Mutants of cultured chinese hamster cells deficient in adenine phosphoribosyl transferase

Spontaneous mutants of cultured Chinese hamster cells (line CHO) deficient in APRT have been isolated by selection in 8-azaadenine (AA). Loss of APRT activity occurs in two discrete steps. In the first step, about 65% of total activity is lost; in the second step, most or all of the remaining activity is lost. Cells totally deficient in APRT are highly resistant to AA and cannot utilize exogenous adenine as a source of purines for cell growth. Cells partially deficient in the enzyme exhibit resistance to AA intermediate to that of wild type and fully deficient cells. Growth of cells partially deficient in APRT is inhibited in medium containing drug by the presence of large numbers of wild type cells, but cells totally deficient in the enzyme are not inhibited by the presence of either partially deficient or wild type cells.Stepwise loss of APRT activity probably has a genetic origin. The mutants exhibit stable phenotypes, and the frequency of fully deficient cells in a partially deficient population is enhanced by treatment with a mutagen. The rate of spontaneous mutation from partial to total deficiency is 3 ± 0.8 × 10^?7 per cell generation, and reversion from full to partial deficiency can occur. Total lack of APRT activity is recessive to its presence, but the specific activity of the enzyme in hybrid cells depends quantitatively upon the specific activities in the two parents.     

Mutants of purine metabolism in cultured mouse and hamster cells

The regulation of purine metabolism in mammalian cells is poorly understood presently. A variety of mutants in hamster and mouse is now available for these studies. It is clear that selective methods provide the potential for isolation of a greater variety and number of such mutants. It is predicted that the methods are now available for extensive study of the purine biosynthetic pathway with respect to both its genetic organization and cellular regulation. These mouse and hamster mutants will undoubtedly facilitate our understanding of this multistep pathway which until recently has been limited to the study of rare human mutants.     

Molecular analysis of deficient class I H-2 antigen expression by mouse lung carcinoma cells

We have continued our investigations of line lung carcinoma cells to understand the molecular basis of decreased expression of class I H-2 Ag and class I Ag induction with DMSO. We show that line 1, a murine lung carcinoma cell line, has low levels of class I Ag (H-2K, D, and L) because it is deficient in both class I and beta 2-microglobulin (B2M) RNA, and that these mRNA can be coordinately induced with DMSO. Evidence presented herein also shows that IFN-gamma can induce surface expression of class I Ag and suggests that it may act through a different mechanism than DMSO in inducing class I Ag. To further evaluate the regulation of class I expression, H-2Dp genes were transfected into line 1 cells. The transfected H-2 genes appear to be constitutively expressed at much higher levels than are the endogenous class I genes because surface expression of the foreign Dp Ag on the transfectants is elevated relative to the endogenous H-2d haplotype class I Ag. Both Dp surface expression and Dp mRNA are induced after treatment with DMSO. In all the Dp transfectants, we observed higher constitutive levels of class I mRNA as well as increased constitutive levels of endogenous B2M mRNA when compared to control or untransfected line 1 cells, however, we could not correlate these constitutive levels with Dp copy number. These results suggest that the regulation of class I and B2M genes is linked and that expression of class I genes can affect the expression of B2M genes.     

Isolation and characterization of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase.

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.     

Mutants of Chinese hamster cells deficient in thymidylate synthetase

Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the "FAT" medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5-7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (Km) for deoxyuridine-5'-monophosphate and inhibition constant (Ki) for 5-fluoro-deoxyuridine-5'-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6-3H]-2'-deoxyuridine 5'-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.     

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.     

H-2 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

The rates of emergence of H-2^b– and H-2^d– variants from an H-2^b+/ H-2^d+ heterozygous mouse leukemia cell line were determined by fluctuation analyses. The results show that the wild type cells throw off the H-2^b– variants at about 10^–5 per cell per generation and the H-2^d– variants at 10^–6 per cell per generation. The reason for this tenfold difference in the rates for the two symmetrical variants is not clear. The fluctuation analyses also indicate that the variants exist in the wild type population prior to exposure to the selective agents.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

In previous studies on the emergence of H-2 antigen loss variants from an H-2^b/H-2^d heterozygous mouse leukemia cell line, it has been shown that stable variants could be obtained after a single-step selection procedure with antibody and complement. Selection against the K-end alleles revealed an asymmetry in the types of variant obtained. This paper deals with the results of selection against theD-end alleles. Once again, an asymmetry is noticed. Selection against the H-2D^d gene product results in the isolation of stable variants that have lost H-2D^d, either alone or in combination with the linked H-2K^d antigens. Selection against H-2D^b, on the other hand, has not, so far, resulted in the isolation of any stable variants. These experiments have not demonstrated unequivocally the mechanism by which these H-2 antigen loss variants are generated. However, certain preliminary considerations suggest the possibility that these variants may not, in fact, be true genetic mutants. The alternative -that these variants arose through some nongenetic, but stable mechanism — must be considered seriously.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An H-2 antigen variant, referred to as ?4 + 31 clone 1, was selected by its resistance to an anti-H-2D^d antiserum (BALB.G anti-BALB/c.H-2 ^g antiH-2 ^d ). When tested by cell-mediated cytolysis, this variant was found to be sensitive to cytolytic T lymphocytes raised in the same donor-host combination as that used in raising the antiserum. Further CML characterization of this variant, reported here, indicates that the cell line is in fact resistant to anti-H-2D^d killer cells raised in a more restricted immunization, viz. BALB.G anti-BALB/cH-2 ^db ,H-2 ^g anti-H-2 ^db . It is, however, sensitive to cytolytic cells raised in (BALB.B xBALB/c-H-2db) F¹ H-2 ^b /H-2 ^db ) against the BALB/c strain. These results suggest that the variant does not express H-2D^d itself, but probably expresses CML target antigens that are missing in theH-2 ^db mutation. This in vitro-isolated variant might thus be the complementary mutation to the in vivoobtainedH-2 ^db mutation.     

Mutants ofPleurotus florida deficient in sporulation

Summary Inheritable sporulation-deficient dikaryons were obtained through pairing compatible monokaryons. The morphological character of centralized stipes of sporulation-deficient fruit bodies was remarkable and enable them to be distinguished from sporulating ones. Light and electron microscopy showed neither spores nor sterigmata on the surface of most basidia. However, in rare cases, some basidia, always clustering in a number of a few to tens, had one abortive spore on each basidium. No mature spores were found. Genetic analysis of sporulation deficiency was carried out by test-crosses. Results suggest that sporulation-deficient mutant inP. florida is rather complex and might be controlled by two recessive genes. The recovery of four mating types in spores obtained from the dikaryotic fruit body used for test-crosses, and the presence of four nuclei on the apex of basidia of sporulation-deficient strain indicated that the process of meiosis had occurred in this sporulation-deficient mutant.

---

Mutants de Pleurotus florida déficients en sporulation

Résumé Des dikaryons à sporulation déficiente héritable ont été obtenus en accouplant des monokaryons compatibles. Le caractère morphologique des tiges centralisées des corps fruitaux déficients en sporulation est remarquable et permet de les distinguer de ceux sporulants. La microscopie optique ou électronique ne montre mi spores ni stérigmates sur la surface de la plupart des basidia. Toutefois, dans quelques rares cas, quelques basidia, toujours agglutinés par groupe de quelques unités à quelques dizaines d'unités, ont une spore avortée sur chaque basidium. On ne trouve pas de spores mûres. L'analyse génétique de la déficience en sporulation a été réalisée par des tests de croisements. Les résultats suggèrent que la mutation de déficience en sporulation chezP. florida est plutôt complexe et pourrait bien être contrôlée par deux gènes récessifs. L'obtention de quatre types d'accouplement chez les spores obtenues du corps fruital dikaryotique utilisé pour les tests de croisements, et la présence de quatre noyaux dans l'apex des basidia de la souche déficiente en sporulation, suggère que le processus de méiose se passe bien chez le mutant déficient en sporulation.

     

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.     

UV sensitivity in thymidine kinase deficient mouse erythroleukaemia cells

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.     

Microtubular system in cultured mouse epithelial cells

Previous observations had shown that colcemid does not affect locomotion of epithelial cells. Nevertheless, cultured mouse kidney cells forming epithelial sheets were found to contain a well-developed microtubular system sensitive to colcemid. The orientation of microtubules in the epithelial cells was not correlated with the stable or active state of the cell edges. It is suggested that microtubular system of the epithelial cells forming coherent sheets, in contrast to that of individually moving fibroblasts, is not essential for stabilization of the lateral cell edges.     

Intraspecies identification of cultured murine cells by using H-2 antigen typing

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.