthesis of DNA:RNA hybrid.

Deoxyribonucleic acid polymerase of Rous sarcoma virus: studies on the mechanism of double-stranded deoxyribonucleic acid synthesis.

The deoxyribonucleic acid (DNA) polymerase of Rous sarcoma virus synthesizes both single- and double-stranded DNA, utilizing the ribonucleic acid (RNA) of the viral genome as the initial template. Results of pulse-chase experiments indicate that the single-stranded DNA serves as unconserved template and precursor for the synthesis of double-stranded DNA. The latter reaction is apparently initiated in association with the viral RNA and may involve a partially double-stranded intermediate form.

Construction of a dominant selective marker useful for gene transfer studies in animal cells.

Biochemically transformed clones of murine, simian and human cells were obtained after transfection with a new dominant selective marker. This marker is a hybrid gene which was constructed with a bacterial neomycin-kanamycin resistance gene coding region and the transcription signals of the herpes simplex virus thymidine kinase gene.

Expression of herpes simplex virus type I thymidine kinase gene in Escherichia coli.

A herpes simplex virus type I DNA fragment containing the sequence coding for thymidine kinase was fused to the very beginning of the Escherichia coli lac Z gene in the three possible reading frames. When the thymidine kinase sequence was in the orientation fit to be transcribed from the lac promoter, functional thymidine kinase was made under lac control in all three cases. Sequences data indicate that translation reinitiation occurs at the 5' end of the thymidine kinase gene after stop signals. Two T+A-rich sequences, which may be part of eukaryotic promoters, are found in the same region.

Late transient expression of human hepatitis B virus genes in monkey cells.

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.

Characterization of the low-molecular-weight RNAs associated with the 70S RNA of Rous sarcoma virus.

Two low-molecular-weight RNAs are associated with the 70S RNA complex of Rous sarcoma virus: a previously described 4S RNA and a newly identified 5S RNA. The 4S RNA constitutes 3 to 4% of the 70S RNA complex or the equivalent of 12 to 20 molecules per 70S RNA. It exhibits a number of structural properties characteristic of transfer RNA as revealed by two-dimensional electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the 4S RNA species. The 5S RNA is approximately 120 nucleotides in length, constitutes 1% of the 70S RNA complex or the equivalent of 3 to 4 molecules per molecules of 70S RNA, and is identical in nucleotide composition and structure to 5S RNA from uninfected chicken embryo fibroblasts. Melting studies indicate that the 5S RNA is released from the 70S RNA complex at the same temperature required to dissociate 70S RNA into its constituent 35S subunits. In contrast, greater than 80% of the 4S RNA is released from 70S RNA prior to its conversion into subunits. The possible biological significance of these 70S-associated RNAs is discussed.