Humanized telomeres and an attempt to express a functional human telomerase in yeast.
The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase. In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA. In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae. We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro. However, there was no evidence for human telomerase activity at yeast telomeres in vivo. Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components. We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains. The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.
Bah
Amadou
A
Department of Microbiology and Infectiology, ARN/RNA Group, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Canada.
Bachand
François
F
Clair
Eveline
E
Autexier
Chantal
C
Wellinger
Raymund J
RJ
D002467:Cell Nucleus / Q000201:enzymology; D005816:Genetic Complementation Test; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D019098:Telomerase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000737:chemistry / Q000378:metabolism
D019098:Telomerase