Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cd...Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cdc13 gene leads to telomere impairment, giving rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature-tolerant mutants called survivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36°C, however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1Δ mutants of S. cerevisiae at 36°C. Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic markers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate overnight, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36°C. Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10<sup>-</sup><sup>5</sup> ± 0.04.展开更多
In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stnlp and Tenlp genetically and physically, and is required for telomere end protection...In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stnlp and Tenlp genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Tenl participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Tenlp in a gelfiltration analysis using purified recombinant Cdc13p and Tenlp. Tenlp itself exhibits a weak DNA-binding activity, but enhances the telomeric TG1-3 DNA-binding ability of Cdc13p. Cdc13p is communoprecipitated with Tenlp. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Tenlp and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Tenlp enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.展开更多
文摘Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cdc13 gene leads to telomere impairment, giving rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature-tolerant mutants called survivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36°C, however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1Δ mutants of S. cerevisiae at 36°C. Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic markers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate overnight, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36°C. Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10<sup>-</sup><sup>5</sup> ± 0.04.
基金Acknowledgments We thank Ms Lu-Xia Xu for the help in antibody preparation, and other members in the Zhou lab. This work is supported by a Chinese Academy of Sciences-Max Planck Society Professorship, and grants from the National Natural Science Foundation of China （NSFC 30630018） and the Ministry of Science and Technology of China （2007CB914502）.
文摘In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stnlp and Tenlp genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Tenl participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Tenlp in a gelfiltration analysis using purified recombinant Cdc13p and Tenlp. Tenlp itself exhibits a weak DNA-binding activity, but enhances the telomeric TG1-3 DNA-binding ability of Cdc13p. Cdc13p is communoprecipitated with Tenlp. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Tenlp and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Tenlp enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.
