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C Heyting

Publications and source records attributed to C Heyting.

At least 19 recordsLinked to original sources

Analysis of male meiotic "sex body" proteins during XY female meiosis provides new insights into their functions.

During male meiosis in mammals the X and Y chromosomes become condensed to form the sex body (XY body), which is the morphological manifestation of the process of meiotic sex chromosome inactivation (MSCI). An increasing number of sex body located proteins are being identified, but their functions in relation to MSCI are unclear. Here we demonstrate that assaying male sex body located proteins during XY female mouse meiosis, where MSCI does not take place, is one way in which to begin to discriminate between potential functions. We show that a newly identified protein, "Asynaptin" (ASY), detected in male meiosis exclusively in association with the X and Y chromatin of the sex body, is also expressed in pachytene oocytes of XY females where it coats the chromatin of the asynapsed X in the absence of MSCI. Furthermore, in pachytene oocytes of females carrying a reciprocal autosomal translocation, ASY associates with asynapsed autosomal chromatin. Thus the location of ASY to the sex body during male meiosis is likely to be a response to the asynapsis of the non-homologous regions [outside the pseudoautosomal region (PAR)] of the heteromorphic X-Y bivalent, rather than being related to MSCI. In contrast to ASY, the previously described sex body protein XY77 proved to be male sex body specific. Potential functions for MSCI and the sex body are discussed together with the possible roles of these two proteins.

Animals↗

Sister chromatid cohesion and recombination in meiosis.

Sister chromatids are associated from their formation until their disjunction. Cohesion between sister chromatids is provided by protein complexes, of which some components are conserved across the kingdoms and between the mitotic and meiotic cell cycles. Sister chromatid cohesion is intimately linked to other aspects of chromosome behaviour and metabolism, in particular chromosome condensation, recombination and segregation. Recombination, sister chromatid cohesion and the relation between the two processes must be regulated differently in mitosis and meiosis. In meiosis, cohesion and recombination are modified in such a way that reciprocal exchange and reductional segregation of homologous chromosomes are ensured.

Animals↗

Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat.

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place.

Animals↗

Association of mammalian SMC1 and SMC3 proteins with meiotic chromosomes and synaptonemal complexes.

In somatic cells, the heterodimeric Structural Maintenance of Chromosomes (SMC) proteins are involved in chromosome condensation and gene dosage compensation (SMC2 and 4), and sister chromatid cohesion and DNA recombination (SMC1 and 3). We report here evidence for an involvement of mammalian SMC1 and SMC3 proteins in meiosis. Immunofluorescence analysis of testis sections showed intense chromatin association in meiotic prophase cells, weaker staining in round spermatids and absence of the SMC proteins in elongated spermatids. In spermatocyte nuclei spreads, the SMC1 and SMC3 proteins localize in a beaded structure along the axial elements of synaptonemal complexes of pachytene and diplotene chromosomes. Both SMC proteins are present in rat spermatocytes and enriched in preparations of synaptonemal complexes. Several independent experimental approaches revealed interactions of the SMC proteins with synaptonemal complex-specific proteins SCP2 and SCP3. These results suggest a model for the arrangement of SMC proteins in mammalian meiotic chromatin.

Amino Acid Sequence↗

RADHA--a new male germ line-specific chromosomal protein of Drosophila.

A new chromosomal protein - RADHA - of Drosophila is described that is specific for the male germ line. It is encoded by a single-copy gene, located in the region 96C-D of D. melanogaster polytene chromosomes. Transcription of the radha gene is restricted to the primary spermatocyte stage. The protein initially accumulates in some of the Y-chromosomal lampbrush loops. After meiosis it is found in the nuclei of spermatids and might be involved in chromatin rearrangement processes in the male germ line. RADHA is a basic protein with a C-terminal leucine zipper region and several segments capable of forming coiled-coil structures.

Amino Acid Sequence↗

Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad.

Transcription controls active at the initial stages of meiosis are clearly key elements in the regulation of germinal differentiation. Transcription of the Sycp1 gene (synaptonemal complex protein 1) starts as early as the leptotene and zygotene stages. Constructs with Sycp1 5' upstream sequences directed the expression of reporter genes to pachytene spermatocytes in transgenic mice. A short fragment encompassing the transcription start (n.t. -54 to +102) was sufficient for stage-specific expression in the adult male and for temporal regulation during development. Upstream enhancer element(s) quantitatively regulating expression were localized in the region between -54 and -260. The gene is normally expressed both in the male and female gonads, but none of the promoter sequences active in the testis allowed the expression of reporter genes during meiosis in the ovary.

Animals↗

Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis.

Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved. Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ). NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref. 3). HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence. HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref. 4), which must be repaired by HR to ensure proper chromosome segregation. How the cell controls the choice between the two repair pathways is not understood. We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref. 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6). Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent. In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway.

Animals↗

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.

Amino Acid Sequence↗

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting.

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene - pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour.

Cell Nucleus↗

Localization of SCP2 and SCP3 protein molecules within synaptonemal complexes of the rat.

SCP2 and SCP3 are major protein components of the lateral elements (LEs) of synaptonemal complexes (SCs) of the rat, with Mrs of 173, 000 and 30,000. We performed a detailed immunocytochemical comparison of the localization of SCP2 and SCP3 within SCs at the electron microscopic level. The ultrastructural localization of SCP2 and SCP3 was analyzed by immunogold labeling of two types of preparations, namely surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. For each of the antisera used, the distribution of immunogold label over SCs in surface-spread spermatocytes differed significantly from the distribution of label on sections. We attributed this difference to artifacts caused by the surface-spreading technique, and therefore we relied on sections for the precise localization of epitopes. On sections, the distribution of label obtained with two antisera against nonoverlapping, widely separated fragments of SCP2 did not differ significantly. There was a small but significant difference between the labeling pattern obtained with an anti-SCP3 serum and the pattern obtained with either of the two antisera against fragments of SCP2; although for all three antisera the peak of the immunogold label coincided with the center of the LE, the distributions of label obtained with the antisera against fragments of SCP2 were asymmetrical, with a shoulder at the inner side of the LE, whereas the distribution of label obtained with anti-SCP3 serum was symmetrical. Furthermore, we observed fuzzy connections between the LEs that were labeled by anti-SCP2 but not anti-SCP3 antibodies. It is possible that labeling of these fuzzy bridges caused the shoulder in the gold label distributions obtained with anti-SCP2 antibodies.

Animals↗

RecA-like proteins are components of early meiotic nodules in lily.

Early meiotic nodules (also called recombination nodules) are proteinaceous structures about 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during early prophase I of meiosis. Although their function is unknown, early nodules may be involved in searching for DNA homology before synaptic initiation. Two potential components of early nodules are Rad51 and Dmc1 proteins. These proteins are important for meiotic recombination in eukaryotes and are homologous to RecA, the major protein that catalyzes homologous pairing and DNA strand exchange in prokaryotes. In addition, Rad51 has been localized by immunofluorescence in abundant foci that may correspond to early nodules in yeast, lily, and mouse. In yeast and lily, Dmc1 and Lim15, the lily homolog of Dmc1, colocalize with Rad51. Here, using electron microscopic immunogold localization to spreads of zygotene and early pachytene SCs from lily, we confirm that RecA-like proteins are components of early nodules. The antibody used was generated to full-length tomato Rad51 protein and binds to both Rad51 and Lim15 in immunoblots of lily primary microsporocyte proteins. The labeled early nodules are heterogeneous in size and are associated with both axial elements and SCs. There are two classes of early nodules, those that are densely labeled with gold and those that are not labeled at all. This result may be due to technical limitations associated with using spread preparations or to differences in the nodules themselves. The presence of Rad51 and/or Lim15 proteins in early nodules supports the hypothesis that early nodules are involved in recombination-related events during meiosis.

Journal Article↗

cDNA cloning and characterization of Npap60: a novel rat nuclear pore-associated protein with an unusual subcellular localization during male germ cell differentiation.

We have cloned and characterized a cDNA, Npap60, encoding a rat nuclear pore-associated protein. The 3-kb cDNA was obtained by antibody screening of a rat testis expression library. The predicted NPAP60 contains 381 amino acids with a composition of 25.6% charged residues and is highly hydrophilic. The Npap60 gene appears to be conserved in mouse, rat, and human. Immunofluorescence studies with anti-NPAP60 fusion protein antibody show that the NPAP60 protein colocalizes with nuclear pore complexes in RAT1A cells. The expression of Npap60 is about 10-20 times higher in rat testis than in somatic tissues. The subcellular localization of NPAP60 protein changes dramatically during male germ cell differentiation, from nuclear pore complex-like staining in spermatocytes to whole nucleus staining in spermatids and finally to a nuclear surface staining in mature spermatozoa. These changes are temporally and spatially related to nuclear reorganization during male germ cell differentiation.

Amino Acid Sequence↗

Human synaptonemal complex protein 1 (SCP1): isolation and characterization of the cDNA and chromosomal localization of the gene.

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes (homologs) during meiotic prophase. They consist of two proteinaceous axes, one along each homolog, that are connected along their length by numerous transverse filaments (TFs). The cDNA encoding one major component of TFs of SCs of the rat, rnSCP1, has recently been isolated and characterized. In this paper we describe the isolation and characterization of the cDNA encoding the human protein homologous to rnSCP1, hsSCP1. hsSCP1 and rnSCP1 have 75% amino acid identity. The most prominent structural features and amino acid sequence motifs of rnSCP1 have been conserved in hsSCP1. Most probably, hsSCP1 is functionally homologous to rnSCP1. The hsSCP1 gene was assigned to human chromosome 1p12-p13 by fluorescence in situ hybridization.

Amino Acid Sequence↗

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Amino Acid Sequence↗

Organization of SCP1 protein molecules within synaptonemal complexes of the rat.

SCP1, a major protein component of synaptonemal complexes (SCs), is probably a constituent of the transverse filaments (TFs). The protein consists of three domains: a short, proline-rich N-terminal part, a stretch of 700 amino acid residues capable of forming an amphipathic alpha-helix, and a C-terminal domain of 240 amino acid residues which is capable of binding to DNA. To analyze the orientation of SCP1 molecules within SCs, we elicited polyclonal antibodies against three non-overlapping fragments of SCP1, which comprise, respectively, the N-terminus, the C-terminus, and a fragment from the middle of the SCP1 molecule. Using these antibodies, we performed immunoelectron microscopy on SCs in two types of preparations, namely, surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. For each of the three antibodies used, the distribution of immunogold label on surface-spread spermatocytes differed significantly from the distribution of label on sections. Masking of SCP1 epitopes within the lateral elements (LEs) and the central element (CE) of SCs in surface-spread preparations and the influence of the surface morphology of the spreads on the labeling pattern were considered as possible explanations for these differences. We therefore relied on the results from sections for the localization of epitopes. On the basis of the distributions of immunogold label in Lowicryl sections and the predicted secondary structure and dimensions of SCP1 molecules, we present the following model: the C-terminus of SCP1 molecules lies in the inner half of the LE, the molecules protrude from the LE through the central region into the CE, and end up with their N-terminus between the center of the CE and the opposite LE, so that the N-termini of SCP1 molecules from opposite LEs overlap. The model has several implications for the assembly of SCs and the possible functions of SCP1.

Animals↗

Synaptonemal complexes: structure and function.

Synaptonemal complexes (SCs) are zipper-like structures which are assembled between homologous chromosomes during the prophase of the first meiotic division. Their assembly and disassembly correlate with the successive chromatin rearrangements of meiotic prophase, namely the condensation, pairing, recombination and disjunction of homologous chromosomes. It was originally thought that SCs created the preconditions for the homologous crossing over of chromosomes by bringing corresponding parts of homologous chromosomes in close apposition. However, this view has been gradually undermined during recent years, and ideas about the roles of SCs have radically changed. SCs are now considered to be structures that both control the number and distribution of reciprocal exchanges between homologous chromosomes (cross-overs) and convert cross-overs into functional chiasmata. How SCs fulfil these roles remains to be elucidated.

Animals↗