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PubMed1999

Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.

BACKGROUND: At M phase, cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed. RESULTS: We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type cyclin B1 and a phosphorylation mutant of cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances cyclin B1 import in vitro and in vivo, in a manner distinct from the previously described import of cyclin B1 mediated by importin beta. CONCLUSIONS: We show that phosphorylation of human cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.

Amino Acid Sequence↗
PubMed2002

Chromosome segregation: playing polo in prophase.

During mitosis, in most eukaryotes, cohesin is removed from chromosomes in two steps. A paper in the March issue of Molecular Cell identifies Polo-like kinase as a key regulator for the first step that releases much of cohesin during prophase.

Animals↗
PubMed2003

Active cyclin B1-Cdk1 first appears on centrosomes in prophase.

Cyclin B1-Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against cyclin B1 that primarily recognise the active form of the complex. We show that cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. We conclude that cyclin B1-Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.

Animals↗
PubMed2006

Reduced meiotic crossovers and delayed prophase I progression in AtMLH3-deficient Arabidopsis.

Characterization of AtMLH3, the Arabidopsis homologue of the prokaryotic MutL mismatch repair gene, reveals that it is expressed in reproductive tissue where it is required for normal levels of meiotic crossovers (COs). Immunocytological studies in an Atmlh3 mutant indicate that chromosome pairing and synapsis proceed with normal distribution of the early recombination pathway proteins. Localization of the MutS homologue AtMSH4 occurs, suggesting that double Holliday junctions (dHjs) are formed, but the MutL homologue AtMLH1, which forms a heterocomplex with AtMLH3, fails to localize normally. Loss of AtMLH3 results in an approximately 60% reduction in COs and is accompanied by a substantial delay of approximately 25 h in prophase I progression. Analysis of the chiasma distribution in Atmlh3 suggests that dHj resolution can occur, but in contrast to wild type where most or all dHjs are directed to form COs the outcome is biased in favour of a non-CO outcome by a ratio of around 2 to 1. The data are compatible with a model whereby the MutL complex imposes a dHj conformation that ensures CO formation.

Arabidopsis↗
PubMed2002

Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers.

The CHFR gene, which was recently cloned by Scolnick and Halazonetis in search for a novel mitotic checkpoint gene with fork-head association motifs, has been suggested to play a key role in the mitotic prophase checkpoint. In this study, we demonstrated tumor-specific aberrant hypermethylation of the promoter region of the CHFR gene in a significant fraction of lung cancers in association with loss of detectable levels of CHFR transcripts. Aberrant hypermethylation was observed in seven of 37 primary lung cancer cases. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored expression of the CHFR gene in lung cancer cell lines exhibiting aberrant hypermethylation and loss of its expression. In contrast, genetic alterations were found to be infrequent in lung cancers. This is the first description of aberrant hypermethylation of the CHFR gene in any type of human cancer, and provides further evidence of the involvement of multiple checkpoint alterations in lung cancer.

Base Sequence↗
PubMed1986

A fractionated cell-free system for analysis of prophase nuclear disassembly.

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.

Animals↗
PubMed1997

Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase.

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

Cell Nucleus↗
PubMed2005

Crossing over is coupled to late meiotic prophase bivalent differentiation through asymmetric disassembly of the SC.

Homologous chromosome pairs (bivalents) undergo restructuring during meiotic prophase to convert a configuration that promotes crossover recombination into one that promotes bipolar spindle attachment and localized cohesion loss. We have imaged remodeling of meiotic chromosome structures after pachytene exit in Caenorhabditis elegans. Chromosome shortening during diplonema is accompanied by coiling of chromosome axes and highly asymmetric departure of synaptonemal complex (SC) central region proteins SYP-1 and SYP-2, which diminish over most of the length of each desynapsing bivalent while becoming concentrated on axis segments distal to the single emerging chiasma. This and other manifestations of asymmetry along chromosomes are lost in synapsis-proficient crossover-defective mutants, which often retain SYP-1,2 along the full lengths of coiled diplotene axes. Moreover, a gamma-irradiation treatment that restores crossovers in the spo-11 mutant also restores asymmetry of SYP-1 localization. We propose that crossovers or crossover precursors serve as symmetry-breaking events that promote differentiation of subregions of the bivalent by triggering asymmetric disassembly of the SC.

Animals↗
PubMed1997

A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2.

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11). Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S), and mutations in three new genes designed SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S). Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks. These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.

Amino Acid Sequence↗
PubMed1985

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis.

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.

Animals↗
PubMed1993

A novel stage-specific differentiation antigen is expressed on mouse testicular germ cells during early meiotic prophase.

A murine cell surface antigen exhibiting stage-specific expression during spermatogenesis was detected with two monoclonal antibodies (mAbs), designated BC7 and CA12. In mouse testis, these mAbs recognized a small population of cells located near the periphery of seminiferous tubules at stages XII and I-VI, and these spermatogenic cells were identified as zygotene and early pachytene spermatocytes. Expression of the antigens was transient and was not detected in germ cells at more advanced stages of spermatogenesis such as late pachytene spermatocytes and round spermatids. Immunoprecipitation and immunoblotting studies showed that both mAbs CA12 and BC7 reacted with the same antigenic molecule, which had an estimated molecular mass of 95 kDa. CA12/BC7 antigen, detected in plasma membrane fraction, was a glycoprotein with sialic acid residues and had affinity with WGA lectin. Furthermore, intraperitoneal injection of mAb BC7 caused an apparent spermatogenic disturbance in prepubertal mice. These results suggested that CA12/BC7 antigen, a novel cell surface glycoprotein, is an essential molecule that plays an important role during early meiotic prophase of spermatogenesis.

Animals↗
PubMed1997

Retinoic acid receptor alpha gene expression in the rat testis: potential role during the prophase of meiosis and in the transition from round to elongating spermatids.

Mutational studies have identified retinoic acid receptor alpha (RAR alpha) as having an essential role in spermatogenesis. The objective of this study was to determine which cells express RAR alpha within the normal rat testis by conducting in situ analyses for mRNA and protein. Characterization of RAR alpha expression revealed the time and location of the vitamin A requirement during spermatogenesis. In situ hybridization analysis of testis from adult rats showed the highest level of transcripts occurring in round spermatids at stage VIII of the spermatogenic cycle. Analysis in the developing testes revealed that the mRNA level was high from 10 to 15 days of age, both in Sertoli cells and in germ cells, and then declined in 20-day-old rats. Consistent with this, immunohistochemical studies on adult testis demonstrated that the protein was present in the nucleus of the elongating spermatids (stages IX-XI) but not in elongated spermatids. The protein was also expressed in germ cells in the prophase of meiosis and at very low levels in Sertoli cells. These results suggest a role for RAR alpha during meiosis, at the transition from round to elongating spermatids, and in Sertoli cells of developing testis.

Aging↗
PubMed2004

A Dicer-like protein in Tetrahymena has distinct functions in genome rearrangement, chromosome segregation, and meiotic prophase.

Previous studies indicated that genome rearrangement involving DNA sequence elimination that occurs at late stages of conjugation in Tetrahymena is epigenetically controlled by siRNA-like scan (scn) RNAs produced from nongenic, heterogeneous, bidirectional, micronuclear transcripts synthesized at early stages of conjugation. Here, we show that Dcl1p, one of three Tetrahymena Dicer-like enzymes, is required for processing the micronuclear transcripts to scnRNAs. DCL1 is also required for methylation of histone H3 at Lys 9, which, in wild-type cells, specifically occurs on the sequences (IESs) being eliminated. These results argue that Dcl1p processes nongenic micronuclear transcripts to scnRNAs and is required for IES elimination. This is the first evidence linking nongenic micronuclear transcripts, scnRNAs, and genome rearrangement. Dcl1p also is required for proper mitotic and meiotic segregation of micronuclear chromosomes and for normal chromosome alignment in meiotic prophase, suggesting that DCL1 has multiple functions in regulating chromosome dynamics.

Animals↗
PubMed1985

RNA and protein synthesis during meiotic prophase in Tetrahymena thermophila.

Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained. We took advantage of this fact to monitor RNA and protein synthesis during meiosis. The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase. We therefore cloned cDNAs corresponding to genes active during this time. The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically. Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene). Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report. The significance of these results is discussed.

Animals↗
PubMed1990

C-band fusion and behaviour of the involved chromosomes during meiotic prophase I of the male domestic pig.

Behaviour of acrocentric chromosome synaptonemal complexes (SCs) and constitutive heterochromatin (C-band) during meiotic prophase I of the male domestic pig was studied using surface spreading and silver staining techniques. At late zygotene C-band regions of complete or incomplete SCs of the acrocentric chromosomes are represented by enlarged structures. At the same time these structures commenced fusion, which is accomplished at pachytene. Using kinetochores and NORs as markers, the pachytene metacentric, acrocentric, and nucleolar SCs can be distinguished. In 96.7% of the pachytene nuclei, either all or some of the acrocentric SCs are associated at their centromeric regions, forming one or two large globular or shapeless masses that represent chromocenters. The SCs are assorted at random in all types of association except in single association of all the SCs. Order of associated SCs varies from one association to another. The C-band region of SC, the portion inside the chromocenter core, is differentiated as thicker and darker structures from the chromocenter mass. A silver-positive core-like structure in meiotic chromosome C-bands, probably a vector for meiotic C-band ectopic pairing, provides faithful representations of C-band behaviour during meiosis.

Animals↗
PubMed1992

RecA-like strand-transfer activity at the meiotic prophase in Bombyx mori.

An ATP-independent strand-transfer activity has been identified in nuclear extracts prepared from Drosophila tissue culture cells and isolated nuclei from Bombyx testes. Extraction of the activity from testes at larval stages where the majority of the cells were in meiotic prophase was only possible when the chromosome scaffold/synaptonemal complex was dissolved by addition of high concentrations of DTT (80 mM). No cross reaction was detected when partly purified extracts were assayed with antibodies against E. coli RecA protein.

Animals↗
PubMed1990

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.

Animals↗
PubMed2003

Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase.

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.

Adaptor Proteins, Signal Transducing↗