Deformed lateral elements in synaptonemal complexes of Phaedranassa viridiflora

An electron microscopical study was made of an intermittent structural deformity in the lateral elements of synaptonemal complexes in pollen mother cells during zygotene of diploid and triploid forms of Phaedranassa viridiflora. The deformed regions, which extended linearly for distances up to 0.6 m in otherwise normal elements, are interpreted as hollow-centred bulges in a structure comprised of protein fibres apparently arranged in the form of a tightly wound helix. It is suggested that the bulging may arise if contraction of the lateral element is out of step with that in the chromosome, during prophase contraction at zygotene/ pachytene, as may perhaps follow under certain environmental conditions in organisms where there is some degree of genetical unbalance. There was evidence which suggested that local precocious duplication may sometimes have occurred in the chromosome axes proximal to the deformities in the lateral elements.     

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.     

Identification of two major components of the lateral elements of synaptonemal complexes of the rat

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.     

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.     

Immunocytogenetics. II. Human autoantibodies to synaptonemal complexes

Synaptonemal complex (SC) autoantibodies are spontaneously produced by patients with various autoimmune diseases. Immunofluorescence staining of pachytene cells localized the antigen to the central element or transverse filaments of the SC but not to the lateral elements. Specific antibody labeling was confined to the SC at synapsis. Cytochemical tests showed that the SC autoantigen is a basic protein possibly bound to DNA. An unusual characteristic of the SC autoantigen is its species specificity. Patients were found whose sera selectively labeled the SCs of other humans, mice, or newts. The combining of anti SC and anti-kinetochore antibodies provides a new immunocytochemical method for the analysis of SC karyotypes. The optimum conditions for preparation of pachytene cells for visualization by indirect immunofluorescence were determined. The nature and functions of the SC antigen, as well as possible applications of SC-specific autoantibodies in cytogenetics and cell biology, are discussed.     

The synaptonemal complexes of Caenorhabditis elegans

Normal synaptonemal complexes (SCs), consisting of two lateral elements and a central element, are present in wild-type, him-4 and him-8 mutant strains in both hermaphrodites and males of Caenorhabditis elegans. Thus, the increase in rate of nondisjunction in the him mutants is not related to aberrant SC morphology. The wild-type hermaphrodite has six SCs, as determined from 3-D reconstruction analysis of serial sections from electron microscopy. Thus, n = 6 and this confirms early reports based on cytological studies with the light microscope. Only one end of the SC is attached to the nuclear envelope while the other end is free in the nucleoplasm and there is no apparent bouquet formation. Either end of the SC can attach to the nuclear envelope. The pairing behavior of the XX bivalent is normal and occurs synchronously with the autosomes. Electron dense bodies, or knobs, are associated with the SC via the central element and displace the chromatin for a distance of 200 nm. Each pachytene nucleus of the wild-type hermaphrodite has six such structures that are randomly dispersed along the bivalents such that some SCs have one or two knobs while others have none. Their function is unknown.     

A new synaptic anomaly: irregular synaptonemal complexes

Summary In this paper we describe a new synaptic anomaly characterized by the presence of irregular synaptonemal complexes (SCs) in two sterile patients.     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Meiotic recombination and synaptonemal complexes in Saccharomyces cerevisiae.

Summary The course of meiotic recombination, gene conversion and crossing-over, was investigated in Saccharomyces cerevisiae. Gene conversion was used as the selected event by removing cells from a medium inducing and promoting meiosis to a vegetative growth medium selective for convertants. Gene conversion started to increase at the same time as DNA synthesis, and nuclei entered a phase where the chromatin appeared as thread-like structures. Crossing-over of linked and unlinked markers also started early but remained at a low level until synaptonemal complexes were formed. However, gene conversion and a limited amount of crossing-over could be completed without synaptonemal complexes. It was concluded that meiotic recombination in yeast can occur as early as during DNA synthesis and does not require the function of synaptonemal complexes. Moreover, the low incidence of crossing-over early in meiosis is attributed to a low frequency of strand isomerization.     

Spreading synaptonemal complexes of B isochromosomes in the grasshopper Omocestus burri.

Meiotic pairing behaviour of one and two B isochromosomes (iso-Bs) in the grasshopper Omocestus burri was analysed by electron microscopy in surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I. Iso-Bs display a peripheral location in the surface-spread nuclei and early pairing relative to that of the long members of the A set. Single iso-Bs undergo foldback pairing to give symmetrical hairpin loops. Two iso-Bs may show interarm pairing, mterchromosome pairing, or combinations of the two. Pericentromeric interarm pairing can be delayed in one or both Bs and this delay is mostly observed in bivalents with pairing partner switches. The iso-B bivalent frequencies observed in the three males analysed were 64, 44, and 41%, respectively; the two latter values were significantly lower than the 66% predicted by the random-end-pairing model. There is a reduction in the frequencies of iso-ring univalents (in 1B males) and bivalents (in 2B males) from pachytene to metaphase I. Similarities and differences between the pairing behaviour of iso-Bs from different species are also discussed.     

Mitotic recombination in the absence of synaptonemal complexes in Saccharomyces cerevisiae.

Summary Mitotic cells of a diploid strain of Saccharomyces cerevisiae with appropriate markers for the detection of mitotic crossing-over and mitotic gene conversion were irradiated with X-rays. Induction of these recombinational events was strong. After irradiation, cells were incubated in a rich growth medium and samples were removed for studying the possible formation of synaptonemal complexes up to a time when most cells had completed the first post-irradiation cell division. No complexes were found during the entire period of sampling, during which mitotic recombination in G₁ (mitotic gene conversion), DNA replication and G₂ (mitotic crossing-over) had occurred. These results are interpreted to mean that synaptonemal complexes are not required for mitotic recombination.     

Histone electrophoretic pattern in the characterization of synaptonemal complexes

The presence of a stoechiometric electrophoresis pattern of histones in carefully isolated synaptonemal complexes is reported. The use of this pattern is suggested as an internal standard of synaptonemal complex purification, in addition to the more generally used electron micrographs. This is especially useful in experiments leading to the characterization of the protein components of SCs. The use of mice of an age at which pachytenes predominate (90%) in the prophase-cell population is also advantageous to improve the final yield in synaptonemal complexes.     

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.     

The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes.

The lateral elements of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (M(r)S) of 30,000 and 33,000. After one-dimensional separation of SC proteins on polyacrylamide-sodium dodecyl sulfate gels, these components show up as two broad bands. These bands contain closely related proteins, as judged from their peptide maps and immunological reactivity. Using affinity-purified polyclonal anti-30,000- and anti-33,000-M(r) component antibodies, we isolated a cDNA encoding at least one of the 30,000- or 33,000-M(r) SC components. The protein predicted from the nucleotide sequence of the cDNA, called SCP3 (for synaptonemal complex protein 3), has a molecular mass of 29.7 kDa and a pI value of 9.4. It has a potential nucleotide binding site and contains stretches that are predicted to be capable of forming coiled-coil structures. In the male rat, the gene encoding SCP3 is transcribed exclusively in the testis. SCP3 has significant amino acid similarity to the pM1 protein, which is one of the predicted products of an X-linked lymphocyte-regulated gene family of the mouse: there are 63% amino acid sequence similarity and 35% amino acid identity between the SCP3 and pM1 proteins. However, SCP3 differs from pM1 in several respects, and whether the proteins fulfill related functions is still an open question.     

Isolation of synaptonemal complexes from hamster spermatocytes

Nuclei from Chinese hamster testicular cells in suspension were prepared in a sucrose gradient. Following the basic procedure of Blobel and co-workers for separating a fibrous lamina-nuclear pore complex, synaptonemal complexes (SCs) from spermatocytes were isolated free of other nuclear structures, except for fibrillar tufts at the attachment plaques in which annuli were observed. All the major morphological components of the SC appeared to be intact, showing that the structure could survive the procedure and was not dispersed by the removal of DNA with DNase and solubilization of membranes and some proteins with Triton X-100. Isolated sex bodies were also well preserved, as were various structures from other cell types in the mixed cell suspension, such as spermatid manchettes, acrosomal ‘ghosts’, axonemes, etc. While no nuclear matrix was found associated with autosomal SCs, a residual material was present in the sex body, in which the X and Y axes were embedded. The results indicate the feasibility of isolating and fractionating SCs from testicular cell suspensions enriched for pachytene spermatocytes. The association between SC attachment plaques and annuli that is seen in spreads of whole nuclei persists through the isolation procedure and implies an integrated structural relationship.     

Surface spreading of synaptonemal complexes in locusts

Details are given of techniques for preparing surface spreads of locust spermatocytes for light and electron microscopy. The pachytene synaptonemal complex (SC) karyotypes of Locusta migratoria and Schistocerca gregaria are analysed and compared. Up to six different SCs can be identified in Locusta migratoria based on lengths, centromere positions, and possession of nucleolar organiser regions, but only two SCs are identifiable in Schistocerca gregaria. The total SC length is significantly greater in Schistocerca gregaria than in Locusta migratoria, and this difference is almost exactly proportional to the difference in the genomic DNA contents of the two species.     

S. pombe genome deletion project: An update

The fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a PCR based strategy. However, deletion of some genes has not been possible using this methodology. Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. The collection of deletion mutants now covers 99% of the fission yeast open reading frames.     

Homolog pairing in S. pombe: the ends are the means

The pairing of homologous chromosomes is a universal feature of meiosis and is important for the accurate segregation of chromosomes in the first of two meiotic divisions. In the March issue of Developmental Cell, report findings in fission yeast which point to telomere clustering and movement as being important determinants of homolog pairing.     

Electron microscopy of spread maize pachytene synaptonemal complexes

The Counce-Meyer microspreading technique for animal synaptonemal complexes (SCs) has been adapted to allow spreading of the SCs of maize pachytene microsporocytes for examination in the electron microscope (EM). The spread nuclei were well dispersed and flattened, and unstained SCs could be seen with light microscope (LM) phase optics. After PTA or ammoniacal silver staining, the SCs and kinetochores were readily recognized in the EM. Variable degrees of asynapsis, stretching of the SCs, and nonhomologous synapsis of lateral elements were noted, and cases of interlocking of lateral elements or SCs were not uncommon. Distinct lens-shaped thickenings of one or both lateral elements were observed at numerous sites along the SC in most nuclei. — The yield of well spread, complete nuclei, although not high, was sufficient to allow karyotypes to be prepared, based on relative SC lengths and arm ratios. The karyotypes agreed well with published EM and LM determinations, establishing the accuracy of the spreading technique for maize. However, considerable variation in absolute lengths of the SCs was noted. To evaluate the utility of the technique for cytogenetic investigations, two paracentric inversions, and two reciprocal translocations were spread and examined in the EM. The breakpoints estimated from measurements of spread SCs were in agreement with LM determinations.     

Two-dimensional spreads of synaptonemal complexes from solanaceous plants. I. The technique

Using beta-glucuronidase the cell walls of tomato and potato primary microsporocytes can be digested. When the resulting protoplasts are exposed to distilled water, they burst, and complete sets of synaptonemal complexes are released to settle on plastic coated slides. After drying and formalin fixation, the synaptonemal complexes can be stained with silver or phosphotungstic acid and observed in the light and/or electron microscope. Silver staining gives better contrast for both light and electron microscopy but stains only lateral elements and kinetochores. Phosphotungstic acid staining gives little or no contrast for light microscopy, but stains both the lateral and central elements of the synaptonemal complex, kinetochores, and structures that are probably recombination nodules for electron microscopy. This technique offers a powerful tool for genome analysis by allowing (1) the determination of relative and absolute lengths of synaptonemal complexes and chromosome arm ratios at pachytene, (2) the analysis of complex patterns of synapsis, and (3) the location of what are probably recombination nodules along the length of synaptonemal complexes.