Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.

Ashman, Charles R.; Jagadeeswaran, Pudur; Davidson, Richard L.

doi:10.1073/pnas.83.10.3356

Cited by 46 publications

(30 citation statements)

References 34 publications

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“…lDneo. Infection of cells was carried out as previously described (1). Line HT1080neo is a population of cells derived from the human fibrosarcoma cell line HT1080 (32) by transfection with the plasmid pSV2Neo (42).…”

Section: Methodsmentioning

confidence: 99%

Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

1994

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Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells.The generation of distinct cell types in multicellular animals only recently has begun to be understood at the genetic and molecular levels. Much emphasis has been placed on factors regulating transcription and on the DNA sequences interacting with such factors. However, the control mechanisms determining which regulatory factors will be active and when they will be expressed are still largely unknown, as are the mechanisms which maintain the differentiated state. Studies on the molecular control of pigment cell differentiation are still at an early stage, since pigment-cell-specific genes were cloned more recently than genes associated with a number of other tissue types.Pigmentation in mammals results from the synthesis of melanin in melanocytes of the skin, hairbulbs, and pigmented epithelium of the eye. Pigmentation is a complex biochemical and genetic process, with, in the mouse, over 50 distinct genetic loci involved (41). The synthesis of melanin in melanocytes requires the enzyme tyrosinase, encoded by the mouse albino (c) locus. In addition, other enzymes which regulate the type of melanin synthesized have recently been identified (for a review see reference 19). Another locus encoding an enzyme related to tyrosinase is the mouse brown (b) locus, which encodes a pigment-cell-specific catalase (18).Tyrosinase cDNAs from both mice (46) and humans (25) have been cloned and sequenced. The cDNA clones map to the albino (c) locus on mouse chromosome 7 (28) and to human chromosome 11 (q14-21) (2). Evidence that these clones encode the authentic tyrosinase enzyme and that mutations therein are the cause of albinism has been provided by (i) synthesis of an enzyme with tyrosinase activity upon transfection of the mouse cDNA clone and expression in cultured cells (28), (ii) identification of m...

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Section: Methodsmentioning

confidence: 99%

Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

1994

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“…The retroviral vector has long terminal repeats, which allow it to integrate into mammalian chromosomes; a simian virus 40 origin of replication, which enables it to be excised from the chromosome in the presence of simian virus 40 large T antigen (introduced through fusion with monkey COS cells, which constitutively produce the antigen) (4); a pBR origin of replication, which allows it to exist as an autonomously replicating plasmid in bacteria; and the bacterial neo gene, which can be used for selection of transformants in bacteria as well as in mammalian cells. In the initial studies on this system, one complicating factor observed was the relatively high rate of spontaneous mutants (around 10-5) (3). A high spontaneous mutation rate reduces the sensitivity of mutagen assays by limiting the lowest detectable frequency of induced mutation.…”

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“…The vector was used to transform bacteria, and the recovered gpt genes were sequenced. Experimental details of the various techniques have been published previously (3).…”

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“…Electrophoretic GPT assays were performed on some revertants with wild-type (B12EH8 and S44EH4) and non-wild-type (S44EH2) sequences and on the parental A912 cells. In all of the revertants, HAT resistance was attributable to the restoration of GPT activity and not to reversion of the endogenous mutant gene of A9 cells that encodes hypoxanthine-guanine The gpt genes were recovered from the revertant lines as described previously (3). DNA base sequencing was carried out with a primer that allowed reading of the sequence in the region in which the original mutation occurred.…”

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Greenspan¹,

Xu²,

Davidson³

1988

Mol. Cell. Biol.

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The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC--AT transitions or AT-*TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT-*GC transitions and for one cell line with a GC-*CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT--GC transitions had mutated back to the wild-type gpt sequence via GC-*AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC->CG transversion had mutated via GC--AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG--GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.Analysis of the molecular mechanisms of mutagenesis in mammalian cells has been hindered by the inability to efficiently recover chromosomal genes from the cells. To overcome this problem, our laboratory recently has developed a system (3) that combines mutagenesis of a gene that is chromosomally integrated in mammalian cells, clonal purification of the mutant cells, excision of the gene, and transfer of the gene into bacteria for DNA sequencing. The key feature of this system is the use of a retroviral shuttle vector as the element in which the gene is located. The target gene for mutagenesis is the Escherichia coli gpt gene, which codes for the enzyme xanthine-guanine phosphoribosyltransferase (GPT). The gene was incorporated into the retroviral vector pZipNeoSV(X)1 (5) and introduced into murine A9 cells, which lack activity of the enzyme hypoxanthine-guanine phosphoribosyltransferase (10), a mammalian enzyme that is similar in function to GPIT. In hypoxanthineguanine phosphoribosyltransferase-deficient cells, transformants that produce functional GPIT can be selected in HAT medium, and those that have lost the ability to produce functional GPIT can be selected with 6-thioguanine. The retroviral vector has long terminal repeats, which allow it to integrate into mam...

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“…It allows selection of cell lines which have taken up DNA by transfection (8). It is also used in a variety of experiments which necessitate counterselection for loss of gpt activity either by inactivation of the gene (1,5,(11)(12)(13) or by deletion of the chromosome carrying the gene (2,10). In all instances, counterselection has been performed with the toxic purine analog 6-thioguanine.…”

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Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

Besnard¹,

Monthioux²,

Jami³

1987

Mol. Cell. Biol.

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Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

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Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.

Cited by 46 publications

References 34 publications

Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

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