SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A new approach to induce directed mutations in genes of study through simple cotransfection of E. coli cells by the mixture of primer and template was developed. This method is based on the use of synthetic phosphotriester analogues of oligonucleotides as site-specific mutagenic primers. The achieved yield of mutant clones was 2-3%.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA. This system also makes possible to determine heterozygotes and homozygotes among the mutants. The yield of mutants in shown to depend on the structure of the DNA heteroduplex region. The yield of deletion mutants is greater than that of insertion mutants. Heterozygotes prevail in mutant colonies (greater than 90%).
A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.
Explore the source record for details and available documents.
We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.
More numbers of mutants were isolated when UV-irradiated cells of Azotobacter vinelandii OP were treated with caffeine for a limited period of time after UV-irradiation. This is encouraging, considering the difficulty in isolating Azotobacter mutants. Post-irradiation treatment with acriflavine, however, yielded comparatively lesser number of mutants.
Determination of chromosomal aberration in the lymphocyte cell culture of the peripheral blood in 50 children with malignant and nonmalignant diseases was the test applied in the investigation of the adverse effects of the cytostatic therapy on the human genome. The study included the cytostatic drugs cyclophosphamide, methotrexat and cytosine arabinoside which are used in the treatment for the autoimmune diseases, in organ and bone marrow transplantations and in malignant diseases. It was confirmed that these cytostatics could be the cause of considerable structural aberrations in chromosomes, particularly so when high dosages and long application are involved. Therefore it is understandable that these drugs as mutagens can be the cause of the secondary cancer in patients treated with cytostatic therapy and also of the congenital malformations in children by mothers treated with these medications.
To test a suggestion that mutation frequency decline (MFD) in UV-irradiated bacteria starved by nitrogen sources is caused by long delay of the first postradiation cycle of DNA replication, we used a strain 1201-05 of Bacillus subtilis 168 (ade6 met5tsdnaC) which does not synthesize DNA at 42 degrees C. In a suspension of UV-irradiated cells of this strain which is incubated at 42 degrees in liquid deprived by nitrogen sources, a decrease of frequencies of Ade+ and Met+ revertants is observed. This shows that the mechanism responsible for MFD normally operates at higher temperature. Such a level of decline is not observed in a suspension incubated in nutritional medium when the DNA replication is blocked by non-permissive temperature for the same period as in starved cells. On the basis of these facts it is concluded that MFD in suspension of bacteria starved by nitrogen sources can not be caused only by delay of postradiation replication of DNA.
By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene. When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC+ and recF+ genes. When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis. This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative. Thus, we conclude that the induced (umuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers. glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9. This uninduced mutagenesis is independent of the umuC+, recF+ and recA+ genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides. The role of recF in UV mutagenesis was tested in two ways. First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent. Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play. Second, UV mutagenesis of E. coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent. We conclude that the mechanism of induced UV mutagenesis of the E. coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference.
Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.
Bacteria live in unstructured and structured environments, experiencing feast and famine lifestyles. Bacterial colonies can be viewed as model structured environments. SOS induction and mutagenesis have been observed in aging Escherichia coli colonies, in the absence of exogenous sources of DNA damage. This cAMP-dependent mutagenesis occurring in Resting Organisms in a Structured Environment (ROSE) is unaffected by a umuC mutation and therefore differs from both targeted UV mutagenesis and recA730 (SOS constitutive) untargeted mutagenesis. As a recB mutation has only a minor effect on ROSE mutagenesis it also differs from both adaptive reversion of the lacI33 allele and from iSDR (inducible Stable DNA Replication) mutagenesis. Besides its recA and lexA dependence, ROSE mutagenesis is also uvrB and polA dependent. These genetic requirements are reminiscent of the untargeted mutagenesis in lambda phage observed when unirradiated lambda infects UV-irradiated E. coli. These mutations, which are not observed in aging liquid cultures, accumulate linearly with the age of the colonies. ROSE mutagenesis might offer a good model for bacterial mutagenesis in structured environments such as biofilms and for mutagenesis of quiescent eukaryotic cells.
Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.
Cells lacking mismatch repair (MMR) exhibit elevated levels of spontaneous mutagenesis. Evidence exists that MMR is involved in repair of some DNA lesions besides mismatches. If some oxidative DNA lesions are substrates for MMR, then the excess mutagenesis in MMR(-) cells might be blocked by dietary antioxidants. Effects of the dietary antioxidants ascorbate, alpha-tocopherol, (-)-epigallocatechin gallate (EGCG) and lycopene on spontaneous mutagenesis were studied using mismatch repair-deficient (hMLH1(-)) human colon carcinoma HCT116 cells and HCT116/ch3 cells, in which normal human chromosome 3 has been added to restore mismatch repair. HCT116 cells have a 22-fold higher spontaneous mutation rate compared with HCT116/ch3 cells. HCT116 cells cultured in 1% fetal bovine serum (FBS) have twice the spontaneous mutation rate of those cultured in 10% FBS, most likely due to reduction in serum antioxidants in the low serum medium. As expected, alpha-tocopherol (50 microM) and ascorbate (284 microM) reduced spontaneous mutagenesis in HCT116 cells growing in 1% serum more dramatically than in cells cultured in 10% serum. The strongest antimutagenic compound was lycopene (5 microM), which reduced spontaneous mutagenesis equally (about 70%) in HCT116 cells growing in 10 and 1% FBS and in HCT116/ch3 cells. Since lycopene was equally antimutagenic in cells growing in low and high serum, it may have another antimutagenic mechanism in addition to its antioxidant effect. Surprisingly, EGCG (10 microM) was toxic to cells growing in low serum. It also reduced spontaneous mutagenesis equally (nearly 40%) in HCT116 and HCT116/ch3 cells. The large proportion of spontaneous mutagenesis that can be blocked by antioxidants in mismatch repair-deficient cells support the hypothesis that a major cause of their excess mutagenesis is endogenous oxidants. Blocking spontaneous mutagenesis, perhaps with a cocktail of antioxidants, should reduce the risk of cancer in people with a genetic defect in mismatch repair as well as other individuals.
DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.
The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4-ethenocytosine (epsilon C) lesions borne on transfected single-stranded M13 vector DNA. All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at epsilon C residues. In contrast to normal cells that show approximately 6% mutagenesis at epsilon C lesions, but approximately 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at epsilon C lesion sites in untreated cells. However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that epsilonC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at epsilonC can also be independently elevated by the SOS pathway.