We have devised a method to detect meiotic recombination in a small interval of a chromosome of Saccharomyces cerevisiae. The ability to detect physically recombined DNA is based on the formation of novel restriction fragments arising by exchange between two parental chromosomes containing pairs of restriction endonuclease recognition site polymorphisms. This method has been used to determine the time of appearance of recombined DNA relative to other meiotic events, including the time of DNA synthesis, the time of commitment to meiotic recombination, and the time of meiotic chromosome segregations. This approach will be used to examine when during meiosis various meiotic-defective mutations are blocked, even when cells fail to yield viable recombinants. In addition, we have detected what appear to be intermediates arising during recombination, including double-strand breaks within a specific region. Further effort will be directed toward characterizing these intermediates and defining the DNA sequences responsible for their creation. We will also look for other types of intermediates, including regions of heteroduplex DNA using methods similar to those we have already developed. A second major effort will be to continue our characterization of DNA sequences that act as stimulators of meiotic recombination. We have already identified two yeast segments, both containing expressed yeast genes, that stimulate meiotic recombination when introduced into other chromosomal locations by recombinant DNA techniques. These elements stimulate recombination even when heterozygous. In vitro mutagenesis will be used to identify the DNA sequences responsible for the stimulation. Further genetic studies will also be carried out to determine if these sequences, when heterozygous, primarily promote recombination in adjacent regions to one side of the element.
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