Hamster Cell Mutants Research Articles

Mutant hamster cells exhibiting a pleiotropic effect on carbohydrate metabolism.

Sixty-seven galactose negative (Gal minus) clones were established from survivors after treatment of Chinese hamster lung (V79) cells with 5-bromodeoxyuridine and black light. The mutational origin of these clonal isolates was inferred on the basis of the previously demonstrated mutagenic action of the combined treatment, persistence of the Gal minus phenotype in cell progeny in the absence of selection, conditional lethality of three isolates, interallelic complementation, and assignment of a homologous human gene to chromosome A2. In contrast to the parental cells, Gal minus mutants could not utilize exogenous galactose, mannose, fructose, galactose 1-phosphate, glucose 1-phosphate, or glucose 6-phosphate. Permeation of galactose into mutant cells appeared unimpaired. In intra- and interspecific cell hybrids, the mutation causing the Gal minus phenotype behaved like a recessive character. The pleiotropic nutritional response was due not to deficiency of any one specific enzyme in the Leloir pathway but to significant reduction in activities of phosphoglucomutase, NADP-dependent isocitrate dehydrogenase, and perhaps other enzymes. In temperature-sensitive Gal minus mutants grown at permissive temperature or in complementing intra- and interspecific cell hybrids, the activities of these enzymes were restored to normal levels, accompanied by a regained ability to use the particular hexoses or hexose monophosphates. We postulate that the change from Gal + to Gal minus phenotype in hamster cells could be due to mutations at a regulatory gene locus, or at a yet unknown locus with enzymic defect that causes secondary metabolic imbalances.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: biochemical analysis of eight mutants.

Purine biosynthesis was studied in 8 mutants of Chinese hamster cells which require purines for growth and in wild-type cells which do not show this nutritional requirement. Of these, 6 mutants, ade-B, ade-D, ade-E, ade-F, GAT-, and AT-, were shown to accumulate metabolic intermediates not accumulated by wild-type cells. These intermediates were shown to be compounds unique to the adenylic acid biosynthetic pathway by the following criteria: (a) their radioisotopic labeling properties, (b) their response to agents which specifically inhibit known enzymatic steps in the pathway, (c) their chromatographic properties, and (d) spectrophotometric analysis. Two mutants, ade-A and ade-C, accumulate no detectable compounds not accumulated by the wild type. These 2 mutants are believed to be defective in steps very early in the purine biosynthetic pathway. The sites of the defects in the other mutants are proposed, and the usefulness of these mutants is discussed.

Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to muta-genesis detections

In vitro enumeration of diploid human cell variants that are resistant to purine analogues is a possible method of detecting mutagenesis. Their incidences can be increased by the known mutagens, X-rays and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG). Usefulness of this method depends on the kinds of hereditary changes that confer analogue-resistance on somatic cells. If resistance usually results from changes in genetic material, in vitro studies could be useful indicators of mutagenic effects on somatic cells and germ cells in vivo . If epigenetic changes are primarily responsible for analogue-resistant variants, their enumeration might not provide information relevant to germinal mutations but would still be a useful way to detect induction of general kinds of stable phenotypic changes that could cause cancer. This article outlines hypothetical epigenetic and genetic causes of somatic cell variation and a prospective genetic analysis of human cell variants that are resistant to 8-azaguanine (AG) or 2,6-diaminopurine ( (DAP). Recent evidences and arguments favoring epigenetic origins of resistance to base-analogues are inconclusive. The often cited high rate of changes causing impermeability to BUdR in hamster cells is based on one improperly executed determination. Comparisons of rates of variation conferring BUdR-resistance on cultured haploid and diploid frog cells included diploid variants that did not behave as mutants and ignored major sources of error in estimating mutation rates. AG-resistance could result from recessive mutations in X-chromosomal genes but comparisons of rates of mutation in hamster cells of different ploidies did not provide information about the numbers of X-chromosomes in the variants. Reports that normal rodent HGPRT reappeared in hybrids of enzyme-deficient rodent cells and HGPRT-containing cells of other species or in the rodent cells alone in response to the conditions of cell hybridization did not include adequate controls for reversions in mutant genes of the rodent cells. Questions about the epigenetic and genetic origins of analogue-resistance are mostly unanswered. It remains possible that some kinds of abnormal epigenetic changes cause somatic disease. Specific methods for detecting their occurrence and responsiveness to environmental factors should be devised by focusing efforts on traits that are normally subject to epigenetic regulation. Derepression of genes on the inactive X-chromosome and of liver phenylalanine hydroxylase production are presented as possible examples of abnormal epigenetic changes that could be quantitatively studied by direct selection in vitro .

Genetics of somatic mammalian cells: biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism.

Studies are presented on the biochemical genetics of 30 adenine-requiring mutants of the Chinese hamster ovary cell which were induced by mutagenesis and selected by the BrdU-visible light technique. Representative experiments conducted with these mutants include: hybridization with each other; hybridization with normal human cells; nutritional analysis; biochemical analysis with radioactively labeled intermediates; and measurement of reversion frequencies to wild-type phenotype occurring spontaneously and under the influence of selected mutagens. All mutants behave as if having point mutations. These experiments provide information relevant to the determination of dominant-recessive relationships, resolution into different complementation classes, localization of the human chromosomes which carry human genes required by the individual mutants, determination of the point of metabolic block for different mutants, and elucidation of the nature of the underlying DNA changes. These experiments illustrate the range of biochemical-genetic studies now possible with such a family of somatic mammalian cell mutants in vitro. Possible application to problems of human genetic disease are indicated.

A temperature-sensitive hamster cell mutant with altered morphology

A hamster cell mutant, ts-542, was isolated after nitrosoguanidine treatment. The mutant grows well at 33 °C and displays a normal, fibroblast-like morphology. It ceases cell division and becomes web-like in morphology after 4 days at 39 °C. No major lesion occurs in nucleic acid or protein synthesis during the first 3 days following transfer to 39 °C, but by the 4th day at this temperature there is a four-fold increase in acid phosphatase activity, an increase in nucleolar number, nuclear blebbing, and frequency of binucleate cells as well as increase in lysosomes. Cell death at 39 °C is independent of the cell density in the dishes or time of subculture. The mutation appears to be stable with a reversion frequency on the order of 10 −6.

Temperature-sensitive mammalian cell line blocked in mitosis.

SEVERAL temperature-sensitive mammalian cell mutants in culture have been isolated1–10, including three defective in protein syntheses6, cytokinesis5 and the processing of ribosomal RNA10, respectively. I have now isolated a hamster cell mutant in which mitosis is blocked in metaphase.

A temperature-sensitive function in a Chinese hamster line affecting DNA synthesis.

AbstractA temperature‐sensitive (ts) mutant of Chinese hamster cells (K12) is blocked at a step late in G1, required for the initiation of DNA synthesis. The ts lesion is recessive in interspecific hybrids.

Genetics of somatic mammalian cells. XVII. Induction and isolation of Chinese hamster cell mutants requiring serine.

AbstractThe induction, isolation and some of the properties of serine‐requiring mutants of Chinese hamster ovary cells have been described. These cells promise to be useful in biochemical genetic studies and cancer research.

Mutagenesis to aminopterin resistance in cultured hamster cells

Spontaneous hamster cell mutants resistant to aminopterin have been shown previously to contain an elevated folate reductase activity, not corrected upon somatic cell hybridization with low enzyme parent lines. In an attempt to isolate other types of aminopterin resistant mutants we have employed the chemical mutagens, ethylmethane sulfonate and nitrosoguanidine. Mutants with low resistance were produced at an increased frequency, but highly resistant clones could not be produced directly with these agents. Highly resistant lines were obtained by a second exposure to mutagens. Unexpectedly, these induced mutants contained no more folate reductase than wild-type cells. Mechanisms of resistance considered possible from work in other systems but excluded here were decreased sensitivity of the reductase to inhibition by drug, increased thymidylate synthetase activity, and decreased drug uptake. Drug catabolism or the metabolism of folate derivatives within the cell may be altered in these mutants. These results underscore the importance of defining the biochemical alterations in induced mutants, and suggest that chemical mutagens may be particularly useful in the production of novel mutants in animal cell systems.

Complementation analysis on virus-fused Chinese hamster cells with nutritional markers.

Cell fusion experiments have been carried out with Chinese hamster cell mutants with different nutritional growth requirements. Conditions have been devised in which approximately 1 to 2 percent of the cell population remaining after fusion are fused, hybrid cells. The all-or-none nature of the genetic markers employed and the extremely low reversion rates insure that no contamination of the hybrid population with parental forms occurs. Hybrids between glycine- and hypoxanthine-requiring mutants are prototrophic, which indicates that both mutations are recessive. Hybrids between a glycine-deficient mutant and a singlestep mutant which requires glycine, hypoxanthine, and thymidine are relieved of the glycine dependency, an indication that the two loci associated with glycine dependence are different. This mutation to the triple-supplement requirement as well as a proline deficiency were also shown to be recessive mutations. The system appears applicable to a variety of genetic problems.

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.

Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline.

Genetics of somatic mammalian cells. IV. Properties of Chinese hamster cell mutants with respect to the requirement for proline.