Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells (original) (raw)
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Somatic Cell and Molecular Genetics, 1988
Pseudodiploid Chinese hamster V79-AP4 cells, functionally diploid at the adenine phosphoribosyltransferase (aprt) locus, were treated with colcemid, a well-known aneuploidizing agent, under various experimental conditions. Aneuploid and tetraploid cells and variants resistant to 10 #g/ml of 2,6-diaminopurine (DAP), which selects for presumptive aprt +/ heterozygotes in the untreated cells, were induced. Many of the induced variants were hypotetraploid with three (rather than four) chromosomes carrying the aprt gene. Dot-blot and Southern analysis of the DNA of these clones confirmed that they had three copies of the aprt gene. Their APR T specific enzymatic activity was 60-80 % of that of wild-type V79-AP4. The results of these and other experiments suggest that in these variants resistance to DAP is due to an altered aprt gene dosage and point to a possible genetic effect of colcemid and other aneuploidizing agents in somatic mammalian cells.
Somatic cell genetics, 1980
Human DNA purified from HeLa cells and from three strains of skin fibroblasts was precipitated with calcium phosphate and added to mouse cells that were deficient in adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT). Selection for cells possessing either of the phosphoribosyltransferases was imposed by blocking de novo synthesis of purine nucleotides with azaserine in a medium supplemented with adenine and hypoxanthine. The frequency of colony formation after selection was 1.7 x 10(-7)-3.3 x 10(-6). Excepting some azaserine-resistant colonies that appeared only in the first experiment and infrequent revertants expressing moust APRT, all characterized clones expressed the human forms of APRT or HPRT according to the criteria of specific immunoprecipitation and electrophoretic mobility. The frequency of transfer of the human APRT gene was much greater than that of HPRT. Transfer efficiency was not significantly reduced when HeLa DNA was sheared...
Cloning and expression of a mouse adenine phosphoribosyltransferase gene
Gene, 1983
indicates that the mouse APRT gene is larger than 1.8 kb and contains at least three introns. ** Present address: Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 49305 (U.S.A.) Tel. (415) 497-5968. ***To whom reprint requests should be sent at the first address. Abbreviations: APRT. adenine phosphoribosyltransferase; bp, base pairs; EtBr. ethidium bromide; kb. kilobase pairs; SDS, sodium dodecyl sulfate: SSC. 0.15 M NaCl. 0.015 M Na,, citrate, pH 7.6.
Somatic cell genetics, 1978
Clonal lines, with either partial or total deficiency of adenine phosphoribosyltransferase (APRT) were derived from the WI-L2 long-term human lymphocyte line by selection for resistance to the adenine analogs 8-azaadenine or 2,6-diaminopurine. Resistance to 8-azaadenine also conferred resistance to 2,6 diaminopurine and vice versa. Cells with 30--40% of wild-type APRT activity were selected by resistance to 0.01 mM 2,6-diaminopurine or 1.40 mM 8-azaadenine. The APRT in the 8-azaadinine-resistant cells exhibited a four- to sevenfold increase in the apparent Km for adenine. Activities of three other purine reutilization and interconversion enzymes in the resistant cells, including hypoxanthine phosphoribosyltransferase (HPRT), adenosine kinase, and adenosine deaminase, were within the range of wild-type activities. The doubling times of the APRT-deficient cells in purine-free medium was not different from wild-type cells. The APRT in the 8-azaadenine-resistant cells did not have an al...
Proceedings of the National Academy of Sciences, 1982
The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low fre...
Somatic Cell and Molecular Genetics, 1989
Southern blot analysis reveals two distinct adenine phosphoribosyltransferase (APRT) alleles in the P-19 mouse teratocarcinoma cell line. One allele is identical to that observed in common laboratory mouse strains (Mus musculus domesticus). The restriction enzyme site variations between the two alleles occur in sequences located both upstream and downstream of the APRT gene, but not within it. Although the P-19 cell line was established from a C3H strain embryo (Mus musculus domesticus), a sixth generation ancestor of this embryo was a feral mouse (Mus musculus musculus). The restriction pattern of the variant APRT allele in P-19 is identical to that of a feral-derived Mus musculus musculus animal, establishing the origin of this allele in the P-19 cell line. A third, distinct APRT allele was found in a Mus spretus feral-derived mouse. Exploiting the differences between the two APRT alleles in the P-19 cell line, we have demonstrated their sequential loss in APRT-deficient clones.
Somatic cell genetics, 1979
Mouse teratocarcinoma stem cells deficient in activity of adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) were obtained in order to have this marker in developmentally versatile cells. Mutagenized stem-cell cultures were selected for resistance to 8-azaadenine and four clonal cell lines were isolated. Three had severe deficiencies of APRT activity (7% or less of wild type) and one had a moderate reduction (73%). The enzyme in the latter clone was found to be an electrophoretic variant with slightly less anodal migration than the wild-type enzyme. Each clone remained stably APRT-deficient for at least 3 1/2 weeks, after subcutaneous inoculation, in the absence of the selective agent. The tumors formed from the inocula comprised a variety of differentiated tissues and thus showed persistence of stem-cell developmental pluripotency despite mutagenesis and selection. All mutants also retained the quasinormal karyotype (X/O sex chromosomal constitution, trisomy-19) of the parent lin...
Somatic Cell Genetics, 1978
A new selective system for isolating somatic celt hybrids, using adenosine kinase as the selective marker, has been developed. The selective medium for forward selection (to select for cells containing adenosine kinase) contains alanosine, adenosine and uridine. To survive in the presence of alanosine, cells must have adenosine kinase in order to utilize exogenous adenosine as the sole source of AMP. Uridine is added to the selective medium to prevent the toxic effects of adenosine on cultured mammalian cells. The selective medium for reverse selection (to select for cells lacking adenosine kinase) contains 2-fluoroadenosine, an analogue of adenosine, which is converted to a toxic nucleotide by the action of adenosine kinase. Mouse mutant cell lines deficient in adenosine kinase have been derived. Human-mouse hybrid cells containing the kinase have been prepared from one of these mutant lines. Karyotype data of these hybrid lines and their adenosine kinase-minus sublines are consistent with assignment by others of the human gene for adenosine kinase on chromosome 10.