RESUMO
BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.
Assuntos
Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/metabolismo , Cromossomos , Endopeptidases , Meiose , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Clonagem Molecular , Primers do DNA , Hibridização in Situ Fluorescente , Mutação , Proteínas de Saccharomyces cerevisiae , SeparaseRESUMO
We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Proteínas Fúngicas/genética , Proteínas de Helminto/genética , Meiose/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas de Schizosaccharomyces pombe , Animais , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona , Sequência Conservada , Imunofluorescência , Proteínas de Helminto/análise , Proteínas de Helminto/metabolismo , Hibridização in Situ Fluorescente , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Filogenia , Proteínas de Saccharomyces cerevisiae , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismoRESUMO
Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, highly repetitive 18S-25S rRNA genes were mapped on Vicia faba chromosomes (2n = 12). The modified method of this horseradish peroxidase based enzymatic detection system gave satisfactory results that are comparable to fluorescent signal detection.
