ABSTRACT
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.
Subject(s)
Genetic Markers , Transfection/methods , Adenosine Deaminase/genetics , Alcohol Oxidoreductases/genetics , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cells, Cultured , Dihydroorotase/genetics , Kanamycin Kinase , Multienzyme Complexes/genetics , Pentosyltransferases/genetics , Phosphotransferases/genetics , Recombinant Proteins/biosynthesis , Tetrahydrofolate Dehydrogenase/geneticsABSTRACT
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.
Subject(s)
Capsid/genetics , DNA Transposable Elements , Mutation , Oligopeptides , Poliovirus/genetics , Amino Acid Sequence , Base Sequence , Capsid/immunology , Codon , Epitopes/analysis , Escherichia coli/genetics , Molecular Sequence Data , PlasmidsABSTRACT
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.
Subject(s)
Primates/genetics , Rodentia/genetics , Transfection , Animals , Cell Line , DNA, Recombinant , Gene Expression Regulation , Hepatitis B Surface Antigens/immunology , Humans , Kanamycin Kinase , Phosphotransferases/genetics , Recombination, Genetic , Species Specificity , Transformation, Genetic , Vero CellsABSTRACT
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.
Subject(s)
Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Transfection , Animals , Cells, Cultured , Chlorocebus aethiops , Hepatitis B Surface Antigens/analysis , Mice , Mice, Inbred BALB CABSTRACT
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.
Subject(s)
Genes , Transformation, Genetic , Animals , DNA, Recombinant , Gene Expression Regulation , Genetic Vectors , Mammals , MethodsABSTRACT
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.
Subject(s)
Gene Expression , Hepatitis B Surface Antigens/genetics , Hepatitis B e Antigens/genetics , Hepatitis B virus/genetics , Animals , Cell Line , Cell Transformation, Viral , Chlorocebus aethiops , Cloning, Molecular , Genes, Viral , Genetic Engineering , Genome, Viral , Haplorhini , HeLa Cells , Humans , Kanamycin Kinase/genetics , Mice , Plasmids , RabbitsSubject(s)
DNA Transposable Elements , Genetic Markers , Phosphotransferases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Cell Line , Cell Transformation, Viral , Drug Resistance, Microbial , Gentamicins/pharmacology , Haplorhini , Humans , Kanamycin Kinase , Mice , Molecular Weight , Operon , Plasmids , Simplexvirus/genetics , Thymidine Kinase/genetics , TransfectionABSTRACT
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.
Subject(s)
Cloning, Molecular , DNA, Recombinant/metabolism , Escherichia coli/enzymology , Simplexvirus/enzymology , Thymidine Kinase/genetics , Animals , Base Sequence , Cell Transformation, Viral , DNA Restriction Enzymes , L Cells/metabolism , Mice , Operon , Plasmids , Protein Biosynthesis , Transcription, GeneticABSTRACT
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.
Subject(s)
Cloning, Molecular , Genes, Dominant , Genes , Genetic Engineering , Simplexvirus/enzymology , Thymidine Kinase/genetics , Animals , Cell Line , Cell Transformation, Viral , Drug Resistance, Microbial , L Cells/physiology , Mice , Plasmids , Transcription, Genetic , TransfectionABSTRACT
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.
Subject(s)
Cloning, Molecular , Genes, Viral , Simplexvirus/genetics , Thymidine Kinase/genetics , Transfection , Animals , Escherichia coli/genetics , Genetic Markers , L Cells , Mice , PlasmidsABSTRACT
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.
Subject(s)
DNA, Recombinant , Genes , Simplexvirus/genetics , Thymidine Kinase/genetics , Antigen-Antibody Reactions , Cell Transformation, Viral , DNA Restriction Enzymes/metabolism , Escherichia coli/genetics , Plasmids , Simplexvirus/enzymology , Thymidine Kinase/immunologyABSTRACT
The Eco RI fragment "b" of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5' quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a "shotgun" approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5' quarter (approximately 500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Econ RI fragment "b" is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150-200 nucleotide long sequence at the 5' end of the ov mRNA similar to the "leader" sequences found at the 5' end of some adenovirus and SV40 mRNAs.
Subject(s)
Genes , Ovalbumin/genetics , RNA, Messenger/genetics , Animals , Base Sequence , Chickens , DNA Restriction Enzymes , DNA, Recombinant , Microscopy, Electron , Nucleic Acid HybridizationABSTRACT
An EcoRI fragment of chicken DNA (fragment 'a') containing a sequence complementary to the 3' half of ovalbumin mRNA has been isolated by molecular cloning. Analysis of the cloned fragment proves conclusively that the chicken ovalbumin gene is split. Fragment 'a' contains no extensive sequence repeated elsewhere in the genome and represents the only type of organisation of this part of the split ovalbumin gene in chicken genome.
Subject(s)
DNA, Recombinant/isolation & purification , Genes , Ovalbumin/genetics , Animals , Base Sequence , Chickens , Chromosome Mapping , Coliphages/genetics , DNA Restriction Enzymes , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
RNA dependent RNA polymerase has been demonstrated in purified Lumbo virus (Bunyavirus) which contains a single stranded segmented RNA. Divalent cations (Mn++ and Mg++) are required for optimal in vitro activity. Reaction products can be specifically annealed with the viral genome.
Subject(s)
Arboviruses/enzymology , Bunyamwera virus/enzymology , RNA Nucleotidyltransferases , RNA-Dependent RNA Polymerase , Cations, Divalent , Dactinomycin/pharmacology , Kinetics , Nucleic Acid Hybridization , RNA, Viral , RNA-Dependent RNA Polymerase/metabolism , Ribonucleases/pharmacology , UridineSubject(s)
Avian Sarcoma Viruses/analysis , RNA Viruses/analysis , RNA, Viral/analysis , Sheep Diseases/microbiology , Animals , Base Sequence , Centrifugation, Density Gradient , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Phosphorus Radioisotopes , Poliovirus/analysis , RNA, Viral/isolation & purification , Sheep , Tritium , Uridine , Visna-maedi virus/analysisSubject(s)
DNA, Viral/analysis , RNA Viruses/analysis , RNA-Directed DNA Polymerase/metabolism , Sheep Diseases/microbiology , Animals , Centrifugation, Density Gradient , Chromatography , DNA, Viral/biosynthesis , Dactinomycin/pharmacology , Deoxyribonucleases , Hydroxyapatites , Molecular Weight , Nucleic Acid Hybridization , RNA, Viral , Sheep , Surface-Active Agents/pharmacology , Templates, Genetic , Thymine Nucleotides/metabolism , Transcription, Genetic , Tritium , Ultracentrifugation , Visna-maedi virus/analysis , Visna-maedi virus/enzymologyABSTRACT
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.
Subject(s)
Avian Sarcoma Viruses/analysis , RNA, Viral/analysis , Adenosine/analysis , Animals , Autoradiography , Avian Sarcoma Viruses/growth & development , Chick Embryo , Culture Techniques , Cytidine/analysis , Electrophoresis , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Guanosine/analysis , Molecular Weight , Nucleic Acid Denaturation , Oligonucleotides/analysis , Phosphorus Radioisotopes , Ribonucleases , Temperature , Uridine/analysisSubject(s)
Avian Sarcoma Viruses/metabolism , DNA, Viral/biosynthesis , RNA, Viral , Transcription, Genetic , Avian Sarcoma Viruses/drug effects , Avian Sarcoma Viruses/enzymology , Base Sequence , DNA, Single-Stranded/analysis , DNA, Viral/analysis , Dactinomycin/pharmacology , Manganese/pharmacology , Molecular Weight , Nucleic Acid Hybridization , Phosphorus Radioisotopes , RNA-Directed DNA Polymerase/metabolism , Surface-Active Agents/pharmacology , Templates, Genetic , Thymine Nucleotides/metabolism , Transcription, Genetic/drug effects , TritiumABSTRACT
Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.
Subject(s)
Avian Sarcoma Viruses/growth & development , RNA, Viral/isolation & purification , Virus Replication , Aspergillus/enzymology , Base Sequence , Cell Nucleus/analysis , Centrifugation, Zonal , Culture Techniques , Cytoplasm/analysis , DNA, Single-Stranded/biosynthesis , Deoxyribonucleases , Dimethyl Sulfoxide , Hydrolysis , Hydroxyapatites , Neurospora/enzymology , Nucleic Acid Denaturation , Nucleic Acid Hybridization , RNA, Viral/analysis , Ribonucleases , Thymidine , TritiumABSTRACT
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.