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D D Sears

Publications and source records attributed to D D Sears.

5 recordsLinked to original sources
PubMed1999

Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

Chromosomes, Artificial, Yeast↗
PubMed1995

Cis-acting determinants affecting centromere function, sister-chromatid cohesion and reciprocal recombination during meiosis in Saccharomyces cerevisiae.

We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role(s) of conserved centromere DNA elements (CDEI, CDEII and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEII delta 31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/--) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I.

Base Sequence↗
PubMed1994

An implanted recombination hot spot stimulates recombination and enhances sister chromatid cohesion of heterologous YACs during yeast meiosis.

Heterologous yeast artificial chromosomes (YACs) do not recombine with each other and missegregate in 25% of meiosis I events. Recombination hot spots in the yeast Saccharomyces cerevisiae have previously been shown to be associated with sites of meiosis-induced double-strand breaks (DSBs). A 6-kb fragment containing a recombination hot spot/DSB site was implanted onto two heterologous human DNA YACs and was shown to cause the YACs to undergo meiotic recombination in 5-8% of tetrads. Reciprocal exchanges initiated and resolved within the 6-kb insert. Presence of the insert had no detectable effect on meiosis I nondisjunction. Surprisingly, the recombination hot spots acted in cis to significantly reduce precocious sister-chromatid segregation. This novel observation suggests that DSBs are instrumental in maintaining cohesion between sister chromatids in meiosis I. We propose that this previously unknown function of DSBs is mediated by the stimulation of sister-chromatid exchange and/or its intermediates.

Base Sequence↗
PubMed1992

Meiotic recombination and segregation of human-derived artificial chromosomes in Saccharomyces cerevisiae.

We have developed a system that utilizes human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to study factors that contribute to the fidelity of meiotic chromosome transmission. Since aneuploidy for the YACs does not affect spore viability, different classes of meiotic missegregation can be scored accurately in four-viable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromatid loss, and meiosis II nondisjunction. Segregation of the homologous pair of 360-kilobase marker YACs was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment. By using this experimental system, a series of YAC deletion derivatives ranging in size from 50 to 225 kilobases was analyzed to directly assess the relationship between meiotic recombination and meiosis I disjunction in a genotypically wild-type background. The relationship between physical distance and recombination frequency within the human DNA segment was measured to be comparable to that of endogenous yeast chromosomal DNA--ranging from less than 2.0 to 7.7 kilobases/centimorgan. Physical analysis of recombinant chromosomes detected no unequal crossing-over at dispersed repetitive elements distributed along the YACs. Recombination between YACs containing unrelated DNA segments was not observed. Furthermore, the segregational data indicated that meioses in which YAC pairs failed to recombine exhibited dramatically increased levels of meiosis I missegregation, including both precocious sister chromatid separation and nondisjunction.

Animals↗