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The yeast motor protein, Kar3p, is essential for meiosis I.

Bascom-Slack CA, Dawson DS - J. Cell Biol. (1997)

Bottom Line: The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown.We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type.These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

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kar3 cells are capable of initiating DSBs. Isogenic  KAR3 (DC99-1) or kar3 (DC99-1C) cultures, homozygous for  the rad50S-K181 allele were pregrown and induced to undergo  synchronous meiosis. Aliquots were removed at the number of  hours indicated after transfer to sporulation medium and DNA  was extracted. The DNA was digested with BglII, subjected to  electrophoresis on a 0.7% agarose gel, and analyzed by Southern  blot hybridization using as a probe the ARG4 sequences indicated in (B). (A) The predominant DSB and parental fragments  are labeled with arrows. (C) Quantification of the DSB products.  The fraction of radioactivity in each lane that was localized to the  DSB products was quantified. The percent of DSB product in  each lane was normalized to the final level of DSB product produced in the KAR3 strain. In the kar3 mutants, the DSBs accumulate to 18.6% of levels detected in the isogenic KAR3 strain.
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Figure 5: kar3 cells are capable of initiating DSBs. Isogenic KAR3 (DC99-1) or kar3 (DC99-1C) cultures, homozygous for the rad50S-K181 allele were pregrown and induced to undergo synchronous meiosis. Aliquots were removed at the number of hours indicated after transfer to sporulation medium and DNA was extracted. The DNA was digested with BglII, subjected to electrophoresis on a 0.7% agarose gel, and analyzed by Southern blot hybridization using as a probe the ARG4 sequences indicated in (B). (A) The predominant DSB and parental fragments are labeled with arrows. (C) Quantification of the DSB products. The fraction of radioactivity in each lane that was localized to the DSB products was quantified. The percent of DSB product in each lane was normalized to the final level of DSB product produced in the KAR3 strain. In the kar3 mutants, the DSBs accumulate to 18.6% of levels detected in the isogenic KAR3 strain.

Mentions: The reduced commitment to recombination could be an indication of a failure to either initiate or resolve recombination events. DSBs in the DNA are the initiating events in meiotic recombination and can be monitored using Southern analysis to detect the appearance of diagnostic restriction fragments. The ability of a kar3 mutant to form DSBs was measured by monitoring the accumulation of DSB products throughout meiosis in the region upstream of the ARG4 gene which contains hot spots for meiotic recombination (Sun et al., 1989). Identification of break products is facilitated by the presence of the rad50S mutation which allows DSBs to form but prevents their processing, resulting in the accumulation of what would otherwise be a transient product (Alani et al., 1990). The interhomologue recombinants measured in the commitment to recombination assay (above) are thought to arise from events initiated at DSBs. Restriction fragments diagnostic of the DSB products were apparent in both a KAR3 wild-type and kar3 mutant strain (Fig. 5 A). In the mutant, the breaks accumulated to 18.6% of wild-type levels (Fig. 5 C). The appearance of DSBs in the kar3 mutant demonstrates that, although this strain is unable to commit to wild-type levels of meiotic recombination, it is capable of initiating substantial levels of DSBs in a rad50S background. To verify that the differences in DSB accumulation are not unique to the ARG4 locus, we quantified DSBs upstream of the ARE1 gene on chromosome III, another hot spot for recombination (Goldway et al., 1993; Baudat and Nicolas, 1997). In a kar3 mutant, breaks accumulate to 33% of the level detected in an isogenic KAR3 strain (data not shown). For both the ARG4 and ARE1 loci, DSBs accumulate to a lesser amount in a kar3 mutant than in an isogenic KAR3 strain in the time span over which the breaks were assayed.


The yeast motor protein, Kar3p, is essential for meiosis I.

Bascom-Slack CA, Dawson DS - J. Cell Biol. (1997)

kar3 cells are capable of initiating DSBs. Isogenic  KAR3 (DC99-1) or kar3 (DC99-1C) cultures, homozygous for  the rad50S-K181 allele were pregrown and induced to undergo  synchronous meiosis. Aliquots were removed at the number of  hours indicated after transfer to sporulation medium and DNA  was extracted. The DNA was digested with BglII, subjected to  electrophoresis on a 0.7% agarose gel, and analyzed by Southern  blot hybridization using as a probe the ARG4 sequences indicated in (B). (A) The predominant DSB and parental fragments  are labeled with arrows. (C) Quantification of the DSB products.  The fraction of radioactivity in each lane that was localized to the  DSB products was quantified. The percent of DSB product in  each lane was normalized to the final level of DSB product produced in the KAR3 strain. In the kar3 mutants, the DSBs accumulate to 18.6% of levels detected in the isogenic KAR3 strain.
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Related In: Results  -  Collection

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Figure 5: kar3 cells are capable of initiating DSBs. Isogenic KAR3 (DC99-1) or kar3 (DC99-1C) cultures, homozygous for the rad50S-K181 allele were pregrown and induced to undergo synchronous meiosis. Aliquots were removed at the number of hours indicated after transfer to sporulation medium and DNA was extracted. The DNA was digested with BglII, subjected to electrophoresis on a 0.7% agarose gel, and analyzed by Southern blot hybridization using as a probe the ARG4 sequences indicated in (B). (A) The predominant DSB and parental fragments are labeled with arrows. (C) Quantification of the DSB products. The fraction of radioactivity in each lane that was localized to the DSB products was quantified. The percent of DSB product in each lane was normalized to the final level of DSB product produced in the KAR3 strain. In the kar3 mutants, the DSBs accumulate to 18.6% of levels detected in the isogenic KAR3 strain.
Mentions: The reduced commitment to recombination could be an indication of a failure to either initiate or resolve recombination events. DSBs in the DNA are the initiating events in meiotic recombination and can be monitored using Southern analysis to detect the appearance of diagnostic restriction fragments. The ability of a kar3 mutant to form DSBs was measured by monitoring the accumulation of DSB products throughout meiosis in the region upstream of the ARG4 gene which contains hot spots for meiotic recombination (Sun et al., 1989). Identification of break products is facilitated by the presence of the rad50S mutation which allows DSBs to form but prevents their processing, resulting in the accumulation of what would otherwise be a transient product (Alani et al., 1990). The interhomologue recombinants measured in the commitment to recombination assay (above) are thought to arise from events initiated at DSBs. Restriction fragments diagnostic of the DSB products were apparent in both a KAR3 wild-type and kar3 mutant strain (Fig. 5 A). In the mutant, the breaks accumulated to 18.6% of wild-type levels (Fig. 5 C). The appearance of DSBs in the kar3 mutant demonstrates that, although this strain is unable to commit to wild-type levels of meiotic recombination, it is capable of initiating substantial levels of DSBs in a rad50S background. To verify that the differences in DSB accumulation are not unique to the ARG4 locus, we quantified DSBs upstream of the ARE1 gene on chromosome III, another hot spot for recombination (Goldway et al., 1993; Baudat and Nicolas, 1997). In a kar3 mutant, breaks accumulate to 33% of the level detected in an isogenic KAR3 strain (data not shown). For both the ARG4 and ARE1 loci, DSBs accumulate to a lesser amount in a kar3 mutant than in an isogenic KAR3 strain in the time span over which the breaks were assayed.

Bottom Line: The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown.We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type.These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.

Show MeSH
Related in: MedlinePlus