Louis Siminovitch

Ouabain-resistant mutants of mouse and hamster cells in culture

Abstract Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5–6 mM K + , or ≥ 0.1 mM with 0.5 mM K + , inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUA R ) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUA R L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42 K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUA R cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUA R clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUA R cells in a wild-type population can be substantially increased, to approximately 10 −4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5–6 × 10 −8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUA R and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

Spleen-Colony Formation in Anemic Mice of Genotype WW

The hemopoietic cells from anemic mice of genotype WW are less able by 200-fold to take part in colony formationin the spleen than cells from the normal littermates of genotype ww. The genetic defect shows itself in the colony-forming cells, since cells from normal littermate mice formcolonies in the spleens of unirradiated mice of genotype WW. Use of animals of genotype WVVWT as recipients improves the spleen-colony method by removing bias resulting fromthe death of irradiated recipients.

Selection and characterization of eight phenotypically distinct lines of lectin-resistant chinese hamster ovary cells

Abstract Clones resistant to the lectins phytohemagglutinin (PHA), wheat germ agglutinin (WGA), the agglutinin(s) from Lens culinaris (LCA), and ricin (RIC) have been selected from parental auxotrophic Chinese hamster ovary (CHO) cells. The sensitivity to other lectins of these cells and of CHO cells resistant to concanavalin A (ConA) has been determined, and their activity of UDP-N-acetyl-glucosamine glycoprotein N-acetyl-glucosaminyltransferase (GlcNAc-T) has been measured. At least 8 different phenotypes have been identified on the basis of this analysis, and complementation between 2 of them has been demonstrated.

Genetics and physiology of defective lysogeny in K12 (λ): Studies of early mutants

Abstract The properties of a number of strains of K12 (λ) which are defective in their ability to form lambda DNA after induction have been examined by genetic, physiological, and biochemical tests. The strains fall into four classes, and include mutants in cistrons N, O, and P, previously identified by Campbell (1961), and a fourth new class, called x, in a region between C I and C II . Mutants in each of the classes can be distinguished genetically, by their relative ability to form the λ-exonuclease, and by their tendency to show curing of their prophage after induction. Mutants in the P and O cistrons form normal levels of λ-exonuclease, whereas mutants in the N cistron form very low levels, and mutants in the x region, relatively high levels of this enzyme. Curing is very efficient for mutants in the x region, less efficient for mutants in the O and P cistrons, and is seldom observed with mutants in the N cistron. These patterns of curing are reflected in the curves for UV survival of the strains carrying defective mutations in the various cistrons. The significance of these findings in respect to the early events in phage development are discussed.

DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression.

Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.

The morphogenesis of bacteriophage lambda: IV. Identification of gene products and control of the expression of the morphogenetic information

Abstract The synthesis of λ-coded proteins has been examined in infected cells in which host protein synthesis was depressed by previous irradiation with uv light. Labeled proteins were analyzed by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate, and the pattern of radioactive bands was visualized by autoradiography of dried longitudinal gel slices. More than 30 viral proteins were identified; 15 of these were assigned to cistrons in the morphogenetic region, three to cistrons in the b region (or silent region) and three were under control of the early genetic region between the att site and cistron N. Among the 15 late proteins assigned to cistrons, nine were structural components of the phage particle. A kinetic analysis of the amounts of gene products, as measured by microdensitometry, indicated that the synthesis of individual late proteins in phage λ seems to be initiated in an orderly fashion, related to the distance of the genes from the late main promoter between genes Q and S. Absence of gene Q product resulted in a sevenfold depression in the synthesis of late proteins, including the late proteins of the b region. Only three polar effects were demonstrated in the left arm. These included the group of cistrons EF, VGTH and LK. The number of copies of gene products synthesized by the different genes in the morphogenetic region was not related in a simple way to the distance of the genes from the main late promoter and may vary by up to a factor of 870. This indicates the presence of a remarkable control mechanism for gene expression of the left arm. Evidence has also been obtained for the conversion of one late protein during λ development, probably by proteolytic cleavage. Measurement of the molecular weights of the assigned late proteins has allowed the construction of a composite genetic, physical and molecular map of the left arm of phage λ. On this basis it is predicted that the gene products of the genes for which no corresponding electrophoretic band has yet been found will be of small molecular weight.

Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells

Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected for resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mix-resistant cells 5–10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mix-resistant phenotype when cultured under nonselectivve conditions.

Complementation between mutants of CHO cells resistant to a variety of plant lectins.

AbstractChinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8–10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (

Mutations in bacteriophage lambda affecting host cell lysis.

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