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Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II.

Sharif WD, Glick GG, Davidson MK, Wahls WP - Cell Chromosome (2002)

Bottom Line: CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I.Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I.In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. WahlsWayneP@uams.edu

ABSTRACT
BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

No MeSH data available.


Related in: MedlinePlus

Structure of the rec12 gene and constructs used. (A) Intron/exon structure. Genomic DNA (D) and total RNA (R) obtained from meiotic cultures of strain WSP0020 were subject to PCR and RT-PCR, respectively, using primers flanking the putative introns (a-d). Products were resolved on a 2% agarose gel stained with EtBr. (B) Primary sequence of Rec12 protein based upon the DNA sequence of a complementing cDNA clone. Residues of Rec12 with at least 50% identity (black boxes) or 50% conservation (gray boxes) relative to other eukaryotic Spo11 family members are indicated. Also shown are the positions of the active site tyrosine (*) and residues conserved among other Spo11 family members, but not conserved in S. pombe (open boxes). Alignments evaluated proteins from S. pombe (P40384), Neurospora crassa (Q9P6Y7), Coprinus cinereus (Q9P4D2), Homo sapiens (Q9NQM7), Mus musculus (Q9QZS1), Arabidopsis thaliana (AAL01152), Drosophila melanogaster (O77205), Caenorhabditis elegans (Q22236), and Saccharomyces cerevisiae (P23179). (C) Structure of constructs. Gene targeting of the endogenous rec12+ locus was used to introduce a  allele (rec12-D15::ura4+) lacking the complete coding region and a point mutation allele (rec12-Y98F) encoding a protein in which the active site tyrosine at position 98 was replaced with phenylalanine. Placing the rec12+ coding region and the rec12+ cDNA into the various pREP plasmids [32] allowed for a wide range of regulated gene expression.
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Figure 1: Structure of the rec12 gene and constructs used. (A) Intron/exon structure. Genomic DNA (D) and total RNA (R) obtained from meiotic cultures of strain WSP0020 were subject to PCR and RT-PCR, respectively, using primers flanking the putative introns (a-d). Products were resolved on a 2% agarose gel stained with EtBr. (B) Primary sequence of Rec12 protein based upon the DNA sequence of a complementing cDNA clone. Residues of Rec12 with at least 50% identity (black boxes) or 50% conservation (gray boxes) relative to other eukaryotic Spo11 family members are indicated. Also shown are the positions of the active site tyrosine (*) and residues conserved among other Spo11 family members, but not conserved in S. pombe (open boxes). Alignments evaluated proteins from S. pombe (P40384), Neurospora crassa (Q9P6Y7), Coprinus cinereus (Q9P4D2), Homo sapiens (Q9NQM7), Mus musculus (Q9QZS1), Arabidopsis thaliana (AAL01152), Drosophila melanogaster (O77205), Caenorhabditis elegans (Q22236), and Saccharomyces cerevisiae (P23179). (C) Structure of constructs. Gene targeting of the endogenous rec12+ locus was used to introduce a allele (rec12-D15::ura4+) lacking the complete coding region and a point mutation allele (rec12-Y98F) encoding a protein in which the active site tyrosine at position 98 was replaced with phenylalanine. Placing the rec12+ coding region and the rec12+ cDNA into the various pREP plasmids [32] allowed for a wide range of regulated gene expression.

Mentions: While rec12+ was reported to encode a protein of 139 amio acids [30], conceptual translation of the surrounding region suggested that rec12+ encoded a larger protein with homology to Spo11 of S. cerevisiae and Top6A of Sulfolobus shibatae [17,21]. Alignments and splicing sequence profiles [31] were used to identify four putative introns (Figure 1A). Oligonucleotide primer pairs flanking each of the putative introns were used for PCR and RT-PCR to amplify genomic DNA and meiotic mRNA, respectively. In each case, the product from mRNA was shorter than that from genomic DNA, confirming the presence of four introns (Figure 1A).


Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II.

Sharif WD, Glick GG, Davidson MK, Wahls WP - Cell Chromosome (2002)

Structure of the rec12 gene and constructs used. (A) Intron/exon structure. Genomic DNA (D) and total RNA (R) obtained from meiotic cultures of strain WSP0020 were subject to PCR and RT-PCR, respectively, using primers flanking the putative introns (a-d). Products were resolved on a 2% agarose gel stained with EtBr. (B) Primary sequence of Rec12 protein based upon the DNA sequence of a complementing cDNA clone. Residues of Rec12 with at least 50% identity (black boxes) or 50% conservation (gray boxes) relative to other eukaryotic Spo11 family members are indicated. Also shown are the positions of the active site tyrosine (*) and residues conserved among other Spo11 family members, but not conserved in S. pombe (open boxes). Alignments evaluated proteins from S. pombe (P40384), Neurospora crassa (Q9P6Y7), Coprinus cinereus (Q9P4D2), Homo sapiens (Q9NQM7), Mus musculus (Q9QZS1), Arabidopsis thaliana (AAL01152), Drosophila melanogaster (O77205), Caenorhabditis elegans (Q22236), and Saccharomyces cerevisiae (P23179). (C) Structure of constructs. Gene targeting of the endogenous rec12+ locus was used to introduce a  allele (rec12-D15::ura4+) lacking the complete coding region and a point mutation allele (rec12-Y98F) encoding a protein in which the active site tyrosine at position 98 was replaced with phenylalanine. Placing the rec12+ coding region and the rec12+ cDNA into the various pREP plasmids [32] allowed for a wide range of regulated gene expression.
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Related In: Results  -  Collection

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Figure 1: Structure of the rec12 gene and constructs used. (A) Intron/exon structure. Genomic DNA (D) and total RNA (R) obtained from meiotic cultures of strain WSP0020 were subject to PCR and RT-PCR, respectively, using primers flanking the putative introns (a-d). Products were resolved on a 2% agarose gel stained with EtBr. (B) Primary sequence of Rec12 protein based upon the DNA sequence of a complementing cDNA clone. Residues of Rec12 with at least 50% identity (black boxes) or 50% conservation (gray boxes) relative to other eukaryotic Spo11 family members are indicated. Also shown are the positions of the active site tyrosine (*) and residues conserved among other Spo11 family members, but not conserved in S. pombe (open boxes). Alignments evaluated proteins from S. pombe (P40384), Neurospora crassa (Q9P6Y7), Coprinus cinereus (Q9P4D2), Homo sapiens (Q9NQM7), Mus musculus (Q9QZS1), Arabidopsis thaliana (AAL01152), Drosophila melanogaster (O77205), Caenorhabditis elegans (Q22236), and Saccharomyces cerevisiae (P23179). (C) Structure of constructs. Gene targeting of the endogenous rec12+ locus was used to introduce a allele (rec12-D15::ura4+) lacking the complete coding region and a point mutation allele (rec12-Y98F) encoding a protein in which the active site tyrosine at position 98 was replaced with phenylalanine. Placing the rec12+ coding region and the rec12+ cDNA into the various pREP plasmids [32] allowed for a wide range of regulated gene expression.
Mentions: While rec12+ was reported to encode a protein of 139 amio acids [30], conceptual translation of the surrounding region suggested that rec12+ encoded a larger protein with homology to Spo11 of S. cerevisiae and Top6A of Sulfolobus shibatae [17,21]. Alignments and splicing sequence profiles [31] were used to identify four putative introns (Figure 1A). Oligonucleotide primer pairs flanking each of the putative introns were used for PCR and RT-PCR to amplify genomic DNA and meiotic mRNA, respectively. In each case, the product from mRNA was shorter than that from genomic DNA, confirming the presence of four introns (Figure 1A).

Bottom Line: CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I.Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I.In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. WahlsWayneP@uams.edu

ABSTRACT
BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

No MeSH data available.


Related in: MedlinePlus