Single-step Selection Research Articles

Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase.

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [3H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.

Cell fusion-impaired variants of a mouse lymphocytic cell line obtained by single-step selection for polyethylene glycol resistance

This report describes an effective method for single-step isolation of cell line variants with reduced intercellular plasma membrane fusion ability. Variants, derived from a subline of L5178Y, were obtained by selection for growth in the presence of a toxic level of PEG 6000. These variants occurred after EMS treatment and spontaneously. Further testing revealed a pleiotropic phenotype, fusion impairment and polyethylene glycol resistance, which has been designated PgfR. Variant sublines, in serial subculture for more than 165 days on nonselective medium, retain a stable PgfR phenotype. PgfR variants have an approximate 7-fold reduction in intercellular fusion competence.

Characteristics of concanavalin A-resistant Chinese hamster ovary cells and certain revertants.

Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes.

Single-step selection of clones of a mouse hepatoma line deficient in aryl hydrocarbon hydroxylase.

The mouse hepatoma line Hepa-1 has high and inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase[benzo[a]pyrene, reduced-flavoprotein:oxygen oxidoreductase (3-hydroxylating), EC 1.14.14.2] activity. Fourteen subclones of a clonal derivative of Hepa-1 were isolated and shown not to display heterogeneity in aryl hydrocarbon hydroxylase activity beyond what could be ascribed to experimental error. Hepa-1 was found to be very sensitive to benzo[a]pyrene (BaP) toxicity and a single-step selection procedure for isolating clones resistant to BaP at 4 microgram/ml was designed. Those BaP-resistant variants tested had much reduced aryl hydrocarbon hydroxylase activities under both inducing and noninducing conditions and they retained their resistance to BaP and low aryl hydrocarbon hydroxylase activities over considerable periods of time of culture in the absence of BaP. The spontaneous rate of origin of the BaP-resistant clones was estimated, by Luria-Delbrück fluctuation analysis, to be 2 X 10(-7) events per cell per generation. It is concluded that the properties of the variants are consistent with the notion that they are mutational in origin and that this system is a promising one for carrying out a somatic cell genetic analysis of aryl hydrocarbon hydroxylase.

Isolation of mutant mammalian cells altered in polyamine transport.

Chinese hamster ovary and rat myoblast cells resistant to the toxic action of methylglyoxal bis guanylhydrazone (MGBG), an antimitotic agent and inhibitor of polyamine synthesis, have been isolated by single step selection. Mutagenesis with ethyl methanesulfonate increases the recovery of the variants at least 30-fold. Intracellular accumulation of MGBG is greatly reduced in resistant cells. This property is accompanied by a 99% decrease in the uptake of all three naturally occurring polyamines. Both the resistant phenotype and the defect in polyamine transport behave recessively in somatic cell hybrids.

Selection and preliminary characterization of cycloleucine-resistant CHO cells affected in methionine metabolism.

Cycloleucine is in vivo a potent inhibitor of S-adenosylmethionine (SAM) biosynthesis and subsequent methylation reactions in somatic mammalian cells. Cycloleucine-resistant (CLr) clones were isolated from CHO cells by single-step selection. Their phenotype was stable when they were grown in the absence of drug. These clones appeared randomly in cultures at the frequency of 5 x 10(-6)/cell/generation, as determined by a fluctuation test. EMS mutagenesis did not significantly increase this frequency. The cycloleucine-resistant phenotype was codominant in intraspecific hybrids. Cycloleucine-resistant clones showed increased SAM pools; on the contrary, methionine pools were not significantly affected in these clones when compared to the wild-type cells. This increased SAM production was correlated with an increase of methionine adenosyltransferase (MAT) SPECIFIC ACTIVITY IN RESISTANT CLONES UNDER VARIOUS GROWm. The mechanism of posttranscriptional control of MAT biosynthesis was not affected in the cycloleucine-resistant clones nor were the kinetic properties of the enzyme modified. The genetic or epigenetic origin of this resistance mechanism is discussed.

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-2Kb or anti-H-2Kd 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-2Kb and those that had lost both H-2Kb and the linked H-2Db. Selection against H-2Kd yielded, exclusively, variants that had lost both the selected antigen and the linked H-2Dd. 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.

Inhibition of the growth of mammalian cells in cuture by amino acids and the isolation and characterization of L-phenylalanine transport.

Raising the concentration of phenylalanine and other amino acids in MEM leads to the inhibition of growth and in some cases to death of A9. Balb 3T3 , SV40 Balb 3T3 (SVT2), CHO, and WI38. All cells tested exhibited some similar senstivities to certain of the amino acids. but there were some unique differences. Phenylalanine-resistant mutants (Pher) of A9 were isolated that had modified phenylalanine-transport properties. These mutants can be isolated by a single-step selection procedure. A Lineweaver-Burk plot of initial rates of phenylalanine uptake by A9 and mutants showed a biphasic curve suggesting two transport systems. The Pher mutants had altered properties of both systems. It is suggested that the selection of clones resistant to high concentration of several of the natural amino acid may be used as a general method for the isolation of mutants affecting the various amino acid transport systems in mammalian cells.

Galactokinase mutants of Chinese hamster somatic cells resistant to 2-deoxygalactose.

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.

Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro.

Clonal isolates of mouse 3T3 cells and primary rat embryo cells, recovered nonselectively after infection by simian virus 40 (SV40), have been tested for tumorigenicity in the immune-deficient nude mice in order to determine the cellular growth properties in vitro specifically correlated with neoplastic growth in vivo. In addition, mouse 3T3 cells transformed by murine sarcoma virus (MuSV, Kirsten strain), and revertants isolated from cells fully transformed by either SV40 or MuSV were also studied. Results suggest that the single cellular property consistently associated with tumorigenicity in nude mice is the acquisition by virus-transformed cells of the ability to proliferate in vitro in the absence of anchorage. Other cellular parameters of virus-induced transformation, such as lack of sensitivity to high cell density and the capacity to grow in low serum concentration, are dissociable from cellular tumorigeneicity. This conclusion is supported further by the demonstration that specific selection in vivo for tumorigenic cells from anchorage-dependent cells results in the isolation of anchorage-independent cells. Conversely, a single-step selection in vitro for anchorage-independent cells from nontumorigenic cells results in a simultaneous selection of highly tumorigenic subclones.

Studies on 1-β- d-arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts : II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK −) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK + cells. Plating of dCK − cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK − hamster line and TK − lines of either mouse or hamster could be isolated in a modified HAT medium (HAT 50dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.

Ouabain-resistant mutants of mouse and hamster cells in culture

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 42K 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.

Derivation of TK- clones from revertant TK+ mammalian cells.

In order to obtain a large collection of Chinese hamster cell clones defective in thymidine kinase (TK(-)), BrdU(r) selection experiments have been performed on wild-type and revertant TK(+) cell lines. No clones (< 10(-9)) were obtained from the wild-type TK(+) cell line by single-step selection. In contrast, revertant TK(+) clones readily gave rise to stable TK(-) derivatives (1 - 2 x 10(-4)). Both wild-type and revertant TK(+) clones spontaneously yielded 8-AG(r) colonies with the same frequency (1 - 5 x 10(-6)), suggesting that the differences between wild-type and revertant cell lines specifically affected selection of the TK(-) phenotype. The increased frequency of TK(-) clones reflects perhaps the number (ploidy) or character of the autosomal TK loci in TK(+) revertants, or perhaps the mechanisms which regulate expression of the TK genes. Several mutagens, EMS, MNNG and UV, stimulated the TK(+) revertants' frequency of TK(-) subclones only slightly (< 3-fold). Biochemical and genetic data indicated that the TK(-) clones derived from one revertant are phenotypically different. The phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.

Transduction with phage PI in Salmonella typhimurium.

Two types of mutants of Salmonella typhimurium LT-2 with increased recipient abilities were isolated by treatment with N-methyl-N′-nitro-N-nitrosoguanidine followed by the transfer of an R factor from Escherichia coli K-12 and its subsequent elimination with acriflavine1. One of them, which was obtained in a single-step selection, was named Rfer1 (fer = fertile) and the other type isolated from Rfer1 was called Rfer2. Rfer1 mutants showed considerably higher recipient abilities than the wild type (Rinfer) (infer = infertile), and Rfer2 mutants acted as better recipients than Rfer1. Rfer1 mutants were considered unrestricted and unmodified and Rfer2 strains were assumed to have mutations of some properties of the cell surface in addition to the Rfer1 mutations because of the rough colonial morphology of Rfer2 mutants. In fact, Rfer2 mutants were found to be sensitive to a number of coliphages, including P1 to which the wild type and Rfer1 strains are resistant. Thus transduction with phage P1 has become possible in S. typhimurium and between E. coli and S. typhimurium.

Streptomycinに可逆的に依存する結核菌株の存在について

Streptomycin-independent strains were isolated in vitro by a single step selection from a large cell population of a streptomycin-dependent strain of the tubercle bacillus designated as 18 b. These strains are not the same as the streptomycin-sensitive ancestor strain H2, but they can easily adapt themselves to higher concentrations of streptomycin. This adaptation is accelerated when Fe and pepton are added to growing environment or the oxygen pressure is lowered in the medium. Glycerol egg media are the far more favourable condition than Kirchner's agar slants for the adaptation. When caffein, 8-azaguanine or acriflavine is incorporated in sub-inhibitory concentrations into streptomycin-containing media, the adaptation is strongly or almost completely inhibited. Reversely, when those streptomycin-independent strains have once completed their adaptation to streptomycin, such purines inhibit their adaptation to streptomycinfree environment. In other words, the streptomycin-independent strains grown on streptomycin-containing media do not show any significant growth when subcultured onto the media free from streptomycin but added with the purines. The strains show a diauxie growth in response to added streptomycin when observation is made of the homogeneous growth in Tween-albumin medium. All these findings can be explained on an assumption that streptomycin-independent strains are neither streptomycin-sensitive norresistant, but they are reversely streptomycin-dependent. In response to the environmental conditions with or without streptomycin, those strains can make phenotypic adaptation expressing one of the two metabolic potentialities, nemely dependent or non-dependent to streptomycin. Caffein or 8-azaguanine can be considered to inhibit this phenotypic adaptation. The present observation are also discussed from the possibility that streptomycin-independent strains are a suppressor mutant.

A modified technique for isolation of bacteriophage from contaminated materials.

This comn~unication describes a modification of well-liuown techniques for the isolation of bacteriophages, fiilethods usually depend on the culturing of given host bacteria with source material, e.g. soil (3), sewage (2), or plant residues (5), in which the presence of phage is suspected. One expects phage particles present to multiply a t the expense of the added host cells so that single plaque isolates may readily be obtained. Such mass-selection methods are appropriate when the aim is to obtain a virulent phage for diagnostic (5) purposes. There exists the possibility that other phages might not be detected if they were, for example, of low virulence, slow to absorb to the host, small burst size, long latent period, or lysogenizing rather than lytic. We tried to detect temperate phage in samples of arable soils by a modification of the massselection technique involving (a) limited opportunities for multiplication of infected bacteria or of already-present phages in the enrichment culture and (b) antibiotic-resistant host bacteria together with appropriate antibiotics to reduce microbial contamination which might otherwise obscure rare plaques in soft agar layer plates (1). One strain each of Rhizobiz~m trifolii (RT2), R. meliloti (RM6), R. leguminosarz~m (RLS), R. plznseoli (RP2), and Xantlzomonas phaseoli (XPS) was used. Medium Y (6) was emploj~ed for the growth of Rhizobiz~wz and antibiotic medium 3 (Difco) for Xantlzomonas. Mutants resistant to 2000 pg/ml of streptomycin (calcium chloride conlplex, fiiIerk) were obtained by singlestep selection on appropriate solid media containing streptomycin. Soil samples were obtained from the legume-crop nursery plots, Central Experimental Farm, Ottawa, and stored in screw-cap jars. In experiments, about 50 g of field soil were enriched with a total of approximately 5 X lo9 bacteria, of one of the mutant strains mentioned above, suspended in 5 ml of antibioticcontaining liquid medium (500 pg/ml streptomycin). This mixture of soil with host bacteria, hereafter referred to as the enriched soil, was incubated for 16 hours to allow absorption and limited multiplication of soil-borne phages. Since we have never found phage titers greater than 105/g, and often much less, after enrichment, we assume that the number of cycles of phage infection, liberation, and reinfection of phage-sensitive cells is probably small. After 16 hours a t room temperature (approximately 2S0 C) 2 g of the enriched soil were suspended in 10 ml liquid medium for 2 hours. Particles of soil and vegetable debris were then sedimented by low-speed centrifugation and the supernatant tested for the presence of phage by plating 0.1-ml and 0.5-ml samples in soft agar layer (1) together with a culture of the appropriate