Biomedical subjects
Publications and source records attributed to P B Moens.
As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory.
The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.
Purified chromosome cores (synaptonemal complexes) of rat pachytene chromosomes, from which the chromatin is removed by extensive DNase II digestion, retain a residual class of DNA, presumably the bases of chromatin loops. This synaptonemal complex-associated DNA, isolated by proteinase digestion and phenol extraction of purified DNase-treated synaptonemal complexes, and cloned in plasmid vector pEMBL18, has a length distribution of 50-500 bp. From a library of these fragments, 21 fragments were sequenced. Present in this sample are short 40-200-bp segments with greater than 80% identity to "long" and "short" interspersed repeated elements (LINE/SINEs), an excess of GT/CA tandem repeats and a number of unidentified sequences. The LINE/SINE segments may play a role in homology vs. nonhomology recognition during meiosis and the alternating purine-pyrimidine sequences have been implicated in genetic recombination. Their enrichment in synaptonemal complexes may be related to the synapsis and recombination functions of meiosis.
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Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial core structure, the synaptonemal complex, SC, from which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.
Surface-spread pachytene chromosomes are several times the length of metaphase chromosomes and the decondensed chromatin loops are attached to a well-defined axis (Weith and Traut, 1980). This arrangement permits detailed DNA sequence localization by in situ hybridization. We show that two probes to low-frequency repeated sequences (20 to 50 copies) which locate the centromere proximal in the mouse X metaphase chromosome between bands A1 and A3 (Disteche et al., 1985) and which map 5.5 cM apart (Disteche et al., 1989), hybridize to two distinct chromatin regions 3 to 5 microns apart on a 25 microns long pachytene X chromosome core.
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At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II - a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation - is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.
Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.
Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66-55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66- to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)
This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.
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Synaptonemal complex (SC) analysis of six laboratory yeast strains showed the SC karyotypes to be repeatable within strains. Chromosomal differences were found between strains. In five of the strains, two SCs insert into the nucleolus. This represents a single bivalent with a nucleolar organizer in a medial position as is suggested by genetic data or two bivalents each with a terminal nucleolar organizer. In the first interpretation, n = 16; in the second, n = 17. Strain Tris has a single nucleolar SC and n = 17. In strains DCx374, DCx416 and x8366a the genetically determined rearrangements of linkage group III could not be identified. Presumably the short SC (0.33 micron) associated with linkage group III cannot accommodate an inversion loop or a translocation configuration. The strains however were found to harbour a reciprocal translocation involving the nucleolar chromosome. Trisomy for one of the longer chromosomes was observed in Tris and spo10. It is concluded that rearrangements of the medium and long but not short yeast chromosomes can be detected cytologically. Measurements of nuclear volumes show SC length to vary with artifactually induced swelling of the nucleus. Linear regression of SC length over nuclear radius indicates that actual SC length is only about one-half the observed length. As a result the DNA packing per SC unit length is higher than previously estimated.
Histone-depleted nuclei were prepared by high-salt extraction of interphase HeLa cell nuclei. A large amount of the nuclear DNA remained associated with a rapidly sedimenting residual nuclear structure including cytoplasmic (intermediate filament) and nuclear (matrix and lamina) proteins. Electron microscopy allowed detection in the insoluble structure of a residual nuclear envelope, nucleolar residues, and an intranuclear network whose correspondence with components of in situ fixed nuclei is discussed. Using three-dimensional electron microscopy, it is further demonstrated that the salt-insoluble structure remaining after histone depletion in 2 M NaCl is highly ordered. This is of the utmost importance when considering the roles reportedly ascribed to this structure in nuclear functions.
Actin and myosin are commonly involved in cell motility systems. This study attempts to identify these two proteins in the synaptonemal complex (SC), a nuclear organelle associated with chromosome pairing during meiotic prophase. The several experimental approaches reported here give no evidence of SC staining with either anti-myosin or anti-actin antibodies. These findings differ from those of other reportings.
An ultrastructural analysis of three yeast rad 6-1/rad 6-1 diploids on sporulation medium for 0, 6, 10, and 24 h shows that arrest occurs at meiotic prophase. Two strains, CL 139 and PU 6, fail to complete chromosome synapsis based on the continued presence of single chromosomal cores in arrested nuclei. A clone derived from CL 139, however, showed complete pairing as evident from the presence of 17 synaptonemal complexes. All three strains underwent spindle pole body duplication but the poles failed to form a proper metaphase I spindle. A revertant Rad 6+ isolated from CL 139 showed normal chromosome behaviour and normal kinetic functions. It is concluded that the absence of meiotic recombination in some Rad 6- strains may result from asynapsis, but that in other strains (e.g., CL 139s) recombination fails in spite of complete synapsis. In all cases the lack of sporulation is adequately explained by failure of the kinetic apparatus to form a metaphase I spindle.
During early meiotic development the yeast Saccharomyces cerevisiae has a characteristic nuclear dense body (NDB). It is shown that the NDB can also be induced in vegetatively growing cells through the inhibition of thymidylate synthetase which causes depletion of the dTMP pool and arrests DNA synthesis. The observations on NDBs and recombination levels suggest that thymidylate-stressed cells may activate parts of the meiotic pathway and, conversely, cells on sporulation medium may sense, among other things, reduced thymidylate levels and respond to the several stimuli by entering the meiotic pathway.