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  • 1
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chinese hamster ovary (CHO) cells selected for resistance to the cytotoxicity of phytohemagglutin (PHA) have been found to exhibit stable alterations at their plasma membranes. The PHA-resistant (Pha R) cells bind markedly less125 I -PHA than do sensitive CHO cells and also exhibit an increased sensitivity to the cytotoxicity of concanavalin A, a lectin of different receptor specificity. Mutagenesis with ethylmethanesulfonate increases the proportion of Pha R cells 20- to 100-fold. PHA-resistant cells maintained for up to 8 months in continuous culture in the absence of the selective agent have retained the PhaR phenotype. These and other characteristics of the experimental system suggest that CHO cells selected for PHA resistance are authentic somatic cell mutants. The Pha marker appears to behave recessively in hybrids formed between PhaR and PhaS cells.
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  • 2
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract 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.
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  • 3
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cytotoxic lymphoid cells derived from in vivo immunization of mice across H2 barriers were utilized in in vitro cytotoxicity assays. The target cells were somatic cell hybrids derived from parental cells differing at the H2 locus. The hybrid cells surviving cytotoxicity were grown to confluent populations and the H2antigens selected against were no longer demonstrable by indirect immunofluorescence. Comparative karyology of hybrid cells expressing both parental H2 types before immunoselection with hybrid cells surviving immunoselection revealed a decrease in the number of murine chromosomes number 17, suggesting that those cells surviving cytotoxicity had spontaneously lost these chromosomes prior to the selection event. The possibility of immunoconstruction of somatic cell hybrids on the basis of their cell-surface antigens is discussed.
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  • 4
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract When thymidine kinase-deficient mouse cells “transformed” by inactivated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 10 6 in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems—eg, exposure of the cells to mutagens or cell fusion—do not affect the frequency with which viral TK is reactivated.
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  • 5
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.
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  • 6
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The terminal phase of DNA replication was studied by autoradiography in hybrids between human lymphocytes and mouse fibroblasts. The hybrids contained on the average only 11 human chromosomes. It was found that the sequence of terminal DNA replication for the human chromosomes in the hybrids was the same as the sequence of terminal replication for the corresponding chromosomes in the human lymphocytes. Furthermore, it was shown that the maintenance of the normal terminal replication sequence of the human chromosomes in the hybrids was not dependent on the presence of any specific human chromosome. The results suggest that the timing of terminal DNA replication is determined independently in each human chromosome.
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  • 7
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT + Chinese hamster or human cells resulted in the appearance of HPRT+ cells. Clones derived from these cells were isolated in HA T medium. Cells in mitosis during incubation with the chromosomes yielded three times more HPRT+ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT+ clones than did the interspecies system using human chromosomes and Chinese hamster recipient cells (5×10−5 and 6×10−6 respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.
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  • 8
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Heterokaryons of chick embryo erythroblasts fused with other avian fibroblasts were studied with regard to globin production. After the incorporation of radioactive amino acids, soluble proteins were separated on SDS-urea polyacrylamide gels. There was a striking increase in radioactivity above background in the globin region from lysates of fusion cultures when compared with fibroblast cultures. This was maximal at 24 hours after fusion, and then declined. Electrophoresis on acid-or alkaline-urea gels further identified the material as globin chains. Tryptic digestion and fingerprinting revealed methionine-labeled peptides characteristic of chick embryo erythroblast globin. An apparent stimulation of globin chain synthesis by heterokaryons compared to erythroblasts was found to be due to a difference in the specific activity of the precursor amino acid pools in the different cell types.
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  • 9
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract When viewed by scanning electron microscopy, the heads of mouse spermatozoa are smaller than those of the hamster. The vitelline microvilli of hamster eggs are longer than those of the mouse egg. Both these factors may contribute to the enhanced interaction of mouse spermatozoa and hamster eggs. Treating unfertilized mouse eggs with Newcastle disease virus causes the vitelline microvilli to elongate, thus improving the interaction between mouse eggs and hamster spermatozoa.
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  • 10
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Variant clones resistant to 40 μg/ml chloramphenicol were isolated from the human cell line VA 2-B after treatment with either ethyl methanesulfonate or N-methyl-N′-nitro-N-nitrosoguanidine. Among 17 clones analyzed, one variant, CAP-23, was investigated in detail. CAP-23 cells in the presence of 40 or 100 μg/ml chloramphenicol grew at essentially the same rate as cells in the absence of the drug; chloramphenicol resistance persisted even after 20 generations in the absence of the drug. No obvious morphological changes in mitochondria were observed by electron microscopy of thin sections of CAP-23 cells. In vivo mitochondrial protein synthesis in CAP-23 cells was inhibited little, if any, by chloramphenicol, and the variant showed partial cross resistance to mikamycin and carbomycin. In vitro protein synthesis in mitochondria isolated from CAP-23 cells showed, likewise, low levels of inhibition by chloramphenicol. This suggests that the drug resistance of the variant CAP-23 is due to altered mitochondria.
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