Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system.

Abstract

Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts. To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3-alleles were placed in a vector. Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3-allele. Many of these events included coconversions in which more than one marker along the allelic sequence was replaced. The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted. From these results the average length of conversion was determined to be approximately 0.5 kllobase. Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers. Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged. The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

“…Second, examination of independent events showed that the frequency with which each of the two HIS3 heteroalleles was converted to the wild type remained nearly constant in all the constructions. (As previously reported [1], a slight preference for conversion of the allele on the side of the plasmid close to the ampicillin marker exists no matter whether the heteroallele is the 5' or 3' member of the pair.) Limitations in homology which would diminish initiation would be expected to decrease the frequency with which the allele nearest the homology border is converted.…”

“…From left to right, the ratios are for the following sets of markers: the SmaI marker in pKD40 versus that in pBYA819 and pBYA918; the SmaI marker in pKD90 versus that in pBYA819 and pBYA918; the second ClaI marker to the 3' side in pBYA229 and pBYA209 versus that in pBYA219 and pBYA239; the Sacl marker to the 5' side in pBYA239 and pBYA209 versus that in pBYA219 and pBYA229; the SmaI marker in pKD400 versus that in pBYA819 and pBYA918; and the first Clal marker to the 3' side in pBYA229 and pBYA209 versus that in pBYA819 and pBYA918. The data for plasmids pBYA819 and pBYA918 are from our previous report (1). the rates, which are measures of exchange within the HIS3 gene, were not clearly diminished by the changes in the position of the homology border suggests that the same number of events were initiated in cases in which borders were closer to flanking markers as in cases with more extended homology.…”

“…Thus, in these plasmids, exchange could take place in either direction between the allelic segments to yield a wild-type HIS3 allele and events involv- b In these events, conversion of one of the heteroalleles occurs, but coconversion of noncontiguous markers on either the same or homologous allelic segment has also occurred. These events have been described as asymmetric discontinuous and symmetric in our previous work (1). The events associated with reciprocal recombination of flanking markers are indicated in parentheses.…”

“…The methodology used in constructing plasmids, the S. cerevisiae strain, SSL204, which contains the his3A200 deletion, the measurement of mitotic exchange rates by fluctuation analyses, and the means by which independent exchange events are analyzed have been described previously (1).…”

“…Current models of exchange postulate that homology is required to synapse two homologous sequences by the formation of heteroduplex DNA in which a double helix is formed from complementary strands of the participating homologs (7,17,27,30). Examination of exchange between sequences of limited homology should define the absolute length of DNA needed to effect gene conversion and to find whether homology must exist on both sides of a marker for it to be included in the conversion event. Previously (1,4) we have constructed plasmids which undergo intramolecular gene conversion and crossing over in S. cerevisiae between heteroallelic copies of the HIS3 gene * Corresponding author.…”