The isolation of a suppressible nonsense mutant in mammalian cells

Capecchi, Mario R.; Haar, Raymond A. Vonder; Capecchi, N. E.; Švéda, Martin

doi:10.1016/0092-8674(77)90113-1

Cited by 71 publications

(18 citation statements)

References 8 publications

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“…Several transformants were examined by hybridization in situ to determine whether the transforming sequences were indeed integrated into host chromosomes. Two of the transformants chosen for this study, Lll and L278, were shown by a Southern transfer analysis to contain approximately 10 How are the head-to-tail concatemers generated? Figure 6 shows three models by which headto-tail concatemers could be generated.…”

Section: Methodsmentioning

confidence: 99%

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Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Folger¹,

Wong²,

Wahl³

et al. 1982

Mol. Cell. Biol.

397

250

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We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk-and RAT-2tk-cells to the TK+ phenotype 20-to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by

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Section: Methodsmentioning

confidence: 99%

“…The methods used for culturing cells, autoradiography, plasmid DNA preparation, and microinjection have been described elsewhere (9,10).…”

Section: Methodsmentioning

confidence: 99%

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Folger¹,

Wong²,

Wahl³

et al. 1982

Mol. Cell. Biol.

397

250

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“…In the first case, Gesteland et al (2) showed that host range mutants of an adenovirus-simian virus 40 hybrid (AD2+ ND1) contain amber and ochre nonsense termination codons that can be suppressed by yeast suppressors in an in vitro cell-free suppression system. A second report described an ochre mutant within the hypoxanthine phosphoribosyltransferase gene (protein) of mouse cells (3). Another report described suppression of the natural amber termination codon between the gag and pol genes of murine leukemia virus RNA (4).…”

mentioning

confidence: 99%

“…and incubated as described (7). To 13 1l of nuclease-treated lysate, [1][2][3] ,ul of total cytoplasmic RNA from HSV-infected cells, prepared as described (7,10), and 15 (2). Schizosaccharomyces pombe strains sup3-e and sup8-e were used to isolate enriched serine and leucine opal (UGA) suppressor tRNAs, respectively (12).…”

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confidence: 99%

In vitro suppression of UAG and UGA mutants in the thymidine kinase gene of herpes simplex virus.

Cremer¹,

Bodemer²,

Summers³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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We have studied the in vitro translation, in nuclease-treated reticulocyte lysates, of mRNA from cells infected with several thymidine kinase-deficient mutants of herpes simplex virus type 1. The addition of suppressor tRNAs from yeast resulted in suppression of the mutant property in the case of two mutants. Synthesis of enzymatically active viral thymidine kinase was restored by serine-inserting amber suppressor tRNA in the case of HSV TK4-and synthesis of the intact, but inactive, thymidine kinase protein was restored by serine-and leucine-inserting UGA suppressor tRNAs in the case of HSV TK43-. Read-through of the normal termination at the end of the thymidine kinase gene was promoted by UGA suppressor tRNAs. We conclude that HSV-TK4-is an amber (UAG) mutant and that HSV-TK43-is an opal (UGA) mutant.The isolation and characterization of nonsense mutants and nonsense suppressors in mammalian cells and their viruses will provide a powerful tool with which to analyze numerous viral biochemical and cellular functions. As yet, however, no nonsense suppressors have been reported in mammalian cells. In this report we describe unique nonsense mutants in a viral gene with a selectable phenotype which should make isolation of nonsense suppressors feasible.A nonsense mutation creates an in-phase polypeptide chain termination codon, such as UAG (amber), UAA (ochre), or UGA (opal), within the coding region of a structural gene. These codons result in the synthesis of shorter polypeptide products and usually loss of enzymatic or other activity. Suppressor mutations have been identified which result in the insertion of an amino acid at the position of the nonsense termination codon, which, in turn, leads to the synthesis of the complete polypeptide chain and restoration of functional activity (1).Several recent reports show that nonsense termination codons in eukaryotic cells or viruses can be suppressed by yeast suppressor tRNAs. In the first case, Gesteland et al. (2) showed that host range mutants of an adenovirus-simian virus 40 hybrid (AD2+ ND1) contain amber and ochre nonsense termination codons that can be suppressed by yeast suppressors in an in vitro cell-free suppression system. A second report described an ochre mutant within the hypoxanthine phosphoribosyltransferase gene (protein) of mouse cells (3). Another report described suppression of the natural amber termination codon between the gag and pol genes of murine leukemia virus RNA (4). Pelham (5) has presented evidence that a natural UAG termination codon in tobacco mosaic virus can be suppressed in vitro by amber suppressor tRNA from yeast.In this report we show that certain thymidine kinase (TK)-deficient mutants of herpes simplex virus (HSV) are nonsense mutants because the mutant property can be suppressed in vitro in a mammalian cell-free translation system using amber and opal suppressor tRNAs from yeast as well as having other properties expected of nonsense mutations. In one case the suppressed protein has enzymatic and immunological properti...

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“…Somatic cell hybridization studies have localized the HPRT gene to regions of the X chromosome ofhuman (2), hamster (3), and mouse (4) and the locus has been extensively studied in somatic cell systems because of the ease of selection for both forward and reverse mutations (1). Several laboratories have identified structural mutations within this gene in cultured cells by peptide mapping, kinetic analysis, thermal sensitivity studies, and immunological changes in the HPRT protein (5)(6)(7)(8)(9)(10)(11). The isolation of cloned sequences of this gene should facilitate the examination of mutations that lead to alterations in gene expression.…”

mentioning

confidence: 99%

Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences.

Brennand¹,

Chinault²,

Konecki³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

111

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Cloned cDNA sequences of the murine hypoxanthine/guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) gene have been isolated by using a mouse neuroblastoma cell line containing increased levels of a variant HPRT protein. We have used these sequences as probes to demonstrate that protein overproduction in this cell line is a consequence of at least a 20-fold increase in HPRT mRNA levels resulting from approximately 50-fold amplification ofHPRT genomic sequences. The largest cDNA insert so far characterized represents about 70% of the HPRT mRNA sequence. This cDNA is shown to possess regions of homology with mRNA and DNA from Chinese hamster, baboon, and human, thus facilitating detailed analysis of this locus in these four species.Hypoxanthine/guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8), a multimeric enzyme catalyzing the conversion of hypoxanthine or guanine to its 5' mononucleotide, is essential for normal purine metabolism in man. A deficiency of it leads to the clinical disorders of Lesch-Nyhan syndrome and gouty arthritis (for review, see ref. 1). Somatic cell hybridization studies have localized the HPRT gene to regions of the X chromosome ofhuman (2), hamster (3), and mouse (4) and the locus has been extensively studied in somatic cell systems because of the ease of selection for both forward and reverse mutations (1). Several laboratories have identified structural mutations within this gene in cultured cells by peptide mapping, kinetic analysis, thermal sensitivity studies, and immunological changes in the HPRT protein (5-11). The isolation of cloned sequences of this gene should facilitate the examination of mutations that lead to alterations in gene expression.The highest tissue levels ofmammalian HPRT occur in brain where it represents 0.2% of the total soluble protein (12); in cultured rodent fibroblasts it represents =0.05% of the protein (13). A mouse neuroblastoma HPRT revertant cell line (NBR4) has been characterized recently (13, 14) which produces 20-to 50-fold increased levels of an altered HPRT protein. In vitro translation ofNBR4 mRNA (13) suggested that its HPRT mRNA level was also increased relative to the wild type. We now report the isolation of cDNA sequences of the HPRT gene from this cell line and demonstrate that these cells contain amplified HPRT genomic sequences and increased levels of HPRT mRNA.

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The isolation of a suppressible nonsense mutant in mammalian cells

Cited by 71 publications

References 8 publications

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

In vitro suppression of UAG and UGA mutants in the thymidine kinase gene of herpes simplex virus.

Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences.

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