UBC Theses and Dissertations
Design and characterization of a system to test synthetic DNA sequences for telomere function in the yeast Saccharomyces cerevisiae Fettes, Mark Thompson
Specialized structures called telomeres are required for the stabilization and replication of eukaryotic chromosome ends, and of other linear DNA ends such as those of linear plasmids in the yeast Saccharomyces cerevisiae. In vitro DNA synthesis techniques were used to synthesize stretches of short, tandemly-repeating sequences similar to those known to be involved in telomere function in vivo. These sequences were cloned in a multiple cloning site in an B. coli circular plasmid, excised with restriction enzymes and used to construct perfect inverted repeats in F. coli/S. cerevisiae shuttle vectors. To avoid the difficulties involved in the propagation of long inverted repeats in conventional E. coli cloning strains, the recBCsbcBrecF strain JC8111 was used for their construction and replication. An inverted 224 base pah-repeat containing two copies of the yeast ARS1 region was faithfully propagated in this strain. However, most plasmids which contained inverted repeats of telomere-type tandemly repeating sequences underwent various degrees of rearrangement within the inverted repeat region. Plasmids which yielded restriction fragments from the inverted repeat region of approximately the right mobility on agarose gels were used to transform S. cerevisiae. DNA was prepared from the transformed yeast clones and analyzed by agarose gel electrophoresis and Southern blotting to determine whether the transforming plasmids were replicating as circular or linear forms. Resolution into stable linear plasmids was observed in several cases where an ARS1 region was immediately adjacent to the tandemly repeating sequences in both halves of the inverted repeat; it was not observed when the ARS1 regions were absent, nor when the telomere-type repeats were present in the opposite orientation to that found in vivo. However, interpretation of the results was hampered by the heterogeneity of the plasmid DNAs propagated in JC8111. For this reason it was concluded that this assay system would not be useful for an extensive study of telomere replication in yeast.
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