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PubMed2005

Visualization of diffuse centromeres with centromere-specific histone H3 in the holocentric plant Luzula nivea.

Although holocentric species are scattered throughout the plant and animal kingdoms, only holocentric chromosomes of the nematode worm Caenorhabditis elegans have been analyzed with centromeric protein markers. In an effort to determine the holocentric structure in plants, we investigated the snowy woodrush Luzula nivea. From the young roots, a cDNA encoding a putative centromere-specific histone H3 (LnCENH3) was successfully isolated based on sequence similarity among plant CENH3s. The deduced amino acid sequence was then used to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea. These data from L. nivea are compared with those from C. elegans, and common features of holocentric chromosomes are discussed.

Amino Acid Sequence↗
PubMed1979

Roberts' syndrome. I. Cytological evidence for a disturbance in chromatid pairing.

In the rare developmental disorder Roberts' syndrome, prophase and metaphase chromosomes display premature sister-chromatid separation, most prominently at certain regions in which the chromatin is composed of highly reiterated base sequences. In addition, interphase nuclei present a striking distortion in their contours. The observed abnormalities are interpreted as evidence for the presence of a genetically determined disturbance affecting the normal mechanisms for pairing and disjoining of sister chromatids.

Abnormalities, Multiple↗
PubMed2006

Cell cycle control by daf-21/Hsp90 at the first meiotic prophase/metaphase boundary during oogenesis in Caenorhabditis elegans.

DAF-21, a Caenorhabditis elegans homologue of Hsp90, is expressed primarily in germline cells. Although mutations in the daf-21 gene affect animal fertility, its cellular roles have remained elusive. To phenocopy daf-21 mutations, we impaired the daf-21 function by RNA interference (RNAi), and found that oocytes skipped the diakinesis arrest and displayed a defective diakinesis arrest, which led to the production of endomitotic oocytes with polyploid chromosomes (Emo phenotype). The same Emo phenotype was also observed with RNAi against wee-1.3. To identify a cause for Emo, we examined the CDK-1 (Cdc2) phosphorylation status in Emo animals, since CDK-1 is a key regulator of the prophase/metaphase transition and is kept inactivated by WEE-1.3 kinase during prophase. We immunostained both daf-21(RNAi) and wee-1.3(RNAi) animals with anti-phosphorylated-CDK-1 antibody and observed no detectable phosphates on CDK-1 in either of the animals. We also examined WEE-1.3 expression in daf-21(RNAi) and found a significant reduction of WEE-1.3. These results indicate that CDK-1 was not phosphorylated in either daf-21(RNAi) or wee-1.3(RNAi) animals, and suggest that daf-21 was necessary for producing functional WEE-1.3. Thus, all together, we propose that DAF-21 indirectly regulates the meiotic prophase/metaphase transition during oocyte development by ensuring the normal function of WEE-1.3.

Animals↗
PubMed1985

Electron microscope autoradiography of DNA synthesis in the replication band of two hypotrichous ciliates.

The synthesis of DNA in two hypotrichous ciliates, Styx sp. and an amicronucleated strain of Oxytricha sp., was studied by high voltage (1000 kV) electron microscopy. High voltage EM permits use of thick sections (0.25-0.40 micron), including serial sections; thick sections produce strong autoradiographic images with relatively short exposure times. The autoradiographs show that DNA synthesis occurs in a narrow part of the rear zone of a replication band in the macronucleus. Macronuclear DNA synthesis occupies a substantial part of the interdivision interval, and micronuclear DNA synthesis in Styx sp. takes place in early prophase at a time when macronuclear DNA synthesis is in its terminal phase.

Animals↗
PubMed1995

The XY pair of the mink (Mustela vision) during different periods of testicular activity.

Synaptonemal complexes of the mink (Mustela vison) were examined during different stages of testicular activity to determine whether the distribution of prophase substages and the configuration of the sex complement are altered during pre-quiescent and regenerative phases compared to those detected during the breeding period. Spermatocytes obtained during pre-quiescence showed no differences from those of breeding season in terms of substage distribution, whereas those from regenerating testes were mainly in zygotene and early pachytene substages, reflecting the high mitotic activity of spermatogonia and their subsequent transit to meiosis. Based on the location of kinetochores on the sex complement, the synapsed segments were identified as the short arm of the X (Xp) and the long arm of the Y (Yq), although pairing of the X and Y beyond the "pseudoautosomal region" was frequently observed. In some spermatocytes, the entire Y chromosome synapsed with the X or split into two strands with only one strand "paired" with the X while the other remained unpaired. It is not clear at present whether the Y chromosome splitting is part of the mechanisms that prevent crossing over in the non-homologous segments of the sex complement that often undergo synapsis or a post-crossover phenomenon unrelated to pairing mechanisms.

Animals↗
PubMed1986

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study.

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.

Animals↗
PubMed1986

An electron microscopic study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis).

Nucleolus-organizing regions (NORs) at some stages of the cell cycle (G0 period, G2 period and mitosis), differing in the level of NOR transcription activity, were studied on ultra-thin serial sections of pig embryo kidney cells. Particular attention was given to the spatial organization of fibrillar centers (FCs) in the nucleoli and to comparing the number of FCs with that of NORs. It was shown that within nucleoli, FCs may be located singly or form groups surrounded by a dense fibrillar component. The overall number of FCs exceeds that of NORs 1.7-fold in the G0 period, and 4.6-fold in the G2 period. At prophase, with progressive condensation of chromosomes and NOR inactivation, the number of FCs decreases and at metaphase corresponds to the number of silver-stained NORs. The data thus obtained make it possible to conclude that in active nucleoli, the number of FCs is higher than that of NORs. Proceeding from these numerical and ultrastructural data, it is suggested that a single NOR of pig embryo kidney cells corresponds to one "fibrillar complex", i.e., to an association of a different number of FCs with the dense fibrillar component. These "fibrillar complexes" contain 1 to 3 FCs in the G0 period and 1 to 12 FCs in the G2 period.

Animals↗
PubMed1963

Chromosomal and nucleolar RNA synthesis in root tips during mitosis.

Comparative rates of RNA synthesis in chromatin and nucleolar fractions during mitosis in root-tip cells of Allium and Nigella were studied by pulse-labeling of cells with tritiated cytidine. Although the rate of RNA synthesis decreases in the condensing chromosomes during prophase, it remains normal in the nucleolar fraction as long as nucleoli are maintained. RNA synthesis stops in mitotic cells lacking distinct nucleoli. In the late telophase or very early interphase cells, RNA synthesis resumes at a faster rate in the pronucleolar bodies than in the chromatin.

Cell Division↗
PubMed1992

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

Animals↗
PubMed2006

Mouse emi1 has an essential function in mitotic progression during early embryogenesis.

For successful mitotic entry and spindle assembly, mitosis-promoting factors are activated at the G(2)/M transition stage, followed by stimulation of the anaphase-promoting complex (APC), an E3 ubiquitin ligase, to direct the ordered destruction of several critical mitotic regulators. Given that inhibition of APC activity is important for preventing premature or improper ubiquitination and destruction of substrates, several modulators and their regulation mechanisms have been studied. Emi1, an early mitotic inhibitor, is one of these regulatory factors. Here we show, by analyzing Emi1-deficient embryos, that Emi1 is essential for precise mitotic progression during early embryogenesis. Emi1(-/-) embryos were found to be lethal due to a defect in preimplantation development. Cell proliferation appeared to be normal, but mitotic progression was severely defective during embryonic cleavage. Moreover, multipolar spindles and misaligned chromosomes were frequently observed in Emi1 mutant cells, possibly due to premature APC activation. Our results collectively suggest that the late prophase checkpoint function of Emi1 is essential for accurate mitotic progression and embryonic viability.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1997

Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes.

Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.

Chromatin↗
PubMed1999

The msh2 gene of Schizosaccharomyces pombe is involved in mismatch repair, mating-type switching, and meiotic chromosome organization.

We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair, S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Alleles↗
PubMed1999

Splicing of the meiosis-specific HOP2 transcript utilizes a unique 5' splice site.

The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5' end of the gene. This intron contains a nonconsensus 5' splice site (GUUAAGU) that differs from the consensus 5' splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5' splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2 pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

5' Untranslated Regions↗
PubMed2002

The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice.

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.

Animals↗
PubMed1987

Pericentric inversion in human chromosome 1 and the risk for male sterility.

A pericentric inversion in chromosome 1 of a severely oligospermic human male is reported. Pachytene analysis in microspread preparations shows an absence of full loop formation in the inversion bivalent and only the rare occurrence of a partial loop. The majority of cells exhibit extensive asynapsis across the inverted segment, or a normal looking synaptonemal complex indicative of heterologous pairing along the length of the inversion. Crossing over is reduced in the No 1 bivalent with only a rare chiasma being seen in the inverted region at metaphase I. Males heterozygous for a pericentric inversion in chromosome 1 appear to be at severe risk for infertility brought about by spermatogenic disturbance. The dearth of full loops at prophase in this patient, and in other pericentric inversion cases studied both in man and other species, raises the question of whether recombinant offspring might be rarer than anticipated on a theoretical basis owing to asynapsis or early heterologous synapsis across inverted segments.

Adult↗
PubMed2004

FISH detection of ribosomal cistrons and assortment-distortion for X and B chromosomes in Dichroplus pratensis (Acrididae).

Assortment-distortion with respect to the X and NOR activity of a rare mitotically stable B chromosome (B(N)), was examined in 16 males of Dichroplus pratensis (Acrididae: Melanoplinae) from Argentine populations. In 1B individuals, the X and B associate preferentially during prophase I reaching a maximum level of association at zygotene. Frequency of X/B association remains relatively high up to diplotene-diakinesis and decreases steeply towards metaphase I. The percent X/B association at each stage is positively influenced by association at the previous stage, and interindividual variability in X/B association decreases as the frequency of association increases. Both chromosomes tended to preferentially orientate toward the same pole at MI (mean ratio of 16 individuals, 1.50:1) which determined an excess of XB and 00 second spermatocytes over X0 and 0B ones (1.39:1). No significant differences occurred between the MI, AI and MII assortment ratios. Fluorescent in situ hybridisation (FISH) confirmed that the B chromosome carries ribosomal genes and helped to establish that, during spermiogenesis, both the B and the normal NOR-bearing chromosome (S8) are clustered near the centriole adjunct region of spermatids. However, FISH failed to reveal the existence of inactive ribosomal cistrons in the X chromosome, as previously suggested, thus providing no support to a simple origin of the B from the X.

Animals↗
PubMed1990

Synaptonemal complex analysis of spermatocytes of Talpa occidentalis (Insectivora, Mammalia): autosomal synapsis and substaging of zygonema and pachynema.

Spermatocytes from the mole, Talpa occidentalis, a species that includes both XX males and intersexes, were surface-spread and silver-stained to substage meiotic prophase from early zygonema through pachynema. In zygonema, only the Z2 and Z3 substages were found. This stage differed in comparison with such species as the Chinese hamster, laboratory mouse, and deer mouse, which belong to orders other than Insectivora. Pachynema, in which five substages were established (P1-P5), seems to be a more homogeneous stage, and remarkable differences with respect to the above-mentioned species were not found. Synaptic adjustment was demonstrated in X-Y pairing. Nonhomologous pairing was evident at the Y-centromeric region and considered likely in the proximal arm of this chromosome. In addition to sequencing the events taking place during zygonema and pachynema in males from a wild population in which some members show sex reversal, our finding represents the first attempt to substage zygonema and pachynema in an Insectivore species, thus contributing to current knowledge of the nature and degree of variability in the mammalian synaptic process.

Animals↗