Genetics Of Somatic Mammalian Cells Research Articles

Genetics of somatic mammalian cells: genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes.

Through hybridization of specific Chinese hamster cell auxotrophs with human cells and selection in media lacking the nutritional supplements required by the former cells, a series of stable hybrid clones can be prepared. These hybrids have genomes consisting of a common part--the complete or almost complete set of Chinese hamster chromosomes, plus a variable part--one or a few human chromosomes. The identity of the human chromosomes can be varied by utilizing different Chinese hamster auxotrophs and the appropriate selective media. The human chromosomes present can be determined by a combination of cytogenetic analysis with chromosome banding and testing for specific human marker genes. Hybrids containing single human chromosomes 11 and 12 and the combination of both 11 and 12 are described. The system appears to lend itself to various studies such as identification of human cell surface antigens, determination of their chromosomal loci, measurement of their distribution among cells of normal human tissues, study of interrelations among syntenic and asyntenic genes, and mutational analysis of the human genome.

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.

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.

Genetics of Somatic Mammalian Cells: Demonstration of a Human Esterase Activator Gene Linked to the AdeB Gene

Prototrophic hybrids formed from an adenine-requiring Chinese hamster cell and human fibroblasts uniformly display new esterase activity that differs from that of either parental cell in electrophoretic mobility and substrate specificity. The hybrids that grew in the selective medium and possessed the new esterase activity had a single extra chromosome that resembled a B-group human chromosome. When clones of such hybrid cells were cultured in nonselective medium, they rapidly reverted to inability to synthesize adenine, disappearance of the new esterase activity, and simultaneous loss of the extra human chromosome. Esterase activity like that of the hybrid is present in cells of various Chinese hamster, but not human, tissues. It is postulated that particular Chinese hamster esterase genes became inactive after longterm cultivation, and that, in the hybrid cell, a human activator gene linked to the adeB gene and located on a human B-group chromosome reactivated expression of these Chinese hamster esterase genes.

Genetics of somatic mammalian cells. XV. Evidence for linkage between human genes for lactic dehydrogenase B and serine hydroxymethylase.

AbstractChinese hamster ovary cells with a deficiency in Serine Hydroxymethylase which produces a specific glycine auxotrophy (gly− A) were fused with human cells from a variety of sources and the resulting hybrids analyzed for human gene linkage. Of 102 hybrid clones examined 65 possessed both glyA and lactic dehydrogenase B markers, 35 possessed neither marker. Two clones were found with altered glycine responses which were not linked to LDH‐B. The data indicate linkage between genes responsible for serine hydroxymethylase activity and lactic dehydrogenase B. Evidence for absence of linkage between these and a variety of other genes is also presented.

Genetics of somatic mammalian cells. XIV. Genetic analysis in vitro of auxotrophic mutants.

AbstractGenetic analysis has been carried out on auxotrophic mutants produced by treatment of Chinese hamster ovary and the Chinese hamster lung cells with mutagenic agents in vitro. Thirty‐six different mutants were subjected to complementation analysis and biochemical tests. The different mutations studied result in growth requirements for proline; glycine; glycine or folinic acid; adenine or several of its precursors; inositol; adenine plus thymidine; and glycine plus adenine plus thymidine.The mutants which require glycine fall into four different complementation classes while those requiring adenine or hypoxanthine form two different complementation classes. The biochemical blocks of the latter two classes both occur somewhere in the steps involved in conversion of 5‐phosphoribosyl‐1‐pyrophosphate to 5‐aminoimidazole 4‐carboxylic acid ribonucleotide.The auxotrophic mutants described exhibit all‐or‐none responses to their specific nutrilite supplements and are stable with respect to reversion. They involve alterations in ten different genes, and hence form a useful set of mutants for a variety of genetic studies. All the auxotrophies produced in a single exposure to a mutagen are due to single gene mutations, even when multiple nutritional requirements were produced. All the mutations studied are recessive.

Genetics of somatic mammalian cells: lethal antigens as genetic markers for study of human linkage groups.

The antigen that causes killing of at least 98% of a human cell population treated with a 1% solution of a specific rabbit antiserum in the presence of complement is a sensitive genetic marker. The rapid loss of human chromosomes in human-Chinese hamster cell hybrids makes possible a convenient test of linkage relationships with this marker. Hybrid clones with and without the lethal antigen were isolated and analyzed. In 76 clones and subclones studied, 41 carried both the lethal antigen and the lactic dehydrogenase-A marker, 35 carried neither, and no clones contained only one of the two markers. In contrast to this clear demonstration of linkage, absence of linkage was found between the lethal antigen and the following markers: Lactic dehydrogenase B, NAD-dependent malic dehydrogenase, NADP-dependent malic dehydrogenase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glutamate oxaloacetate transaminase, indophenol oxidase, glucose phosphate isomerase, proline, inositol, hypoxanthine B, and glycine A. This lethal antigen appears to be carried on a single human autosome.

Genetics of somatic mammalian cells. XII. Mutagenesis by carcinogenic nitroso compounds.

AbstractFour known carcinogenic compounds, all of which contain the nitroso group, have been found to be effective in producing cell killing, chromatid breaks, mutagenesis and chromatid rearrangements in Chinese hamster ovary cells. Single cell survival curves revealed these agents to exhibit a 50,000‐fold difference in their mean lethal dose (Do) values. However, all agents yielded constant values for their efficiencies of production of chromatid breaks and single gene mutagenesis, but not chromatid rearrangements, when these were expressed in terms of the Do value of each agent. Three of the carcinogens produced single‐hit and one produced a multiple‐hit survival curve. The system offers distinct advantages in study of the interrelationships between mutagenic and carcinogenic actions in mammalian cells.

Genetics of somatic mammalian cells: linkage studies with human-Chinese hamster cell hybrids.

Genetics of Somatic Mammalian Cells: Linkage Studies with Human–Chinese Hamster Cell Hybrids

Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents.

AbstractThe system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N‐methyl‐N1‐nitro‐N‐nitrosoguanidine, hydroxylamine, an acridine mustard (ICR‐191), caffeine and ultraviolet‐ and X‐irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water‐soluble carcinogen N‐nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.

Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells.

Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells.

Genetics of somatic mammalian cells, VI, use of an antimetabolite in analysis of gene multiplicity.

Genetics of somatic mammalian cells, VI, use of an antimetabolite in analysis of gene multiplicity.

Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants.

The preceding paper of this series described genetic studies on a Chinese hamster cell which exhibited a specific growth requirement for proline. The nutritional deficiency was shown to be a mutation causing lack of the enzyme converting glutamic acid to the 'y-semialdehyde. The character was demonstrated to be stable, exhibiting a reversion frequency of 10-6 per generation as expected of a single gene mutation.1 The present study was undertaken in order to develop methods for the detection and isolation of additional nutritionally deficient mutants for the purpose of location of genes on chromosomes, measurement of linkage, and study of gene regulatory phenomena. The principle employed, which has been widely used in microbial genetics, involves placing a mixed cell population in a limited medium in which nutritionally deficient forms cannot grow, and applying an agent which will kill only dividing cells.2 Two agents were studied in these experiments. The first was H3-thymidine, which can readily be incorporated in lethal amounts into the DNA of mammalian cells.3 DNA synthesis has previously been shown to be dependent on protein synthesis.4 Experiments demonstrated that differential incorporation of H3-thymidine into proline-sufficient and -deficient cells could readily be achieved in proline-free media, with a consequent enrichment of the deficient mutanit. However, the large amounits of radioactivity required for the execution of such experiments on a routine basis made this method objectionable. Consequently, another approach was adopted, in which cells were incubated with 5-bromodeoxyuridine (BUdR) for a period sufficient to ensure its incorporation into DNA.5 Thereafter, the cells were exposed to light in the hope that those cells which had incorporated BUdR into their DNA would become light-sensitive in the or near-visible (sometimes referred to as visible) region and be killed as occurs in bacteriophage.6 Hence, if a mixed cell population is so treated in a minimal medium, onie may expect more BUdR incorporation and subsequent killing among the nutritionally sufficient cell population than among the deficient forms, which should be concentrated among the survivors. This paper describes experiments which quantitate the response of mammalian cells to BUdR followed by visible light and which also demonstrate that proline-defi

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.

Growth and genetics of somatic mammalian cellsin vitro

Journal of Cellular and Comparative PhysiologyVolume 52, Issue S1 p. 287-311 Article Growth and genetics of somatic mammalian cells in vitro† Theodore T. Puck, Theodore T. Puck Department of Biophysics, Florence R. Sabin Laboratories, University of Colorado Medical Center, DenverSearch for more papers by this author Theodore T. Puck, Theodore T. Puck Department of Biophysics, Florence R. Sabin Laboratories, University of Colorado Medical Center, DenverSearch for more papers by this author First published: December 1958 https://doi.org/10.1002/jcp.1030520414Citations: 18 † Contribution No. 75 from the Department of Biophysics, Florence R. Sabin Laboratories, University of Colorado Medical Center. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume52, IssueS1Supplement: Symposium on Genetic Approaches to Somatic Cell VariationDecember 1958Pages 287-311 RelatedInformation

Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects.

A methodology designed to eliminate mitotic inhibitor action and involving use of pretested fetal calf serum and careful pH and temperature control has been described by which cells from normal human and animal tissue can be maintained in active growth for long periods in vitro without development of aneuploidy. By means of this procedure, it is possible reliably to establish cell cultures from minute skin biopsies which can be taken from any individual. Clones of mammalian cells with chromosomal markers have been isolated by this means from x-irradiated non-irradiated cell cultures. Application of these techniques to chromosome delineation in large numbers of human subjects; determination of chromosomal sex in patients; spontaneuos and induced genetic changes in somatic mammalian cells in vivo and in vitro; comparison of metabolic differences between normal and cancerous cells and other problems have been indicated.

Genetics of somatic mammalian cells. II. Chromosomal constitution of cells in tissue culture.

A convenient, reliable method for chromosome delineation of animal cells grown as monolayers on glass has been applied to human, opossum, and Chinese hamster cells. Tissue cultured cells from 5 different, normal organs of 7 different human subjects uniformly displayed the expected chromosome number of 46 and showed no variations in morphology or number other than the expected sex differences and a small incidence of polyploidy. The chromosomes of normal cells from the American opossum were as uniform as those of human cells. Cells of the inbred Chinese hamster demonstrated appreciable karyotype variability, the cause of which is under investigation. The chromosome number and morphology of cells from normal human tissues have remained constant after more than 5 months of continuous, rapid growth in tissue culture involving scores of vessel transfers and a number of generations equivalent to many billions of progeny. By the use of routine recloning, even cells of malignant, aneuploid constitution have been maintained in active growth for 3 years and hundreds of generations, with stable chromosomal and metabolic characteristics. The cells of the American opossum and Chinese hamster which possess only 22 chromosomes have been established in vitro and are especially suitable for genetic studies. The readily recognizeable Y and X chromosomes of the male opossum are particularly favorable as cytological markers. Photomicrographs of the chromosomes of the various cells employed are presented.

The Genetics of Somatic Mammalian Cells ,

New quantitative techniques have been described for rapid plating of isolated single cells from a wide variety of human tissues under conditions such that almost every cell produces a macroscopic colony. These methods make possible new kinds of studies of the growth and genetics of mammalian cells. Application of these methods to problems of growth rate analysis, cell morphology, mutant production and isolation, analysis of the action of ionzing radiations, study of virus-cell interactions, and to certain problems in clinical medicine has been indicated.