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1.
5-Azacytidine (5-AzaC) induced mutation in the TK+/− human lymphoblastoid line, TK6, at both the thymidine kinase (tk) locus as measured by resistance to trifluorothymidine (F3TdR), and the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, as measured by resistance to 6-thioguanine (6TG). F3TdRR and 6TGR mutant fractions induced by 5-AzaC were observed after a normal phenotypic expression time and remained stable. Interestingly, 5-AzaC was 5–10 times more mutagenic at the tk locus than the hgprt locus. However, F3TdRR colonies from 5-AzaC-treated cultures behaved like TK-deficient mutants induced by other chemical mutagens.

The TK or HGPRT phenotype had no effect on the toxicity of 5-AzaC, thus eliminating differential toxicity as a potential cause for the observed higher mutability at the tk locus. 5-AzaC did not induce F3TdRR cells in the parental TK+/+ lymphoblastoid line, indicating that 5-AzaC-induced F3TdRR variants were not due to a dominant alteration in gene expression. 5-AzaC did not induce chromosomal aberrations in TK6 cells, eliminating clastogenic events as a potential cause for the higher mutability at the tk locus.

5-AzaC was also found to be mutagenic in a forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium.  相似文献   


2.
Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt+/−aprt−/−) was 1.3 × 10−5/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.  相似文献   

3.
Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.  相似文献   

4.
D Clive  R Krehl 《Mutation research》1991,260(4):409-413
The phenotypic stability of over 2000 large- and small-colony trifluorothymidine-resistant (TFTres) variants of L5178Y/tk(+/-)-3.7.2C cells has been examined. All except 4 of 488 spontaneously arising small-colony variants analyzed (0.8%) retained the TFTres phenotype when rechallenged with TFT after growth for several generations in its absence. All of 558 spontaneous large-colony variants, and 440 small-colony or 487 large-colony variants arising from 13 different mutagens showed similar stability. These results attest to the completeness of TFT selection in the mouse-lymphoma assay when used at 1 microgram/ml in Fischer's medium supplemented with heat-inactivated serum and, together with previous cytogenetic and molecular studies, justify considering essentially all such TFTres variants as stable mutants. The implications of these results for those versions of the mouse lymphoma assay that fail to optimize the recovery and scoring of small-colony mutants is discussed.  相似文献   

5.
Molecular characterisation of the Stc mutation of Escherichia coli K-12   总被引:3,自引:0,他引:3  
R. Misra  P. Reeves   《Gene》1985,40(2-3):337-342
The previously described Stc - (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF to OmpF+. Restriction mapping of chromosomal DNA from Stc + and Stc strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc phenotype involves increase in ompF expression.  相似文献   

6.
Juvenile hormone (JH) involvement in male reproduction is poorly understood. In Drosophila melanogaster adults, JH deficiency has been shown to result in lowered protein synthesis in male accessory glands. To probe additional roles, we have examined males homozygous for a null allele of Methoprene-tolerant (Met). This gene is involved in the action of JH, possibly at the JH receptor level, and Met27 null mutants reflect a diminution of JH action. Met27 males were found to have reduced protein accumulation in male accessory glands and to court and mate wild-type females much less avidly than do either Met+ or Met27; Met+ transgenic males. Exposure of Met27 males to methoprene partially rescued the courtship deficiency. However, sperm transfer as reflected by fertility of Met27 fathers was found to be similar to that of Met+. Taken together with previous work examining the JH-deficient mutant apterous, these results corroborate JH involvement in protein synthesis in the male accessory glands and suggest a role for JH in promoting male mating behavior in these flies.  相似文献   

7.
Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO AL). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59 and CD59+ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.  相似文献   

8.
The effect of guanidinium chloride solutions on the circular dichroism of native (ZnZn-) and apophospholipase C (Bacillus cereus) indicated marked protein unfolding at denaturant concentrations of 1.4–1.8 M and 0.1–0.6 M, respectively. With the apoenzyme near u.V. region circular dichroism bands remained even after all ordered structure appeared to have been lost. Apophospholipase C bound two equivalents of Ni2+, Cd2+, Co2+, Mn2, Pb2+ or Cu2−, with only the latter metal causing marked changes either in circular dichroism or protein fluorescence relative to the native enzyme. Stability in guanidinium chloride for the metalloforms of phospholipase C decreased in the order: ZnZn->ZnCo->NiNi->CoCo->PbPb->CdCd->MnMn-apoenzyme.  相似文献   

9.
The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq+) and the mutants chr, frq1, frq7, and frq8. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4-9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq1. A loss of pH homeostasis of the period, however, was observed in the mutants frq7 and frq8, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H2PO4- ion has been found, indicating that the uptake of H2PO4- may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq7 and frq8 mutants may be related to less easily degradable FRQ7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ7 protein degradation increases and should lead to shorter period lengths. (Chronobiology International, 17(6), 733-750, 2000)  相似文献   

10.
The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the “guardian of the genome”. Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53−/− and p53+/− mice. Six male mice from each genotype (p53+/+, p53+/−, and p53−/−) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53+/+ and p53+/− or between p53+/+ and p53−/− at the level of p ≤ 0.05. Both genes with increased expression and decreased expression were identified in p53+/− and in p53−/− mice. Most notable in the gene list derived from the p53+/− mice was the significant reduction in p53 mRNA. In the p53−/− mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.  相似文献   

11.
Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O2), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG+ HP-I catalase), mutation by NUV was reduced to wild-type (sodA+ sodB+) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O2 suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H2O2 induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG+ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH. radicals, possibly by generating excess H2O2.  相似文献   

12.
13.
Mitomycin C (MC) was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 4 dikaryons of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on MC-induced killing and ad-3 mutation. These dikaryons were homokaryotic for uvs-2+ (H-12), homokaryotic for uvs-2 (H-59), and heterokaryotic for uvs-2/uvs-2+ (H-70 and H-71). MC induced killing and ad-3 mutation in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a great increase in the killing and mutagenic activities of MC. This increased sensitivity to MC-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is a different effect than that noted by others for a defect in nucleotide excision-repair in Escherichia coli and Salmonella typhimurium or in human cells. The dikaryons heterokaryotic for uvs-2/uvs-2+ had the same sensitivity to MC as H-12, indicating that for MC-induced killing and ad-3 mutation uvs-2 is recessive to uvs-2+.  相似文献   

14.
G. Duc  A. Messager   《Plant science》1989,60(2):207-213
Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod), or with ineffective nodules (nod+, fix) were obtained, whilst in the other 4 hypernodulated mutants (nod++) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod), (nod+, fix) and (nod++, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.

Grafting experiments showed the (nod) and (nod+, fix) phenotypes are associated with the root genotypes and that (nod++, nts) phenotype is associated with the shoot genotype.  相似文献   


15.
A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occuring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30°C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30°C but not at 37°C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37°C and shows a temperature conditional UV-induced respiration shutoff response at 30°C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37° This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.  相似文献   

16.
Brian Sauer  Nancy Henderson 《Gene》1988,70(2):331-341
The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox2 plasmids) are cyclized in Cre+ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox2 plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox2 plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre+ E. coli recBC strains allow the efficient recovery of lox2 plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.  相似文献   

17.
N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O6-methylguanine (O6-MeG) and O4-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O6-MeG is repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O6 position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O6-benzylguanine (O6-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O6-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O6-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O6-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O6-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10−6 and 240 × 10−6, respectively; in the cells with abundant AGT activity, these values were 10 × 10−6 and 20 × 10−6, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.  相似文献   

18.
The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD+C+ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD+C+ plasmids. pBR322-derived umuD+C+ plasmids inhibited the induction of the SOS response of lexA+ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA+ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA+ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD+C+ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.  相似文献   

19.

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·103 M−1·s−1 and a Ki that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa3−O2 system a biphasic reduction of cytochrome c occurs corresponding to an initial Ki of 0.8 μM and a final Ki of about 0.1 μM for the cytochrome aa3−cyanide reaction.

3. 3. The inhibited species (a2+a33+HCN) is formed when a2+a33+ reacts with HCN, when a2+a32+HCN reacts with oxygen, or when a3+a33+HCN (cyano-cytochrome aa3) is reduced. Cyanide dissociates from a2+a33+HCN at a rate of 2·10−3 s−1 at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a2+a33+ than to a3+a33+.

Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine  相似文献   


20.
The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA+ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.  相似文献   

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