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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 mutants fail to produce haploid meiotic products.  Isogenic kar3 (TC12) and KAR3 (DJ1 + pD154.12) cultures heterozygous for ade2, cyh2, and can1 were pregrown and induced  to undergo meiosis as described in Materials and Methods. At the  times indicated, aliquots were removed and plated onto YPD and  CM-Arg + cyclohexamide + canavanine. The parameter plotted  is the fraction of Ade−, Canr, Cyhr CFUs at each time point relative to the total CFUs on YPD plates. For the kar3 mutants, at  least 13,000 cells were plated at each time point. Ade−, Cyhr,  Canr colonies were never observed for the kar3 mutants.
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Figure 3: kar3 mutants fail to produce haploid meiotic products. Isogenic kar3 (TC12) and KAR3 (DJ1 + pD154.12) cultures heterozygous for ade2, cyh2, and can1 were pregrown and induced to undergo meiosis as described in Materials and Methods. At the times indicated, aliquots were removed and plated onto YPD and CM-Arg + cyclohexamide + canavanine. The parameter plotted is the fraction of Ade−, Canr, Cyhr CFUs at each time point relative to the total CFUs on YPD plates. For the kar3 mutants, at least 13,000 cells were plated at each time point. Ade−, Cyhr, Canr colonies were never observed for the kar3 mutants.

Mentions: To quantitatively measure whether the meiotic block in kar3 mutants was absolute, we designed a strain that would allow us to measure the efficiency with which a kar3 mutant produces viable haploid meiotic products compared to a KAR3 strain. Briefly, a homozygous kar3Δ::HIS3 diploid strain (TC12) was heterozygously marked for both canavanine and cyclohexamide resistances (CAN1/can1, CYH2/ cyh2). In both cases, the sensitive allele is dominant; therefore, with the exception of rare double recombinants, these diploids are unable to grow on medium containing canavanine and cyclohexamide. One quarter of the spores produced by such a diploid will inherit both the can1 and cyh2 genes and will grow in the presence of both compounds. In addition, the diploid strain is heterozygous for the ADE2 gene (ADE2/ade2) and produces white colonies, but haploid cells that have inherited only the recessive ade2 allele will produce red colonies. The haploidization frequency of this kar3 diploid was measured by inducing a culture to undergo synchronous meiosis and plating cells at timed intervals to measure the ratio of Ade−, Cyhr, Canr cells to the total number of viable cells (Fig. 3). An isogenic strain carrying a single copy number plasmid with the wild-type KAR3 gene (DJ1 + pD154.12) was used as the wild-type control. The wild-type culture began to produce Ade−, Canr, Cyhr cells at 8 h, and by 48 h 9.18% of the cells had this phenotype (100% sporulation would be expected to yield ∼12.5% Canr, Cyhr, Ade− cells). No Ade−, Canr, Cyhr colonies were ever detected for the mutant even after 3 d in sporulation medium (Fig. 3), indicating that the kar3 defect causes a robust block of the meiotic process.


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

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

kar3 mutants fail to produce haploid meiotic products.  Isogenic kar3 (TC12) and KAR3 (DJ1 + pD154.12) cultures heterozygous for ade2, cyh2, and can1 were pregrown and induced  to undergo meiosis as described in Materials and Methods. At the  times indicated, aliquots were removed and plated onto YPD and  CM-Arg + cyclohexamide + canavanine. The parameter plotted  is the fraction of Ade−, Canr, Cyhr CFUs at each time point relative to the total CFUs on YPD plates. For the kar3 mutants, at  least 13,000 cells were plated at each time point. Ade−, Cyhr,  Canr colonies were never observed for the kar3 mutants.
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Figure 3: kar3 mutants fail to produce haploid meiotic products. Isogenic kar3 (TC12) and KAR3 (DJ1 + pD154.12) cultures heterozygous for ade2, cyh2, and can1 were pregrown and induced to undergo meiosis as described in Materials and Methods. At the times indicated, aliquots were removed and plated onto YPD and CM-Arg + cyclohexamide + canavanine. The parameter plotted is the fraction of Ade−, Canr, Cyhr CFUs at each time point relative to the total CFUs on YPD plates. For the kar3 mutants, at least 13,000 cells were plated at each time point. Ade−, Cyhr, Canr colonies were never observed for the kar3 mutants.
Mentions: To quantitatively measure whether the meiotic block in kar3 mutants was absolute, we designed a strain that would allow us to measure the efficiency with which a kar3 mutant produces viable haploid meiotic products compared to a KAR3 strain. Briefly, a homozygous kar3Δ::HIS3 diploid strain (TC12) was heterozygously marked for both canavanine and cyclohexamide resistances (CAN1/can1, CYH2/ cyh2). In both cases, the sensitive allele is dominant; therefore, with the exception of rare double recombinants, these diploids are unable to grow on medium containing canavanine and cyclohexamide. One quarter of the spores produced by such a diploid will inherit both the can1 and cyh2 genes and will grow in the presence of both compounds. In addition, the diploid strain is heterozygous for the ADE2 gene (ADE2/ade2) and produces white colonies, but haploid cells that have inherited only the recessive ade2 allele will produce red colonies. The haploidization frequency of this kar3 diploid was measured by inducing a culture to undergo synchronous meiosis and plating cells at timed intervals to measure the ratio of Ade−, Cyhr, Canr cells to the total number of viable cells (Fig. 3). An isogenic strain carrying a single copy number plasmid with the wild-type KAR3 gene (DJ1 + pD154.12) was used as the wild-type control. The wild-type culture began to produce Ade−, Canr, Cyhr cells at 8 h, and by 48 h 9.18% of the cells had this phenotype (100% sporulation would be expected to yield ∼12.5% Canr, Cyhr, Ade− cells). No Ade−, Canr, Cyhr colonies were ever detected for the mutant even after 3 d in sporulation medium (Fig. 3), indicating that the kar3 defect causes a robust block of the meiotic process.

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