Isolation of mutants of CHO cells resistant to 6(p-hydroxyphenylazo)-uracil I. A novel BrdU cross-resistant phenotype

Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPU ^R A exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.     

Action of 6-(p-hydroxyphenylazo)-uracil on bacteriophage IG 1

Summary IG 1, a temperate phage ofBacillus subtilis, is strongly induced from its lysogens by 6-(p-hydroxyphenylazo)-uracil, an azopyrimidine known as selectively inhibiting theB. subtilis DNA polymerase III. IG 1 phages originated either by induction or infection multiply, abundantly, in the presence of that azopyrimidine, in spite of the drastic decline of cell viability. Chloramphenicol completely suppresses the induction effect and also blocks the formation of spontaneously induced phage particles.     

Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP

From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growthinhibitory effects of 8-Brcyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased K_a for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased K_a for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.     

Mutants of CHO cells resistant to the protein synthesis inhibitor emetine: Genetic and biochemical characterization of second-step mutants

Second-step mutants highly resistant to the protein synthesis inhibitor emetine (Emt^RII) have been selected from emetine resistant (Emt^RI) Chinese hamster ovary cells described earlier. The frequency of the Emt^RII mutants was increased 50- to 75-fold after mutagenesis, and none of these highly resistant mutants could be selected in one step using wild-type cells. Like the Emt^RI mutants, the increased resistance of Emt^RII mutants results from another lesion in the polyribosomal fraction, as measured by the effects of emetine in fractionated extracts. As with the first-step mutants, the Emt^RII isolates behave recessively in somatic cell hybrids. Segregation studies have shown that the Emt^R lesions are not on the X chromosome, and in at least one isolate there is evidence that the Emt^RI and Emt^RII mutations may occur at different sites.     

Lectin-resistant CHO cells: Selection of seven new mutants resistant to ricin

In attempts to isolate new CHO glycosylation mutants, selection protocols using plant lectins that bind galactose residues of cell surface carbohydrates were applied to mutagenized CHO populations. The lectins were used alone or in combination to obtain seven ricin-resistant phenotypes. Each mutant had distinctive properties compared with previously described ricin-resistant CHO cells. One of the new phenotypes was dominant in somatic cell hybrids, and the others were recessive. Complementation analyses between related lectinresistant (Lec^R) phenotypes indicated that each new isolate represented a novel genotype. Five of the mutants had properties typical of new CHO glycosylation mutants. The remaining two mutants were not readily categorized. Although they did not appear to be ricin-internalization or protein-synthesis mutants, they also did not display the marked alterations in sensitivity to several lectins of different sugar specificity expected for glycosylation mutants. The seven new Lec^R mutants described in these studies brings the total number of different Lec^R CHO mutants isolated by this and other laboratories to about 40. Criteria for identifying new Lec^R mutations in CHO cells are discussed.     

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

Chinese 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 (Lec^R) mutants behave recessively in somatic cell hybrids. One ricin-resistant class ( behaves dominantly. Test for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complementation groups can be delineated among the phenotypically recessive mutants.     

Genetic and biochemical characterization of mutants of CHO cells resistant to the protein synthesis inhibitor trichodermin

Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Tri^r) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunit from Tri^r cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-¹⁴C]trichodermin. The lesion in Tri^r mutants therefore seems to have affected this ribosomal subunit. Tri^r × Tri^s hybrids are sensitive to trichodermin indicating that the Tri^r mutation behaves recessively to Tri^s in hybrids. The Emt^r and Tri^r markers segregate independently from hybrid cells showing that the Tri^r mutation is probably not linked to the Emt^r locus, which as we have shown earlier affects the 40S ribosomal subunit.     

Isolation and characterization of a CHO amino acid transport mutant resistant to melphalan (l-phenylalanine mustard)

A mutant of Chinese hamster ovary cells, CHY-2, was isolated on the basis of its reduced ability to grow on a limiting concentration of leucine and was found to be defective in uptake of leucine via the sodium-independent L system. Consistent with published reports that the L system can mediate melphalan uptake, the D₁₀ of the mutant for melphalan was increased threefold under conditions designed to limit drug uptake to the L system (brief exposure in sodium-free medium). Unlike a previously described melphalan-resistant CHO mutant (CH^r), CHY-2 displays no cross-resistance to colchicine or puromycin. It differs from a second melphalan-resistant CHO mutant, mel_r, in its sensitivity to melphalan in the presence of high Na⁺, and from a melphalan-resistant mouse leukemic cell in possessing normal levels of intracellular glutathione. Thus, CHY-2 represents a new melphalan-resistant mutant class. The effect of the CHY-2 mutation is pleiotropic, involving significant reductions in amino acid uptake via the L, A and Ly⁺ (but not ASC) systems. The primary defect is unknown; however, the mutant possesses normal intracellular concentrations of Na⁺ and K⁺ and normal membrane fluidity. The growth rate of the mutant in standard medium is greatly reduced (generation time of 60 h vs. 24 h), although it can be improved by the addition of a supplement containing high concentrations of leucine, proline, and peptides.     

Isolation of mutants of Candida glabrata resistant to miconazole

Elucidation of the mode of action of azole antifungals would be aided by studying resistant mutants. It is difficult to obtain mutants of Candida albicans in the laboratory, and there have only been a few studies on clinical isolates which seem to be resistant because of impaired drug uptake. C. glabrata, unlike C. albicans, is haploid and more likely to give rise to resistant variants. Over 30 mutants have been isolated by selection with miconazole on solid medium and have MICs of miconazole about ten times that of the parental strain. One such mutant has a reduced growth rate and final cell yield. In intact cells, ergosterol biosynthesis is tenfold less sensitive to miconazole than in the parent. However, uptake of [3H]miconazole by cells is identical in both strains. The significance of these observations is discussed.     

Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes.

Attempts to isolate auxotrophic mutants of Legionella pneumophila have been hampered by the complex nutritional composition of the media used to cultivate this organism. We developed a semidefined medium, designated CAA, to facilitate the isolation and characterization of Legionella auxotrophs. Unlike previously described chemically defined media for this organism, L. pneumophila formed colonies on CAA agar. Using this medium, we isolated several independent tryptophan auxotrophs of strain Philadelphia-1 after ethyl methanesulfonate mutagenesis and penicillin enrichment. Trimethoprim selection was used to isolate several independent thymidine-requiring mutants of the same strain. The thymidine auxotrophs exhibited a marked decrease in viability when they were deprived of thymidine. The results of monocyte infection experiments with both the tryptophan and thymidine auxotrophs indicated that the thymidine auxotrophs were incapable of intracellular survival or multiplication. In contrast, the tryptophan auxotrophs grew well in monocyte cultures. The isolation of additional auxotrophic mutants will facilitate the study of the nutritional requirements of L. pneumophila for growth in human mononuclear phagocytes.     

Isolation and characterization of Entamoeba histolytica mutants resistant to emetine

Isolation of emetine-resistant mutants of Entamoeba histolytica is described. Spontaneous rate of mutation, obtained from a Luria-Delbrück fluctuation test was 2.5 X 10(-7). The mutagenesis frequency obtained from the number of colonies grown in the presence of emetine, divided by the number of viable trophozoites inoculated in semisolid agar was less than or equal to 10(-7) and it was increased from 10 to 100 fold with ethyl methanesulfonate. Two types of clones were isolated: one was cross-resistant to colchicine and the other was not, indicating that mutation took place at different loci. Protein synthesis in the presence of emetine remained unaltered for colchicine-sensitive mutants, suggesting that the molecular lesion lay in the protein synthesis machinery. Colchicine-resistant mutants showed a lower level of [35S]methionine incorporation, probably due to alterations in the molecular transport of some substances across the membrane. This mutant clone was also deficient in red blood cell adhesion, suggesting membrane alterations.     

Isolation and characterization of spontaneously arising auxotrophic and Kanagawa phenomenon-negative mutants of Vibrio parahaemolyticus.

As a first step toward developing a system of genetic exchange between Vibrio parahaemolyticus strains, spontaneously arising auxotrophic and Kanagawa phenomenon-negative (KP-) mutants were isolated and characterized. Auxotrophic mutants were selected by nalidixic acid enrichment of parental cultures. Some Cys- and Arg- mutants of a KP+ strain were found to be KP-. Reversion to prototrophy by these strains was not accompanied by a return to the parental KP+ phenotype. Additionally, two prototrophic KP- mutants were isolated. No detectable levels of vibriolysin were found in supernatant extracts of KP- mutants by slide gel immunodiffusion analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or assay for lethal activity in mice. All Cys-, Arg-, and Pur- mutants tested reverted to a different auxotrophy (phenotypic interconversion) as well as to prototrophy. The possible role of insertion sequence-like elements in vibriolysin production and phenotypic interconversion is discussed.     

Use of cis-Pt(II)-uracil for electron microscopic cytochemistry of rat brain nucleic acids.

Cis-Pt(II)-uracil staining reveals nucleic acids in a relatively specific manner when applied alone for 1 h to rat brain tissue specimens. Poor contrast and resolution are observed when glutaraldehyde-fixed and epoxyembedded thin sections are post-stained with cis-Pt(II)-uracil alone. Counterstaining thin sections with uranyl acetate decreases staining specificity by revealing many tissue proteins. Synapses did not stain, which suggests that they do not contain significant amounts of large RNA molecules. Technical procedures must be carefully regulated to avoid artifactual staining of cellular components other than nucleic acids.     

Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts

Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fom^r) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10^–6. Cross-resistance studies with these mutants show that the Fom^r mutants exhibit increased resistance to all adenosine analogs (N- and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fom^r mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N⁷-(²-isopentenyl) formycin A for similar inhibition of [³H]adenosine phosphorylation. These results indicate that AK from the Fom^r mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fom^r mutants may thus be directly affecting the structural gene for AK.     

A screening method for isolating DNA repair-deficient mutants of CHO cells

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV), ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D₃₇ dose of 1.0 J/m² compared to 7.0 J/m² for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m². Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of⁶⁰Co -rays.Notice: This report was prepared as an account of work sponsored by the United States Government. Neither the United States nor the United States Department of Energy, nor any of their employees, nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights.     

Isolation and characterization of mutants of Trichosporon adeninovorans resistant to 2-deoxy-D-glucose

We isolated stable mutants from Trichosporon adeninovorans resistant to 2-deoxy-D-glucose. All mutants were altered in the C-catabolite repression and produced different amounts of glucoamylase and β-glucosidase when grown on several carbon sources. In addition to the C-catabolite derepression of these extracellular enzymes, a repression defect of isocitrate lyase was found.     

Isolation and properties of mutants of Streptomyces noursei resistant to benzyl alcohol

Stable mutants resitant to benzyl alcohol (= Bal^r) were selected from natural colony populations of several strains of S. noursei JA 3890 b. Irrespective of the absence of aerial mycelium formation the majority of the Bal^r mutants produced the same amount of nourseothricin as their parental strains. The Bal^r derivative 2/8 showed a higher production of biomass and lipids than its ancestor BF32 under standard conditions of growth. In the presence of increasing concentrations of benzyl alcohol in the medium (0–0.2%), growth of this Bal^r strain was more resistant to the toxic agent, and the lipid content dropped more slowly concomitant with aberrant alterations of the composition of fatty acids. The results suggest that Bal^r strains represent regulatory mutants whose occurrence in natural populations has been caused either by changes at the level of pleiotropic gene functions or by several, independent genetic alterations.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to T-2 toxin

Summary T-2 toxin, a trichothecene mycotoxin, inhibits the growth of Saccharomyces cerevisiae. We have isolated nine spontaneous S. cerevisiae mutants resistant to this toxin. The mutants were distinguished from the wild type according to their degree of resistance to T-2 toxin on media with dextrose or glycerol as the carbon source. Generation time, mutation stability and level of cross-resistance to roridin A, another trichothecene, were determined for each mutant. The T-2 toxin resistant mutants were further characterized by subsequent tests involving cross-resistance and collateral sensitivtiy to chlorampenicol, neomycin, paromomycin, ethidium bromide and thiolutin. Mutants have been placed into three subgroups and the mechanism of T-2 toxin resistance in each group has been postulated. Mutant HK1 is the first S. cerevisiae isolate resistant to roridin A. One particular isolate, mutant HK1 l , carries a single recessive nuclear mutation. This mutation was termed ttt (for T-2 toxin resistant).     

Bacteriophage interference in Streptococcus pyogenes. II. A25 mutants resistant to prophage-medicated interference.

On the basis of the host ranges of the interference-resistant A25 mutants h1 and h2, which recombine with high frequency, five group A streptococcal strains carrying prophages interfering with wild-type A25 can be subdivided into three groups, a classification which is consistent with that made previously on a different basis [Behnke, D., and Malke, H. (1978) Virology85, 118–128]. Mixed infection of appropriate lysogens with the two A25 mutants does not result in either rescue of the inhibited phage or suppression of the resistant one, suggesting that A25 does not form diffusible products which are involved in the interference process. Mutation h2 causing resistance to interference mediated by prophages P6240, P13234mo, and R58/14820 increases the sensitivity of A25 to P5004 interference and suppresses h1, which when present alone renders A25 resistant to the latter mechanism. Since A25h2 is still inhibited on an interference-deficient P5004-lysogenic strain which supports growth of A25, it is possible that P5004 determines two distinct interference mechanisms, one acting on A25 and the other on A25h2. Prophage-mediated interference and classical phage restriction/ modification are found to act side by side in strain 5004, the natural host of P5004.     

Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

The dihydrofolate (FH₂) reductase specified by the T4 frd gene (previously called the wh gene) is apparently nonessential for phage growth on Escherichia coli because the bacterial FH₂ reductase can partially substitute for the phage enzyme. To study the effect in vivo of folate analogs which specifically inhibit the T4 FH₂ reductase, the enzyme must be made essential for phage production. Partial inhibition of the E. coli FH₂ reductase with the folate analog trimethoprim strongly inhibits phage production by a T4 frd mutant and slightly inhibits wild-type phage production. Adding an additional T4-specific folate analog, a chlorophenyl triazine, strongly inhibits wild-type phage production without preventing the uninfected E. coli cells from multiplying.We have isolated spontaneous mutants of T4 which are capable of producing progeny in the presence of chlorophenyl triazine and another folate analog pyrimethamine. These mutants are designated folate analog resistant (far) and have been separated into two general classes. Class I mutants induce T4-specific FH₂ reductases which are less sensitive to the action of the folate analogs than the normal FH₂ reductase. Class II mutants induce an unaltered FH₂ reductase but contain mutations in a variety of T4 genes. Some class II mutants induce more phage FH₂ reductase activity than wild-type T4 and appear to be regulatory mutants.