The left telomere on chromosome iii in Saccharomyces cervisiae isolation and characterization

• Main Author: Button, Linda Louise
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/26778

Telomeres, the molecular ends of eukaryotic chromosomes, are essential for chromosome stability and complete replication of the termini. A yeast Saccharomvces cerevlslae telomerlc region was Isolated by chromosome walking from HML∝, the most distal known gene on the left end of chromosome III. The terminal Sail restriction fragment on IIIL (chromosome III left end) mapped 8.6 kb distal to HM∝ and was cloned in a circular vector to generate a probe for the MIL telomere.This Sail fragment on the end of chromosome IIIL was heterodisperse In length , having an average size of 3.3 kb and a length distribution of +/- 0.2 kb. Southern blot and DNA sequence analyses Indicated that the IIIL end conforms to the T-X class, rather than the T-Y'-X class, of yeast telomeres. The terminal (T) region on IIIL consists of 0.6 kb (+/- 0.2 kb) of 5'-C₁₋₃A-3' tandem repeat sequence, adjacent to the 1.2 kb type X ARS region. There is no homology to the type Y' ARS region between HML∝ and the IIIL telomere. The telomerlc probe from IIIL hybridized to multiple genomic restriction fragments; both heterodisperse and defined length bands were observed in Southern hybridization analyses and these mapped to chromosome ends. Heterogeneity of the length of the terminal fragment on a given chromosome end was observed among various yeast strains and this length variation was localized to the T region. The cloned fragment from the IIIL telomere lacked the terminus due to the cloning method used to Isolate It, however this T-X fragment functioned as a telomere on linear plasmids in yeast. Plasmids constructed in vitro with IIIL ends were maintained as linear molecules in yeast, and were more stable mitotlcally than linear plasmids constructed with Tetrahymena rDNA termini. Plasmids constructed with IIIL end fragments that had the entire T region deleted were replicated as circular molecules In yeast. Addition of Y" regions did not occur on any of the linear plasmids constructed with fragments from the IIIL telomere during replication in yeast, but T region was presumably added to the equivalent extent as that observed on the natural chromosome IIIL end [0.6 kb (+/- 0.2 kb)]. A rationale is discussed for the existence of two telomerlc classes in yeast, T-X and T-Y'-X, and for the addition of T region to chromosome ends in replication. The region adjacent to HM∝, on IIIL, which extends 8.6 kb toward the telomere, is homologous to an alternate telomeric region In the genome of haplold yeast strains. Southern hybridization analyses with HML∝ distal probes revealed that at least the terminal 6.5 kb of this region is retained In some circular chromosome III strains that were expected to have the IIIL and IIIR telomeric regions deleted. Northern hybridization indicated that an unidentified RNA transcript is homologous with a fragment from the HML∝ distal region. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate