Showing papers on "Mutant published in 1972"

Phage P22-mutants with increased or decreased transduction abilities.

Abstract: The properties of mutants of the Salmonella phage P22 are described which show decreased or increased frequencies for generalized transduction. It is shown that not only the markers used for detection of the mutants are affected by the mutation but also other markers tested. Evidence is presented that the altered T/P-ratios reflect changes in the actual numbers of transducing particles and that other factors like integration in the recipient cells cannot account for these alterations. Quantitative estimations of the amount of bacterial DNA converted to transducing fragments are shown for several phage mutants. Difficulties in the isolation procedure are discussed.

Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon.

Abstract: Three temperature-sensitive (ts) mutants of simian virus 40 (SV40) in complementation group A (tsA7, tsA28, tsA30) have been isolated and characterized in permissive and restrictive host cells. At 41 C in the AH line of African green monkey kidney cells, the mutants are deficient in an early function required to produce infectious viral deoxyribonucleic acid (DNA). Temperature-shift experiments and analysis of SV40 viral DNA replication by gel electrophoresis have provided strong evidence that the ts gene product of the three mutants is directly required to initiate each new round of viral DNA replication but is not required to complete a cycle which has already begun. The synthesis of mutant DNA molecules themselves can be initiated by a nonmutant gene product in viral complementation studies at 41 C. The cell, however, cannot substitute a host function to provide the initiator required for the replication of free viral DNA. The viral initiator is also required to establish the stable transformation of 3T3 cells.

Mutation Rate and Dominance of Genes Affecting Viability in DROSOPHILA MELANOGASTER

Abstract: Spontaneous mutations were allowed to accumulate in a second chromosome that was transmitted only through heterozygous males for 40 generations. At 10-generation intervals the chromosomes were assayed for homozygous effects of the accumulated mutants. From the regression of homozygous viability on the number of generations of mutant accumulation and from the increase in genetic variance between replicate chromosomes it is possible to estimate the mutation rate and average effect of the individual mutants. Lethal mutations arose at a rate of 0.0060 per chromosome per generation. The mutants having small effects on viability are estimated to arise with a frequency at least 10 times as high as lethals, more likely 20 times as high, and possibly many more times as high if there is a large class of very nearly neutral mutations.—The dominance of such mutants was measured for chromosomes extracted from a natural population. This was determined from the regression of heterozygous viability on that of the sum of the two constituent homozygotes. The average dominance for minor viability genes in an equilibrium population was estimated to be 0.21. This is lower than the value for new mutants, as expected since those with the greatest heterozygous effect are most quickly eliminated from the population. That these mutants have a disproportionately large heterozygous effect on total fitness (as well as on the viability component thereof) is shown by the low ratio of the genetic load in equilibrium homozygotes to that of new mutant homozygotes.

Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping.

Abstract: SUMMARY: Non-smooth mutants of Salmonella typhimurium strain LT2 with known lipo-polysaccharide (LPS) defects were tested for sensitivity to smooth-specific phages (P22 and P22h and a newly isolated phage, 9NA, active on P22 lysogens); Felix O phage; and several rough-specific phages including C21. Smooth strains were sensitive only to the smooth-specific and Felix O phages. Six rfb mutants (unable to make O chains) were sensitive to Felix O and all the rough-specific phages except C21 (pattern R-sens). Of nine rfa mutants (presumed to have LPS core defects) four were R-sens, six were resistant to Felix O and some rough-specific phages (pattern R-res-1), and one was also resistant to phage Br2 (pattern R-res-2). The phage sensitivity of phosphomannoisomerase (pmi) mutants was the same as that of rfb mutants, except that they were partly sensitive to P22h. UDPgalactose-epimerase-negative mutants were sensitive to C21 and various rough-specific phages in-cluding Br2 (pattern Epi-i). An rfc mutant (unable to polymerize O repeat units) was sensitive only to Felix O and P221 (pattern Zsr). A part-rough mutant of class D (with abnormally few O chains) was incompletely resistant to smooth-specific phages, resistant to Felix O but sensitive to all rough-specific phages except C21 (pattern D-i). Spontaneous and mutagen-induced non-smooth mutants were isolated from lt2 strains with appropriate markers by selection with Felix O and/or P22 phage. (One parent strain used was non-lysogenic for Fels 2, for which lt2 wild-type is lysogenic. Lysogeny for Fels 2 did not affect sensitivity to the other phages.) Some mutants gave new sensitivity patterns. Mutants of these and of previously unmapped classes were crossed with smooth Hfr strains. The rfc loci of two mutants and pmi loci of two others were located in the gal-trp segment. Three mutants of pattern R-sens yielded O-specific hapten but mapped near his; they are believed to be unable to transfer O chains from antigen carrier lipid to the LPS core as a result of mutation at rfbT. Six mutants of pattern R-sens were smooth in cultural and serological properties; they mapped near his and are probably leaky rfb mutants. Many mutants had the class D part-rough phenotype, divisible by phage sensitivities into patterns D-1, D-2 and D-3. Mutants of all three classes mapped near xyl; they are likely to be rfa mutants, perhaps leaky, with LPS core defects which hinder but do not prevent attachment of O chains. Two classes were sensitive to C21 (WilkinsonS and rfaG mutants, resistant to Br2 (pattern Epi-2), unable to add the proximal glucose unit. Both loci mapped in or near the strA-xyl-metA segment. Several non-smooth mutants did not grow in the presence of bile-salts. Three mutants (rfaF) made LPS deficient of the distal heptose unit; one mutant (rfaE) was unable to add the proximal heptose unit. Both these loci mapped in or near the strA-metA segment.

Dominant Mutations (lex) in Escherichia coli K-12 Which Affect Radiation Sensitivity and Frequency of Ultraviolet Light-Induced Mutations

Abstract: Three mutations, denoted lex-1, -2 and -3, which increase the sensitivity of Escherichia coli K-12 to ultraviolet light (UV) and ionizing radiation, have been found by three-factor transduction crosses to be closely linked to uvrA on the E. coli K-12 linkage map. Strains bearing these mutations do not appear to be defective in genetic recombination although in some conjugational crosses they may fail to produce a normal yield of genetic recombinants depending upon the time of mating and the marker selected. The mutagenic activity of UV is decreased in the mutant strains. After irradiation with UV, cultures of the strains degrade their deoxyribonucleic acid at a high rate, similar to recA(-) mutant strains. Stable lex(+)/lec(-) heterozygotes are found to have the mutant radiation-sensitive phenotype of haploid lex(-) strains.

Conditional Lethality of recA and recB Derivatives of a Strain of Escherichia coli K-12 with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I

Abstract: We have isolated a strain of Escherichia coli K-12 carrying a mutation, polA12, that results in the synthesis of a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I. The double mutants polA12 recA56 and polA12 recB21, constructed at 30 C, are inviable at 42 C. About 90% of the cells of both double mutants die after 2 hr of incubation at 42 C. Both double mutants filament at 42 C and show a dependence on high cell density for growth at 30 C. In polA12 recB21 cells at 42 C, DNA and protein synthesis gradually stop in parallel. In polA12 recA56 cells, DNA synthesis continues for at least 1 hr at 42 C, and there is extensive DNA degradation. The results suggest that the primary lesion in these double mutants is not in DNA replication per se.

Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid.

Abstract: Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.

Selection and Preliminary Characterization of Temperature-Sensitive Mutants of Type 5 Adenovirus

Abstract: Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wild-type strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1%. All eight mutants had reversion frequencies of 10(-5) or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3- to a 3,000-fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25% of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).

Mutant tRNA His ineffective in repression and lacking two pseudouridine modifications

Abstract: TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons1–4. Histidyl-tRNAHis has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNAHis as the wild type4, and in the one example sequenced, contains tRNAHIS with a structure identical to that of the wild type8. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNAHIS in vitro2. The amounts of charged and uncharged tRNAHis present in vivo during physiological derepression of the wild type, and in the six classes of regulatory mutants, have been determined9. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNAHis in vivo, and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNAHis. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNAHis), and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNAHis which these mutants contain.

Mutations in R factors of Escherichia coli causing an increased number of R-factor copies per chromosome.

Abstract: A mutant of the repressed R factor R1a and two mutants of the derepressed R factor R1drd-19 showing a two- to fourfold increase in resistance to all of the antibiotics to which the wild-type R factors mediate resistance were studied. The increased resistance was due to a two- to fourfold increase in the number of R-factor copies per chromosome. The production of drug-metabolizing enzymes was linearly correlated to the gene dosage. There was also a linear correlation between resistance to the drugs and the production of the corresponding enzymes. The mutations were also expressed in Proteus mirabilis PM1. In Proteus, R factors are split into two plasmids, resistance transfer factor and the resistance part. The mutation in one of the mutant R factors seems to be located in the resistance part. A second fi+ R factor (R100) was introduced into strains already carrying R1drd-19 or the mutant R factor R1drd-19B2. In the first case, R100 and R1drd-19 segregated with equal probability when the bacteria were grown on antibiotic-free medium, whereas, in the second case, R100 was rapidly and preferentially excluded.

Absence of Laccase from Yellow-spored Mutants of Aspergillus nidulans

Abstract: SUMMARY: Green-spored strains of Aspergillus nidulans contain a p-diphenol oxidase (laccase) which is only formed during sporulation and is absent from yellow-spored mutants. The enzyme was assayed colorimetrically using N,N-dimethyl-p-phenylenediamine as substrate. Other substrates oxidized were pyrogallol, gallic acid and 2,6-dimethoxyphenol. Quinol, catechol and p-cresol were not detectably oxidized. Experiments with a temperature-sensitive yellow-spored mutant showed that the yA locus of A. nidulans is a structural gene for at least a component of the enzyme, while indirect evidence suggests that the enzyme also contains copper. Yellow-green-spored mutants of the ygA locus produce an inactive enzyme whose activity can be restored by dialysis against copper salts. Under certain conditions the enzyme itself can diffuse from a wild-type colony to a yellow-spored mutant colony and there act to give green spore pigment. From this it is deduced that the normal site of action of the enzyme may be external to the cell membrane. A similar enzyme has also been found in conidiating cultures of Aspergillus niger and Penicillium brevicompactum.

Reconstitution of Diphtheria Toxin from Two Nontoxic Cross-Reacting Mutant Proteins

Abstract: The isolation of a new type of mutant Corynephage beta, which carries a missense mutation in the structural gene for diphtheria toxin synthesis is described. The lysogenic C7(8)(beta(197))(tox-crm+) strain of Corynebacterium diphtheriae produces a nontoxic, extracellular protein of molecular weight 62,000. This protein is immunologically indistinguishable from toxin itself but inhibits the action of toxin on HeLa cells, probably by competing for attachment sites on the cell membrane. In contrast to fragment A derived from diphtheria toxin, fragment A(197) is unable to catalyze the inactivation of eucaryotic polypeptidyl-transfer RNA-transferase II. When mixtures of the two nontoxic mutant proteins, enzymically active crm(45) protein and enzymically inactive crm(197) protein, are subjected to mild treatment with trypsin in the presence of a thiol and then allowed to reoxidize after dialysis to remove excess thiol, "diphtheria toxin" is reconstituted in high yield.

A mutant sex factor of Pseudomonas aeruginosa.

Abstract: A mutant with the properties of a recipient has been isolated from the P. aeruginosa donor strain PAT (FP 2 + ) following treatment with the acridine-mustard ICR-191. While this strain displays the properties expected of a female or recipient in a number of genetic tests, the FP 2 determined property of mercury resistance is retained by the strain, suggesting that it may carry the FP2 factor in a mutated form. Treatment of the donor strain PAT (FP2 + ) with acridine-mustard has produced mutant male strains with the ability to form recombinants with other male strains at frequencies similar to that obtained in FP2+ × FP2 − matings. This characteristic has been shown to be due to a mutation in the FP2 factor which is dominant to the wild-type function. The isolation of stable male strains carrying both the mutant and wild type forms of the sex factor suggests that more than one copy of the FP 2 factor occurs in P. aeruginosa strain PAT donors.

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

Abstract: The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts.

Abstract: The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10-6 to 19×10-6 and the mean value was 4.1×10-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10-6 to 1.8×10-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.

New small polypeptides associated with DNA-dependent RNA polymerase of Escherichia coli after infection with bacteriophage T4.

Abstract: Four new small polypeptides are associated with DNA-dependent RNA polymerase from E. coli after infection with T4 phage. The new polypeptides are easily detected in RNA polymerase from E. coli cells labeled with amino acids after phage infection. Their molecular weights range from 10,000 to 22,000, as detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All four polypeptides are found after infection with either wild-type T4 phage or T4 early amber mutants in genes 44, 42, 47, and 46. None of the polypeptides is labeled significantly before 5 min after infection at 30°. When two maturation-defective amber mutants in gene 55 of T4 phage are used for infection, a polypeptide with a molecular weight of 22,000 is absent. When a maturation-defective amber mutant in gene 33 of T4 phage is used, another small protein is absent.

Sporulation in Bacillus subtilis. Characterization of oligosporogenous mutants and comparison of their phenotypes with those of asporogenous mutants.

Abstract: SUMMARY: An investigation was undertaken to determine to what extent the properties of oligosporogenous (Osp) mutants allow them to be considered as a separate class of sporulation mutant, distinct from asporogenous (Sp-) mutants. Of thirty Osp mutants examined, seventeen at least had a phenotype which had previously been identified with a Sp- mutant. The majority of cells in an Osp culture either reached a particular stage in the sporulation process and then stopped, or in some cases went on to produce aberrant forms. Some of these aberrant forms have their counterparts in Sp- mutants described by other authors, but some present new features. The morphological and biochemical sequences were linked so that if the majority of cells were blocked at a certain stage, then the biochemical sequence stopped accordingly. The general similarity in behaviour between the two types of mutant is consistent with the assumption that at least some of the Osp mutants have leaky mutations in genes where mutation can also give rise to Sp- phenotypes. Evidence is presented to suggest that the ability of a cell of an Osp mutant to overcome its block, and so go on to form a spore, is a chance event when that stage in the process is reached. A mutant has been obtained in which the spores are octanol-resistant yet contain no measurable dipicolinate. In several other mutants the spores contained well-developed coat layers, but the cortex was poorly formed or completely missing.

Temperature sensitive mutants of BHK 21 cells.

Abstract: Temperature sensitive mutant cells were isolated from animal cells by multiple culturing in the presence of 5-fluoro-2′-deoxyuridine. Properties of the mutants are described.

Mutants of the biological clock in chlamydomonas reinhardi

Abstract: A genetic analysis of the biological clock in Chlamydomonas reinhardi has been initiated. Of six wild-type strains tested (3 mt+ and 3 mt-), five had periods close to 24 hr whereas one had a 21-hr period. Mutants with altered clock period have been isolated. The periods of 3 of these variant strains are temperature compensated. Genetic crosses involving a long-period mutant suggest that a single gene confers the long-period character, and in general clock-period length seems to be a useful phenotypic measure of alterations in the clock due to genetic differences. One phase mutant was found but its behavior was variable and the phase of the rhythm, relative to a light–dark transition which initiates the rhythm, does not seem to be reliable as a parameter of clock differences. No markers have yet been mapped.

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

Abstract: A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.

Involvement of a bacterial factor in morphogenesis of bacteriophage capsid.

Abstract: A new bacterial factor has been found necessary for the activity of T4 gene 31, the only catalytic factor in the early stage of phage head formation, to process the assembly of head precursor proteins. In a mutant missing this factor, the precursors of phage head aggregate on the bacterial membrane.

Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus.

Abstract: Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10−3 or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Abstract: A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polA1 by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polA1 mutation was suppressed during mutagenesis by introduction of the suppressor, su7+, into the parental strain. The mutant, HMS83 polA1 polB1, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 42°, and supports the growth of bacteriophages T4, T7, lambda, ϕX174, 186, P2, and fd. The polB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.

Ammonium Repression of Extracellular Protease in Aspergillus nidulans

Abstract: Summary: Extracellular protease of Aspergillus nidulans is repressible by ammonium. A mutant (xpr D i) selected for derepression of protease in the presence of ammonium is also derepressed for nitrate reductase, xanthine dehydrogenase and glutamate uptake; it retains the ability to use ammonium as sole nitrogen source. The mutant is partially dominant in heterozygous diploids. The mutation does not confer methyl-amine resistance, nor are methylamine-resistant (meaA) strains derepressed for protease.