Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes.
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Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes. (+info)
Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition.
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Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit. (+info)
Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation.
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The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation. (+info)
Homologous chromosome pairing in wheat.
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Bread wheat is a hexaploid (AABBDD, 2n=6x=42) containing three related ancestral genomes, each having 7 chromosomes, giving 42 chromosomes in diploid cells. During meiosis true homologues are correctly associated in wild-type wheat, but a degree of association of related chromosomes (homoeologues) occurs in a mutant (ph1b). We show that the centromeres are associated in non-homologous pairs in all floral tissues studied, both in wild-type wheat and the ph1b mutant. The non-homologous centromere associations then become homologous premeiotically in wild-type wheat in both meiocytes and the tapetal cells, but not in the mutant. In wild-type wheat, the homologues are colocalised along their length at this stage, but the telomeres remain distinct. A single telomere cluster (bouquet) is formed in the meiocytes only by the onset of leptotene. The sub-telomeric regions of the homologues associate as the telomere cluster forms. The homologous associations at the telomeres and centromeres are maintained through meiotic prophase, although, during leptotene, the two homologues and also the sister chromatids within each homologue are separate along the rest of their length. As meiosis progresses, first the sister chromatids and then the homologues associate intimately. In wild-type wheat, first the centromere grouping, then the bouquet disperse by the end of zygotene. (+info)
Bloom's syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes.
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Bloom's syndrome (BS) is a rare autosomal recessive disorder of humans characterized by severe pre- and postnatal growth deficiency, immunodeficiency, genomic instability, and a predisposition to a wide variety of neoplasms. The genomic instability is evidenced in BS somatic cells as a high incidence of gaps and breaks, chromatid exchanges, chromosome rearrangements, and locus-specific mutations. BS arises from a mutation in BLM, a gene encoding a protein with homology to the RecQ helicase family. Men with BS are sterile; women have reduced fertility and a shortened reproductive span. The current immunocytological study on mouse spermatocytes shows that the BLM protein is first evident as discrete foci along the synaptonemal complexes (SCs) of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. BLM foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema. BLM colocalizes with the single-stranded DNA binding replication protein A, which has been shown to be involved in meiotic synapsis. However, there is a temporal delay in the appearance of BLM protein along the SCs relative to replication protein A, suggesting that BLM is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase. In late pachynema and into diplonema, BLM is more dispersed in the nucleoplasm, especially over the chromatin most intimately associated with the SCs, suggesting a possible involvement of BLM in resolution of interlocks in preparation for homologous chromosome disjunction during anaphase I. (+info)
Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.
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An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination. (+info)
Characterization of human zona pellucida glycoproteins.
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The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy. (+info)
A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.
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Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination. (+info)