Genetic analysis of lon mutants of strain K-12 of Escherichia coli

Summary Following UV irradiation of AB1157 31 mucoid ultraviolet light UV sensitive mutants were isolated. These were all induced to form filaments by UV irradiation, i.e. they had all the phenotypic properties of Lon mutants. These lon mutants fell into two phenotypic classes based on their sensitivity to UV. The gene determining UV sensitivity and mucoidy in all mutants of both Class A and Class B was cotransducible with proC. Intra-class crosses by Pl transduction yielded no UV resistant recombinants. Inter-class crosses yielded UV resistant nonmucoid recombinants, the frequency depending on the direction of the cross. The data imply two adjacent blocks in the lon region of E. coli and the order of markers in this region is probably proC tsx lon Class A lon purE Class B.This work was carried out under Public Health Service Grant CA 05687-08 from the National Cancer Institute.Recipient of a Public Health Service Career Development Award.     

Complementation between uvr mutants of Streptococcus pyogenes

Summary From the group A streptococcal strains K 56 and 56 188, respectively 13 and 6 nitrosoguanidine-induced uvr mutants were isolated and used in complementation experiments employing strain 56 188 and its derivatives as donors in phage A 25-mediated heterologous transductions. When A 25 propagated on wild type or on 2 of the six 56 188-derived uvr mutants was used to infect 4 of the uvr recipient strains, a substantial increase in survivors of UV irradiation was found over those observed in selfing experiments or control experiments without phage. Less than 1% of the UV survivors had stably integrated the uvr ⁺ allele. The remaining 4 uvr donors failed to complement the 4 above-mentioned recipients, indicating that the strains in question fell into 2 complementation groups.Nine of the 13 K 56-derived mutants, which in contrast to the others were characterized by non-reversibility to UV resistance, did not even show an increase in UV survivors when infected with phage grown on wild type. The possibility is discussed that these strains might carry second mutations affecting UV sensitivity which, however, did not appear to be of the rec type.     

Rescue by chloramphenicol and rifampicin of UV-irradiated lon, exrA and recA mutants of Escherichia coli K12

The effect of temporary treatment with chloramphenicol or rifampicin on the survival of UV irradiated cells of selected Escherichia coli K12 radiation sensitive mutants was examined. Increased survival resulted for both exrA and recA mutants, and also for the unsuppressed lon mutant, but cells of the parent strain and the recB mutant were not rescued. This contrasts with our earlier finding that after exposure of the bacteria to γ-rays, chloramphenicol treatment rescued the exrA and lon mutants but not the recA mutant. We now report that an exrA recA double mutant was rescued by chlramphenicol after UV radiation, but not after anaerobic ionizing radiation. Inclusion of inhibitors of uvrA governed repair, caffeine and 8-methoxypsoralen (8-MOP), in the incubation medium containing chloramphenicol, did not reduce the rescue of the exrA or recA mutants, although caffeine eliminated rescue of the lon mutant, which was itself unaffected by 8-MOP. However it is concluded that chlormaphenicol rescue of the exrA and recA mutants after UV radiation was not entirely independent of the excision-repair process, since the uvrA recA and uvrA exrA double mutants were not rescued by this treatment.     

The utilization of paramecia by the carnivorous plant Utricularia gibba

Summary A method has been devised to ensure capture of large numbers of live paramecia within a short period of time under controlled conditions by the bladders of Utricularia gibba. The method permits a direct evaluation of the role of entrapped animals in the nutrition of this carnivorous plant. Paramecia captured by the bladders of plants growing in a near optimal inorganic medium do not cause an increase in number or length of internodes. In contrast, feeding paramecia to plants grown in a poorly balanced or incomplete medium does result in an increase in both number and length of internodes produced. Feeding paramecia to Utricularia also results in an increase in number of bladders.This study was supported in part by an Undergraduate Research Participant stipend from Public Health Service, grant number 2-TIHE 5 303-09, to the first author and in part by Agricultural Research Service, U.S. Department of Agriculture, Grant No. 12-14-100-7981 (34) to the second author, administered by Crops Research Division, Beltsville, Maryland.     

Studies on the xanthine oxidase activity of mammalian cells

Xanthine oxidase in man is confined to but a few tissues and is absent from cultured cell strains. In rodents, however, the enzyme is more widely distributed among the tissues and can be demonstrated in most cell lines. Rodents possess the enzyme uricase and are therefore able to carry purine catabolism one step further than man. Preliminary results suggest that uricase is restricted to but a few rodent tissues and is absent from cultured rodent cells. Hence it may be that in each species only the final enzyme of purine catabolism is tissue restricted. In other experiments, mammalian cells were grown in the presence of compounds known to induce xanthine oxidase in a eukaryotic fungus (Aspergillus nidulans). These compounds did not induce the enzyme in mammalian cells.Supported by program project grants 1-PO-GM 15419 and GM 18153-01, National Institutes of Health, United States Public Health Service.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

Phosphoglucomutase electrophoretic variants in the mouse

The phosphoglucomutase (PGM) electrophoretic phenotype of the mouse (Mus musculus) consists of several distinct components which can be grouped into two major zones designated PGM-1 and PGM-2. Evidence presented here indicates that each zone is controlled by a single genetic locus denoted Pgm-1 and Pgm-2, respectively. Two variant forms segregated at the Pgm-1 locus. They were codominantly expressed and inherited as alleles at an autosomal locus. The alleles were termed Pgm-1 ^a (fast) and Pgm-1 ^b (slow). These alleles were separately fixed in a number of inbred strains of mice. Preliminary evidence based on wild mouse phenotypes indicates that variant forms also exist for PGM-2 which are inherited as alleles at an autosomal locus. Genetic linkage relationships have not been determined for these loci. PGM-1 variants and PGM-2 were expressed in mouse fibroblasts in vitro.Supported by U.S. Public Health Service grants GM-09966 and GM-07249 from General Medical Sciences and 5 F2 HD-35,531 from Child Health and Human Development; and Atomic Energy Commission contract AT(30-1)-3671.Postdoctoral Fellow of the U.S. Public Health Service.     

Metabolic suppressors of a regulatory mutant in Neurospora

The synthesis of a number of enzymes of sulfur assimilation in Neurospora crassa is controlled by the sulfur source. Mutants in one regulatory gene, cys-3, are unable to make any of the enzymes. This locus is thought to specify a macromolecule that is required for the expression of the structural genes. A mutant, scon ^c, of another regulatory gene is nonrepressible for the synthesis of the enzymes. We report here the isolation of suppressed scon ^c strains which are actually the double mutant, scon ^c,cys-3. These strains are phenotypically indistinguishable from single mutants at the cys-3 locus. Thus cys-3 is epistatic to scon ^c. Evidence that the expression of the cys-3 gene is itself controlled is also presented.This work is supported by a Public Health Service grant, GM-08995, from the National Institute of General Medical Sciences. One of us (R.L.M.) is supported by a Public Health Service Career Development Award.     

Glucose-induced resistance to methyl methanesulfonate in Escherichia coli

Summary The sensitivities to inactivation by methyl methanesulfonate (MMS) of repairproficient and deficient strains of Escherichia coli K-12 and B grown to the stationary phase in nutrient broth (NB) or in glucose-enriched nutrient broth (GNB) have been compared. GNB-grown Rec⁺ and B/r cells are much more resistant to MMS at low exposures than are such cells grown in NB. Rec^– cells, whether Uvr⁺ or Uvr^–, do not exhibit this glucose-induced resistance (GIR). Strains B_s-1 and B_II also do not exhibit GIR. Caffeine added to the posttreatment plating agar at non-lethal concentrations abolishes GIR. It is suggested that growth in GNB enhances, at low exposure, the type of repair controlled by the rec genes.Supported in part by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. This is report No. COO-1686-20.Supported in part by the United States Public Health Service Training Grants No.'s 1 RH 00080-03 and T01 EC 00053.     

Genetic variation in mouse salivary amylase rate of synthesis

Heterozygotes from matings of the mouse strains YBR/Cv and C3H/As have about 3 times more YBR-amylase than C3H-amylase in the saliva. The determinant for this quantitative effect is located on linkage group XVI close to or within the structural gene for salivary amylase. The quantitative effect is the result of an increase in the rate of synthesis of YBR-amylase, and the determinant is cis acting. Studies of other mouse strains suggest that regulatory genetic elements may modulate salivary amylase production.This work was supported by the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521).     

Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data

Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

Probability that two diffusing molecules will collide

A formula is derived for the probabilityP(r ₁) that two molecules, originally at a distancer ₁ from each other and moving in an infinite scattering and absorbing medium, will eventually collide with each other. Public Health Service Research Fellow of the National Cancer Institute.     

Genetics of human-mouse somatic cell hybrids: Linkage of human genes for isocitrate dehydrogenase and malate dehydrogenase

Based on somatic cell genetic analysis, autosomal gene linkage is reported for the supernatant enzymes of human isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) in human-mouse cell hybrids. The IDH, MDH linkage was not linked to the X and E ₁₇ chromosomes or to 12 additional human enzyme markers.This work was supported in part by grants from the U.S. Public Health Service (Child Health and Human Development) and the United Health Foundation of Western New York.     

Thermal Resistance of Salmonellae Isolated from Dry Milk

Salmonella anatum, S. binza, S. cubana, S. meleagridis, S. newbrunswick, and S. tennessee isolated from dry milk, and S. senftenberg 775W were studied for heat resistance to determine whether these organisms would survive pasteurization as recommended by the 1965 Pasteurized Milk Ordinance of the U.S. Public Health Service. Thermal inactivation determinations were made on washed cells of the test microorganisms suspended in sterile whole milk. Excluding S. senftenberg, D values ranged from 3.6 to 5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C, and from 0.28 to 0.52 sec at 68.3 C. Corresponding values for S. senftenberg were 34.0, 10.0, 1.2, and 0.55 sec for respective exposure temperatures of 65.5, 68.3, 71.7, and 73.9 C. The present milk pasteurization processes as recommended by the Public Health Service will inactivate all seven strains of salmonellae studied, provided that the initial concentration does not exceed a calculated 3 × 10¹² salmonellae per ml of milk.     

The effect of cis-dichlorodiammineplatinum(II) on Escherichia coli B. The role of fil, exr and hcr markers

The effects of cis-dichlorodiammineplatinum(II) (cis-Pt(II)) upon the colony forming ability (c.f.a.) and growth of Escherichia coli B was studied with strains mutated in loci fil, hcr and exr.The growth in the presence of cis-Pt(II) remained unaffected for 2 h even in the most sensitive strains grown at concentrations that reduced the c.f.a. by five orders of magnitude. In the given set of mutants, the sensitivity of the c.f.a. to cis-Pt(II) went roughly parallel with the sensitivity to UV. However, the relative role of mutations in individual markers was different. Of the loci investigated here, the most important one for the survival measured by the ability to form colonies was the exr locus, whose mutation raised the sensitivity 13–23 times. The hcr locus affected the sensitivity by a factor of 2–5. The fil locus controlled cell elongation and the preservation of c.f.a. during growth in the presence of cis-Pt(II), although it had little effect on the survival of resting cells under our experimental conditions.We believe that the sensitivity to cis-Pt(II) depends to a great extent on the pleiotypic response of the cells.     

Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells

Summary The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification. This research was supported by Public Health Service grant AR 27130 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, and National Research Service postdoctoral fellowship ES 05336 from the National Institute of Environmental Health Sciences, Research Triangle Park, NC.     

Deficiency of dihydroxy acid dehydratase in the mito-chondria of the iv-1 mutants of Neurospora crassa

The isoleucine-valine requiring mutants of Neurospora crassa which map at the iv-1 locus lack, or have a very low level of activity for, the enzyme dihydroxy acid dehydratase in the mitochondrial fractions derived from them. This enzyme is, however, present in the soluble fractions of the mutant homogenates. The enzyme is present in both mitochondrial and soluble fractions from homogenates of wild-type and from homogenates of iv mutants blocked at other steps in the isoleucine-valine pathway.The work reported here was supported in part by grants GM 12323 and 5TO1-GM-00337-09 from the National Institutes of Health, United States Public Health Service, and by a grant from the Robert A. Welch Foundation.Recipient of Research Career Award 4-K-6-GM-18,383 from the National Institutes of Health, United States Public Health Service.     

Iron requirement for longevity ofPseudomonas cultures

Cultures of five out of six strains ofPseudomonas species grown in shaken flasks at 37 C needed tenfold more iron for longevity than for growth. At 30 C, the iron requirement was present in only two, and at 25 C in only one, of the six strains. The metal acted during early stationary phase and, to function, required protein synthesis. In a variety of microorganisms, qualitative and quantitative trace metal requirements for secondary metabolism and culture longevity are identical; we propose that successful completion of the former is a prerequisite of the latter.This work was supported in part by grant HD-03038-02 from the National Institute of Child Health and Human Development, U. S. Public Health Service.We express our appreciation to Miss Patsy Linn Person for performing preliminary experiments. A portion of the material was presented at the Annual Meeting of the American Society for Microbiology in Miami Beach, Florida, 4–9 May, 1969.