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PubMed1982

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant.

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.

Chromosome Mapping↗

PubMed1994

Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.

Acetates↗

PubMed1968

Analysis of sporulation mutants. II. Mutants blocked in the citric acid cycle.

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.

Acetates↗

PubMed2004

Complementation in cells cotransfected with a mixture of wild-type and mutant human immunodeficiency virus (HIV) influences the replication capacities and phenotypes of mutant variants in a single-cycle HIV resistance assay.

The impact of cotransfection of mixtures of mutant and wild type (WT) virus on the observed phenotype and replication capacity (RC) in a single-cycle human immunodeficiency virus (HIV) phenotypic assay has been investigated by cotransfecting mutant HIV clones expressing the firefly luciferase expression gene with a WT clone expressing Renilla luciferase. Four mutant constructs with different genotypes displayed <1% RC when transfected alone. Cotransfection of as little as 9% of the WT clone resulted in an 18- to 33-fold increase in the RC of the mutant clones. In addition, the 50% inhibitory concentration (IC(50)) of lopinavir against seven mutant clones decreased by up to 97% after incremental cotransfection of 9 to 50% of the WT clone. The enhancement of RC and decrease in IC(50) for mutant variants following cotransfection with the WT variant appear to be due to complementation rather than genetic recombination. These findings suggest that the RC and susceptibility of plasma isolates from patients who are off therapy or not adherent to treatment, in which WT virus may expand to significant levels, should be interpreted with caution.

Animals↗

PubMed1974

Temperature-sensitive mutants of vesicular stomatitis virus: comparison of the in vitro RNA polymerase defects of group I and group IV mutants.

When tested in vitro, certain temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) belonging to complementation groups I and IV appear to have defects in the virion-bound polymerase. To obtain further information concerning the nature of these defects, representative mutants were dissociated by the method of S. Emerson and R. Wagner (1972), and their supernatant (S) and pellet (P) fractions were tested for transcriptase activity when combined with the P and S fractions, respectively, of VSV-HR virions. It was found that the S fractions from group I mutants tsW4, 11, 14, 15, and 28 were defective in transcriptase activity, whereas their P fractions were as active as those of VSV-HR. On the other hand, the P fraction derived from virions of the group IV mutant tsW16B showed reduced activity at 25 C and very little activity at 38 C. These results suggest that our group I mutants, like those examined by D. Hunt and R. Wagner (1974), have a defect in the soluble transcriptase enzyme, whereas mutant tsW16B (group IV) has a defect in a sedimentable component required for transcriptase activity, possibly in the ribonucleoprotein template.

Animals↗

PubMed1977

Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated.

A procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of Bacillus subtilis phage SPO1. A modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest. The applicability of these procedures to other viral systems is discussed. The mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from that of the heat-sensitive mutations. As a preliminary to the identification of initiation-deficient mutants, the mutants were divided into three classes on the basis of their ability to synthesize DNA after a shift to nonpermissive temperature. We also report two incidental results: (i) the SPO1 dUMP hydroxymethylase, like the T4 dCMP hydroxymethylase, may be part of a multifunctional complex; and (ii) mutants were isolated that were replication positive but lysis deficient and failed to complement one of the replication-deficient mutants.

Bacillus subtilis↗

PubMed1995

Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase.

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca(2+)-mediated signaling in many cells. Yeast cells lacking functional calcineurin (cna1 cna2 or cnb1 mutants) display growth defects under specific environmental conditions, for example, in the presence of high concentrations of Na+, Li+, Mn2+, or OH- but are indistinguishable from wild-type cells under standard culture conditions. To characterize regulatory pathways that may overlap with calcineurin, we performed a synthetic lethal screen to identify mutants that require calcineurin on standard growth media. The characterization of one such mutant, cnd1-8, is presented. The CND1 gene was cloned, and sequence analysis predicts that it encodes a novel protein 1,876 amino acids in length with multiple membrane-spanning domains. CND1 is identical to the gene identified previously as FKS1, ETG1, and CWH53, cnd1 mutants are sensitive to FK506 and cyclosporin A and exhibit slow growth that is improved by the addition of osmotic stabilizing agents. This osmotic agent-remedial growth defect and microscopic evidence of spontaneous cell lysis in cnd1 cultures suggest that cell integrity is compromised in these mutants. Mutations in the genes for yeast protein kinase C (pkc1) and a MAP kinase (mpk1/slt2) disrupt a Ca(2+)-dependent signaling pathway required to maintain a normal cell wall and cell integrity. We show that pkc1 and mpk1/slt2 growth defects are more severe in the absence of calcineurin function and less severe in the presence of a constitutively active form of calcineurin. These observations suggest that calcineurin and protein kinase C perform independent but physiologically related functions in yeast cells. We show that several mutants that lack a functional vacuolar H(+)-ATPase (vma) require calcineurin for vegetative growth. We discuss possible roles for calcineurin in regulating intracellular ion homeostasis and in maintaining cell integrity.

Amino Acid Sequence↗

PubMed1978

Temperature-sensitive mutants of simian virus 40. I. Isolation aand preliminary characterization of B/C gene mutants.

Sixteen temperature-sensitive mutants of simian virus 40 were isolated after treatment of wild-type virus with UV light, nitrous acid, or nitrosoguanidine. All of the mutants were assigned to the B complementation group on the basis of quantitative complementation analyses. Characterization of several representative mutants revealed that under restrictive conditions such mutants produced immunologically identifiable tumor and viral capsid antigens, induced the synthesis of cellular DNA, and replicated infectious viral DNA. In addition, mutant particles produced at 33 degrees were more heat-labile than wild-type virus. This collection of mutants will be useful in an analysis of SV40 particle morphogenesis.

Animals↗

PubMed2006

Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years.

Essential thrombocythemia (ET) is heterogeneous with respect to natural history, X-chromosome inactivation patterns (XCIPs), and presence of the V617F mutation in Janus kinase 2 (JAK2). We studied 111 patients with ET; 39% were JAK2 mutant positive, and clone size (percentage mutant JAK2) was concordant with XCIP when constitutive T-cell patterns were taken into account. JAK2 mutant clones were present in both clonal and polyclonal cases as determined by XCIP, and the former had higher mutant JAK2 levels (median 26% versus 16%; P=.001). No change was observed in serial XCIP analysis of 14 polyclonal patients over a median follow-up of 61 months. Furthermore, 18 of 19 mutant-positive patients showed no significant change in mutant JAK2 level over a median follow-up of 47 months. These results suggest that, in many cases of ET, a small stable clone containing a JAK2 mutation can be maintained as a subpopulation for many years.

Cell Survival↗

PubMed1979

[Biochemical mutants of Actinomyces griseus, a producer of the antibiotic grisin. The isolation of the mutants and a study of the level of grisin antibiotic synthesis].

Biochemical mutants of Actinomyces griseus producing grisin were obtained under the action of chemical mutagens. The mutants were divided into 2 groups. The mutants with impaired synthesis of amino acids of the aspartic acid family, i.e. lysine, homoserine and methionine were included into the 1st group. The 2nd group included the mutants with impaired synthesis of the other amino acids, i.e. histidine, arginine, tyrosine, phenylalanine and valine. The antibiotic production level in the biochemical mutants was studied. It was found that the level of the antibiotic production was decreased in most of the biochemical mutants. A necessity for increased fonds of lysine and arginine for biosynthesis of grisin was shown.

Amino Acids↗

PubMed2001

[Genetic analysis of mutant genes and RAPD marker of mutant gene of narrow leaflet in soybean].

Mutant E182 with "narrow leaflet-4 seeded pod" was selected from descendents of EMS-treated seeds of soybean variety Lu Dou No. 4 (LD4) with "ovate leaflet-without 4 seeded pod". Genetic analyses of F2 individuals of crossing between mutant E182 and parent LD4 indicated that segregation ratio between ovate and narrow leaflet was 3:1, so was segregation ratio between "without 4 seeded pod" and "4 seeded pod". Segregation ratios of four character types in F2 population of 1,654 individuals were beyond 9:3:3:1 of two pairs of independent gene, showing linkage inheritance. Reccmbinant ratio between mutant genes of narrow leaflet and 4 seeded pod was 11.24% +/- 0.81%. On the other hand, mutant E182, parent LD4 and F2, F3 individuals of their hybrids were analyzed by means of RAPD technique. The marker OPY6-1300 linked with the mutant gene of narrow leaflet was generated, and genetic distance of the marker and mutant gene was 8 cent Morgan (cM), being 10 cM nearer than RFLP marker of narrow leaflet generated by shoemaker.

Genetic Markers↗

PubMed1990

Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes.

The majority of the p53 genes derived from human colorectal carcinomas contain point mutations. A significant number of these mutations occur in or around amino acids 143, 175, 273, or 281. Experiments presented here demonstrate for the first time that p53 DNA clones containing any one of these mutations cooperate with the activated ras oncogene to transform primary rat embryo cells in culture. These transformed cells produce elevated levels of the human p53 protein, which has extended half-lives (1.5-7 h), as compared to the wild-type human p53 protein (20-30 min). The p53 mutant with an alteration at residue 175 (p53-175H) binds tightly to the cellular heat shock protein, hsc70. In contrast, the p53 mutants possessing mutations at either residue 273 or 281 (p53-273H/281G) do not bind detectably to this heat shock protein and generally are less efficient at forming transformed foci in culture. The transformed cell lines are tumorigenic in nude mice. Thus, two classes of p53 mutant proteins can be distinguished: p53-175H, which cooperates with ras efficiently and binds to hsc70, and p53-273H/281G, which has a reduced efficiency of transformed foci formation and does not bind hsc70. This demonstrates that complex formation between mutant p53 and hsc70 is not required for p53-mediated transformation, but rather it facilitates this function, perhaps by ensuring sequestration of the endogenous wild-type p53 protein. The positive effect on cell proliferation by these mutant p53 proteins is consistent with a role for activated p53 mutants in the genesis of colorectal carcinomas.

Animals↗

PubMed1990

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.

Amino Acid Sequence↗

PubMed1989

Ketoconazole-resistant mutants of Microsporum gypseum. II. Characteristics of mutants.

The presented study deals with the properties of two wild strains of dermatophyte Microsporum gypseum and of eleven mutants resistant to ketoconazole. The growth rate of spontaneous mutants is greater than that of the wild strain, the group of UV-induced mutants manifests in general lower growth rate. The resistance level varies in the interval 1.1-1.6 (spontaneous mutants), resp. 2.7-5.5 (UV-induced mutants). The phenomenon of cross-resistance to other imidazole drugs (miconazole, clotrimazole) has been observed in mutants. There is further given the characteristic of ergosterol synthesis influence by the three used antifungal drugs. The data obtained are discussed from the point of view of possible biochemical processes resulting in the formation of resistance to ketoconazole.

Clotrimazole↗

PubMed1987

[Transmission of amber mutants of bacteriophage T4. III. Thermostability of the replication of amber mutants in cells of a non-permissive host is typical for the majority of phage tail genes].

The article deals with determination of the spreading of the earlier discovered phenomenon of the temperature sensitivity of multiplication of T4 phage amber mutants. On the basis of the study of the dependence of multiplication of 50 amber mutants in 22 genes of T4 phage tail in the cells of non-permissive host on the incubation temperature in the range of 15-41 degrees C, the following conclusion is drawn: temperature sensitivity of multiplication of amber mutants appears to be gene-specific and is widely spread among T4 phage genes, i.e. in the case of amber mutants the burst size decreases, even for 14 tail genes, by several orders with the increase in incubation temperature. Temperature sensitivity of multiplication is typical of amber mutants in the genes whose proteins are either of small number in a phage particle (several molecules) or play the role of catalytic factors. Moreover, genes, amber mutants of which possess temperature sensitivity of multiplication, map in defined clusters.

Escherichia coli↗

PubMed1977

[Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants].

Riboflavin deficient mutant Pichia guilliermondii MS1 which requires approximately 1000-fold lower concentration of exogenous vitamin B2 for growth when compared with a non-adapted riboflavin deficient mutants of this species was isolated by means of of UV-irradiation. The growth of the mutant was strongly inhibited by actinomycin D and L-canavanine. The revertant MS8 and MS14 which synthesized riboflavin were selected from the strain MS1. These revertants posses a multiple sensitivity to actinomycin D, rifamycin, euflavine, mitomycin C, antimycin A, 8-azaadenine, 8-azaguanine, L-canavanine and 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine. The ability to utilized glycerol and ethanol as a sole carbon source for growth was impaired in these mutants. The mutants which can utilize glycerol were isolated from the strain MS14. Such mutants were resistant to actonomycin D. Mutation (s) which determines a multiple sensitivity and inability to utilized glycerol was recessive.

Anti-Bacterial Agents↗

PubMed1995

Testing homology modeling on mutant proteins: predicting structural and thermodynamic effects in the Ala98-->Val mutants of T4 lysozyme.

Backgound. Current approaches to homology modeling predict how amino acid substitutions will alter a protein's structure, primarily by modeling sidechain conformations upon essentially immobile backbone frameworks. However, recent crystal structures of T4 lysozyme mutants reveal significant shifts of the mainchain and other potentially serious problems for sidechain rotamer-based modeling. This paper evaluates the accuracy of structural and thermodynamic predictions from two common sidechain modeling approaches to measure errors caused by the fixed-backbone approximation. Results. Tested on a series of T4 lysozyme mutants, this sidechain rotamer library approach did not handle mainchain shifts well, correctly predicting the sidechain conformations of only two of six mutants. By contrast, allowing sidechains to move more flexibly appeared to compensate for the rigidity of the mainchain and gave reasonably accurate coordinate predictions (rms errors of 0.5-1.0 &angst; for each mutated sidechain), better on average than 90% of possible conformations. The calculated packing energies correlated well with experimental stabilities (r2=0.81) and correctly captured the cooperative interactions of several neighboring mutations. Conclusion. Mutant modeling can be relatively accurate despite the fixed-backbone approximation. Mainchain shifts (0.2-0.5 &angst;) cause increased sidechain coordinate errors of 0.1-0.8 &angst;, torsional errors of 10-30°, and exaggerated strain energy for overpacked mutants, compared with the same calculations performed with the correct mutant backbones.

Journal Article↗

PubMed1983

Thyroid graft rejection between C57BL/6 and H-2Kb mutant mice. Differences in the immunogenic expression of the mutated molecule on thyroids of six different mutants.

Mice that possess mutations in the H-2 region were selected by means of skin grafting. We tested the survival time of thyroid grafts transplanted between C57BL/6 mice and their descendent H-2Kb mutant mice and compared the kinetics of skin versus thyroid graft rejection in each of the mouse strain combinations tested. The results demonstrated that reciprocal thyroid grafts between C57BL/6 mice and bm1, bm3, bm4, bm5, and bm6 mutants are accepted for long periods. The bm8 mutant appeared to be an exception because a small fraction of reciprocal thyroid grafts were rejected between bm8 and C57BL/6 mice. Transplantation of C57BL/6 mice with skin grafts from bm1, bm3, bm4, and bm8 donors induced rejection of subsequent thyroid grafts syngeneic with the skin. Thyroid grafts of bm5 and bm6 were not rejected by C57BL/6 mice even after preimmunization with bm5 and bm6 skin grafts. In contrast to skin grafts, innoculation of C57BL/6 mice with 50 X 10(6) spleen cells from mutant mice failed to immunize the recipients and induce rejection of mutant thyroid grafts. The results indicate that skin graft rejection assays and in vitro cell-mediated lympholysis (CML) typing can not always predict the consequence of transplantation of organs such as thyroid, and also that alterations in the H-2Kb molecule as a result of spontaneous mutations can give rise to mutated molecules that differ in their immunogenic and antigenic properties when expressed on thyroid cells, as compared with skin.

Animals↗