Humanizing the yeast telomerase template.
Saccharomyces cerevisiae contains an irregular telomere sequence (TG1-3)n, which differs from the regular repeat (TTAGGG)n found at the telomeres of higher organisms including humans. We have modified the entire 16-nt template region of the S. cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n repeats, the human telomere sequence. Haploid yeast strains with the tlc1-human allele are viable with no growth retardation and express the humanized gene at a level comparable to wild type. Southern hybridization demonstrates that (TTAGGG)n repeats are added onto the yeast chromosome ends in haploid strains with the tlc1-human allele, and sequencing of rescued yeast artificial chromosome ends has verified the addition of human telomeric repeats at the molecular level. These data suggest that the irregularity of the yeast telomere sequence is because of the template sequence of the yeast telomerase RNA. Haploid strains with the tlc1-human allele will provide an important tool for studying the function of telomerase and its regulation by telomere-binding proteins, and these strains will serve as good hosts for human artificial chromosome assembly and propagation.
Henning
K A
KA
Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-4442, USA.
Moskowitz
N
N
Ashlock
M A
MA
Liu
P P
PP
D015139:Blotting, Southern; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000235:genetics; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D004247:DNA; D019098:Telomerase