RESUMO
Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.
Assuntos
Dano ao DNA , Desoxirribonuclease (Dímero de Pirimidina) , Proteínas de Escherichia coli , Genes Fúngicos , Peróxido de Hidrogênio/farmacologia , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Bleomicina/farmacologia , Eletroforese em Gel de Campo Pulsado , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Teste de Complementação Genética , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Paraquat/farmacologia , Saccharomyces cerevisiae/genéticaRESUMO
Patients with hereditary non-polyposis colorectal cancer (HNPCC) have a DNA mismatch repair defect (MMR) in their tumor tissue that results in instability of microsatellite DNA sequences (MSI). Thus, MSI analysis may effectively indicate this form of cancer that should be then proved by analysis of germline mutations in MMR genes. The aim of this study was to identify HNPCC suspected patients in the Slovak population by investigating microsatellite instability in colorectal tumor tissues. MSI was studied at 5-11 loci in matched tumor and normal DNA using radioactively labeled PCR products separated on sequencing gels. High microsatellite instability (MSI-H) was present only in patients younger than 50 years, in 100% of patients having two affected relatives by colorectal cancer and in 67% of patients with only one affected relative. In both groups of patients colorectal cancer was present in two successive generations. No MSI-H was found in the group of patients older than 50 years, even if they had positive family history for colorectal cancer. Among all markers used, the BAT26 mononucleotide repeat (100%), DI0S197 and D13S175 (62.5%) dinucleotide repeats were the most frequently altered in the tumor tissues. Retrospective analysis revealed that some of the patients having MSI-H tumors have had clinicopathological characteristics frequently reported to HNPCC. The family members of those patients with MSI-H are enrolled in preventive health care program until mutational analyses will enable to select carriers from non-carriers of mutated MMR genes.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Adulto , Fatores Etários , Idoso , Pareamento Incorreto de Bases , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo do DNA/genética , Diagnóstico Diferencial , Saúde da Família , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Linhagem , EslováquiaAssuntos
Alquilantes/toxicidade , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Mutagênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Etilnitrosoureia/análogos & derivados , Etilnitrosoureia/toxicidade , Metanossulfonato de Metila/toxicidade , Metilnitronitrosoguanidina/toxicidade , O(6)-Metilguanina-DNA Metiltransferase , Saccharomyces cerevisiae/genética , Fatores de TranscriçãoRESUMO
Construction of E. coli-yeast shuttle plasmids containing the neo selection gene is described. The protein-coding regions of the E. coli ada or recA genes under the control of the ADH1 promoter and terminator were ligated into the SphI unique site of pNF2 to produce pMSada and pMSrecA, respectively. The plasmids were used for transformation of the haploid and diploid pso4-1 strains of S. cerevisiae and their corresponding wild types. Transformants were obtained by selection for geneticin (G418) resistance. Crude protein samples were extracted from the individual transformants. Both the RecA and Ada proteins were present in all strains containing the recA and ada genes on plasmids, respectively. Thus the geneticin selection system was successfully used for the preparation of model yeast strains.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Vetores Genéticos , Saccharomyces cerevisiae/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Expressão Gênica , Genótipo , Gentamicinas/farmacologia , Mutação , O(6)-Metilguanina-DNA Metiltransferase , Plasmídeos/genética , Recombinases Rec A/biossíntese , Recombinases Rec A/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fatores de Transcrição , Transformação GenéticaRESUMO
The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceding mitotic conversion events in yeast.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Saccharomyces cerevisiae/genética , Álcool Desidrogenase/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Diploide , Escherichia coli/efeitos da radiação , Conversão Gênica , Mitose/genética , Mitose/efeitos da radiação , Regiões Promotoras Genéticas , Recombinases Rec A/genética , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
The authors investigated the age of onset of the menopause by random selection, using a questionnaire, in 344 women aged 43 to 90 years in relation to smoking. They evaluated 107 non-smokers, 112 passive smokers, and 118 active smokers. The mean age of onset of the menopause in non-smokers was 49.8 +/- 4.1, in passive smokers 49.1 +/- 4.9 and in active smokers 48.1 +/- 4.3 years. Active smokers had their menopause on average by 1.7 years sooner than non-smokers (p less than 0.01). This difference depended on the duration of smoking and increased with the number of smoked cigarettes to as much as 2.4 years in smokers of greater than 20 cigarettes per day and to three years when also the mother of the investigated woman was a smoker (p less than 0.01).
