INTRODUCTION

During early prophase of meiosis in most sexually reproduc-ing organisms, each chromosome develops a longitudinal axialcore to which the chromatin loops are attached (Fig. 1). Sub-sequently, the cores of each pair of homologous chromosomesbecome aligned in parallel, thereby forming the lateralelements of the synaptonemal complex (SC). Later, the coresseparate while remaining attached at a few points, presumablythe sites of crossovers (von Wettstein, 1977; von Wettstein etal., 1984). Meiotic functions of the SCs have been detected inthe yeast,

Saccharomyces cerevisiae, where the Hop1 proteinof the lateral element facilitates between-homologue recombi-nation (Hollingsworth et al., 1990), the Zip1 protein of the SCfunctions in the pairing of homologues (Sym et al., 1993), andtopoisomerase II as a component of the SC functions in chro-mosome segregation at meiosis (Rose et al., 1990; Klein et al.,

1992). In mammals, a number of SC-specific and SC-associ-ated proteins have been identified (Heyting et al., 1987, 1988;Smith and Benevente, 1992; Chen et al., 1992; Moens et al.,1992; Moens and Earnshaw, 1989). In the absence of gene dis-ruption experiments, their functions can be inferred indirectlyfrom known functions, e.g. topoisomerase II, from knownamino acid sequence motifs (Meuwissen et al., 1992), fromtheir location (Heyting et al., 1987), or from assays that detectSC protein-protein or SC protein-DNA interactions. As thesecharacteristics become known for several SC proteins, a com-prehensive picture of SC function can be assembled.

In an attempt to determine the occurrence and organizationof meiosis-specific chromosome proteins, we constructed acDNA expression library from hamster spermatocytes. Thislibrary was screened with sera from rabbits and mice inocu-lated with hamster SCs to identify clones producing SCantigens (Moens et al., 1992). Here we report characteristics

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We have used polyclonal antibodies against fusion proteinsproduced from cDNA fragments of a meiotic chromosomecore protein, Cor1, and a protein present only in thesynapsed portions of the cores, Syn1, to detect the occur-rence and the locations of these proteins in rodent meioticprophase chromosomes. The 234 amino acid Cor1 proteinis present in early unpaired cores, in the lateral domains ofthe synaptonemal complex and in the chromosome coreswhen they separate at diplotene. A novel observationshowed the presence of Cor1 axial to the metaphase I chro-mosomes and substantial amounts of Cor1 in associationwith pairs of sister centromeres. The centromere-associ-ated Cor1 protein becomes dissociated from the cen-tromeres at anaphase II and it is not found in mitoticmetaphase centromeres. The extended presence of Cor1suggests that it may have a role in chromosome disjunctionby fastening chiasmata at metaphase I and by joining sisterkinetochores, which ensures co-segregation at anaphase I.Two-colour immunofluorescence of Cor1 and Syn1 demon-strates that synapsis between homologous cores is initiatedat few sites but advances rapidly relative to the establish-ment of new initiation sites. If the rapid advance of synapsisdeters additional initiation sites between pairs of homo-

logues, it may provide a mechanism for positive recombi-nation interference. Immunogold epitope mapping of anti-bodies to four Syn1 fusion proteins places the aminoterminus of Syn1 towards the centre of the synaptonemalcomplex while the carboxyl terminus extends well into thelateral domain of the synaptonemal complex. The Syn1fusion proteins have a non-specific DNA binding capacity.Immunogold labelling of Cor1 antigens indicates that thelateral domain of the synaptonemal complex is about twiceas wide as the apparent width of lateral elements whenstained with electron-dense metal ions. Electronmicroscopy of shadow-cast surface-spread SCs confirmsthe greater width of the lateral domain. The implication ofthese dimensions is that the proteins that comprise thesynaptic domain overlap with the protein constituents ofthe lateral domains of the synaptonemal complex morethan was apparent from earlier observations. This arrange-ment suggests that direct interactions might be expectedbetween some of the synaptonemal complex proteins.

Key words: synaptonemal complex, immunocytochemistry, cDNAlibrary, fusion protein, rodent spermatocyte, chromosome disjunction

SUMMARY

Synaptonemal complex proteins: occurrence, epitope mapping and

chromosome disjunction

Melanie J. Dobson*, Ronald E. Pearlman, Angelo Karaiskakis, Barbara Spyropoulos and Peter B. Moens†

Department of Biology, York University, Downsview, Ontario, M3J 1P3, Canada

*Present address: Department of Biochemistry, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada†Author for correspondence

Journal of Cell Science 107, 2749-2760 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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of two meiotic chromosome components whose encodingcDNAs were isolated with this approach. The SYN1 geneencodes a protein found only where the chromosome cores aresynapsed. Accordingly, this protein is named Syn1 (meioticchromosome Synaptic protein). The COR1 gene encodes aproduct that is a component of the chromosome core in themeiotic prophase chromosomes, referred to here as Cor1protein (meiotic chromosome Core protein). In this report,immunofluorescent and immunogold labelling are used todetermine the occurrence and locations of Syn1 and Cor1antigens in rodent spermatocytes.

MATERIALS AND METHODS

cDNA library The construction of a Syrian golden hamster (Mesocricetus auratus)spermatocyte cDNA expression library has been reported previously(Moens et al., 1992). Briefly, the library was constructed from thepoly(A)+ mRNA fraction of purified spermatocytes of young (25 day)males in the

λZap II vector (Stratagene, La Jolla, CA), and wasscreened (Heyting and Dietrich, 1991) with SC-reactive serum fromtwo rabbits and a mouse that had been inoculated with purifiedhamster SCs.

Because the sera could contain antibodies against antigens otherthan meiotic chromosome proteins, the immunoreactive λ clones werescreened by affinity elution (Sambrook et al., 1989). The eluted anti-bodies were immune tested on spermatocytes in order to detect thosephage clones that have a cDNA insert that produces meiotic chromo-some antigens. The inserts of choice were excised in vivo as Blue-script plasmids following the manufacturer’s protocol (Stratagene).Double- and single-stranded DNA isolated from these Bluescriptplasmids or suitable subclones were sequenced in their entirety withthe dideoxy-chain termination method of Sanger et al. (1977).

Production of SC fusion proteinsHamster SC antigens were produced in Escherichia coli by sub-cloning partial cDNAs in the expression vector pGEX-2T (Pharmacia)to give a translational fusion of the cDNA fragment at the carboxylterminus of the glutathione S-transferase (GST) protein. Fusionproteins derived from the SYN1 gene are named Syn1a, -b, etc. andthose derived from the COR1 gene are named Cor1a, -b, etc.Expression of the fusion protein in these vectors is under the control

of the highly inducible tac promoter. Expression and purification ofthe GST-SC fusion proteins were carried out according to the manu-facturer’s protocol: transformed Escherichia coli cultures were grownto an A600 of 0.7 in YT medium supplemented with 50 µg/ml ampi-cillin before gene expression was induced for 1 hour at 37°C by theaddition of 0.1 mM IPTG. Cultures were then chilled to 4°C,harvested by centrifugation, washed with cold phosphate bufferedsaline (PBS) and resuspended in cold PBS to which the following hadbeen added: 50 mM EDTA, 10 mM DTT, 0.5 mM PMSF, 0.5 µg/mlleupeptin, 0.01 TIU/ml aprotinin. Triton X-100 was sometimes addedto the extraction buffer at a final concentration of 1% but it was notfound to aid the solubilization of any of the fusion proteins. Cells werelysed by sonication and a freeze-thaw cycle. The lysed mixture wascentrifuged for 10 minutes at 4°C at 12,000

g. The supernatant fromthis spin was taken as the soluble protein and the pellet was washeda further time in the lysis buffer before being resuspended in lysisbuffer as the insoluble protein. For injection into mice or rabbits, theinsoluble protein from SC-positive bacterial extracts was washedtwice in cold PBS and resuspended in this same buffer at a concen-tration of 0.5 mg/ml.

Analysis of fusion proteinsExtracts of E. coli induced with IPTG and containing GST fusionproteins were analysed for the presence of SC antigens by westernblotting. Soluble protein, insoluble protein or total protein wereseparated by SDS-PAGE (Laemmli, 1970) in 10% running gels with4% stacking gels and with a 29:1 (w/w), acrylamide:bisacrylamideratio. Of duplicate gels, one was stained with Coomassie Blue andprotein molecular masses were estimated from the relative mobilitiesof standard proteins (Bio-Rad Low Molecular Weight Markers). Theduplicate gels had Amersham Rainbow Molecular Weight Markersand they were transferred to nitrocellulose membranes for stainingwith anti-SC antibodies by standard protocols (Sambrook et al.,1989). Transfers were incubated with the primary anti-hamster SCserum originally used to detect the SC-positive clones in the Strata-gene λZap expression library. SC-positive bacterial extracts wereused to generate antibodies to fusion proteins in mice and rabbits.Prior to use, the SC-positive serum from mice injected with bacterialfusion proteins was incubated with E. coli extract (Promega) toremove anti-E. coli antibodies. These primary antibodies were dilutedeither 1/1000 or 1/3,000 in antibody dilution buffer (ADB) (10%, v/v,goat serum; 3%, w/v, BSA; 0.05%, v/v, Triton X-100 in PBS).Secondary antibody was a 1/3000 dilution of goat anti-rabbit or goatanti-mouse conjugated to alkaline phosphatase and the positivepolypeptides were identified with the NBT-BCIP colour reaction(Sambrook et al., 1989).

For Southwestern analysis, SDS-PAGE gels were electrophoresedas usual, washed with gentle shaking for 30 minutes in a large volumeof transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 20%methanol) to remove the SDS and partially renature the proteins priorto transfer to the nitrocellulose membrane. After transfer, filters werewashed twice for 15 minutes in blocking-binding-wash buffer, BBW(3 g/l Ficoll, 3 g/l polyvinylpyrollidone, 10 mM NaCl, 20 mM Tris,pH 8) and were then incubated with 32P-radiolabelled probe DNA(1×108 cpm/µg) for 1 hour at room temperature in 1 ml of BBW at107 cpm/ml as described by Mellor et al. (1985). Filters were subse-quently washed for 2 hours at room temperature in BBW before beingexposed to X-ray film. Probes were end-labelled by a fill-in reactionusing Klenow polymerase according to Sambrook et al. (1989).Binding was stable up to 100 mM NaCl.

Production of antibodies to fusion proteinsMice and rabbits with SC-negative preimmune serum were inoculatedtwo or more times with a 1:1 mixture of 50 µg insoluble SC-positiveE. coli extract (inclusion bodies; Meuwissen et al., 1992) withadjuvant per mouse per intraperitoneal injection, or 150 µg proteinextract with adjuvant per rabbit per subcutaneous injection. Cor1a

M. J. Dobson and others

Fig. 1. A diagrammatic representation of the conventional image of asynaptonemal complex, SC. Meiotic prophase chromosome cores(co) with the attached chromatin (ch) become parallel aligned duringchromosome synapsis and are then referred to as the lateral elements(le) of the SC. There are transverse filaments (tf) between the lateralelements and there is a median central element (ce). The SCs areusually attached to the nuclear envelope (ne).

ne ch

tf

co

cele

2751SC proteins

protein extract proved toxic to mice at this level but was toleratedwhen injections were reduced to 5 µg doses. To enhance antibody pro-duction, Syn1 fusion proteins were incubated with rabbit anti-SCantibody for injection into rabbits and Cor1 fusion proteins with theoriginal mouse anti-SC antibody for injection into mice. The immunesera were reacted with E. coli extract to remove anti-bacterial proteinantibodies. The immune sera were tested at dilutions of 1/1,000 and1/10,000 on surface-spread hamster, rat and mouse testicular cells, oncryosections of various body tissues, on western blots of spermato-cyte nuclear proteins, and on western blots of bacterial extractproducing GST protein to assure specificity for meiotic chromosomeproteins.

ImmunocytologyThe procedures used for immunocytology of meiotic prophase chro-mosomes have been reported previously (Moens et al., 1987; Moensand Earnshaw, 1989). Briefly, for epifluorescence microscopy, testic-ular cells collected in MEM were surface-spread on a hypotonic saltsolution, picked up on a glass multiwell slide, fixed in 1%paraformaldehyde, pH 8.2, twice for 3 minutes, rinsed in 0.4% Photo-Flo wetting agent, pH 8.0, briefly air-dried, washed in PBS with 10%ADB, and incubated with various primary antibodies on the individ-ual wells overnight at 4°C. After washing, the material was exposedto secondary antibody for 1 hour at 37°C or a few hours at 20°C. Afterwashing the material was covered with an antifading mounting agentcontaining 5 ng/ml of DAPI or propidium iodide and a coverslip.

For electron microscopy, cells were picked up on plastic-coatedglass slides. Treatment with 1 µg/ml of DNase I in MEM for 20minutes at 20°C was found to improve accessibility of SC antigens.After immunostaining, the plastic film was floated off and EM gridswere placed on the film. Contrast was enhanced by post-fixation in1% osmium tetroxide after the cells (on nickel grids) were thoroughlydried for a day. For comparisons of SC dimensions in sectioned andsurface-spread spermatocytes, the same staining protocol was used forboth (saturated uranyl acetate, 10 minutes; lead citrate, 5 minutes).

Double labelling of SC antigens was performed using mouse serumreactive with one of the antigens and rabbit serum specific for theother. The secondary antibodies were conjugated with either FITC orrhodamine for detection by epifluorescence or confocal microscopy.For electron microscopy, the antigens were differentiated with 5 nmand 15 nm gold-conjugated secondary antibodies. For anti-centromereantibody, we used a 1/1000 dilution of the centromere positive serumfrom a scleroderma (CREST) patient from a local rheumatologyclinic. Secondary antibody was goat anti-human FITC conjugated ata 1/1000 dilution.

Epitope mappingThe positions of about 500 5-nm immunogold grains were determinedfor each of the antibodies to the fusion proteins. Electron micrographswere printed at a magnification of ×150,000 to ×170,000. The dimen-sions of the SC were measured at each gold grain position with adigitizer and entered in a computer. The measurements include thewidth of the osmium tetroxide-stained lateral elements, the total SCwidth, and the position of the grain relative to the SC. The computa-tions accumulated the gold grain position in 5 nm classes across anSC of standardized size (40 nm wide lateral elements and an 80 nmdistance between them) (Moens et al., 1987). The distribution ofgrains was printed as smoothed curves by Harvard Graphics software.

Shadow-castingSurface-spread DNase-treated hamster spermatocytes on a plasticfilm- (<80 nm) covered glass slide were exposed to platinum or gold-palladium evaporated in vacuum from a glowing tungsten wire at anangle of about 7° to the plane of the slide. A fine grain was achievedby placing the slide on a metal block at −80°C with some insulationto delay cooling of the slide until the vacuum is established. Theplastic film was floated off the glass slide and recovered on EM grids.

Electron micrographs were printed at ×150,000 magnification andwidth measurements were made with a digitizer.

RESULTS

SYN1 and COR1 cDNAsSequence analysis of the hamster cDNAs encoding Syn1a-dfusion proteins (accession number L32978; Moens et al., 1992)proved that these represent overlapping cDNAs of the hamsterhomologue of the rat SCP1 gene that encodes a 125 kDa SCprotein (Meuwissen et al., 1992). The hamster and rat proteinsare 90% identical. We therefore adopt for Syn1 the numberingof the SCP1 deduced amino acids and the designation of othermotifs. Our various cDNA clones of SYN1 are the result ofinternal priming on A-rich sequences in the mRNA during theconstruction of the library. Clones a to d correspond to clones15, 14, 17 and 16, respectively, in the report on the isolationof those clones (Table 1) (Moens et al., 1992).

Three co-terminal overlapping cDNAs encoding the hamstermeiotic chromosome core protein, Cor1, were isolated with thefollowing lengths, 680 bp (COR1a), 795 bp (COR1b) and 1070bp (COR1c). The longest open reading frame in this cDNAbeginning at the first ATG and terminating with two adjacentTGA codons (Fig. 2) encodes a putative 234 amino acid proteinwith a predicted Mr of 27,134. This size is consistent with datafrom western blots of hamster SC proteins in which the anti-Cor1a antibody reacts with a polypeptide of approximately 30kDa molecular mass. The longest cDNA also contains 81 5′untranslated nucleotides, 260 3′ untranslated nucleotides withtwo potential poly A addition signals (AATAAA) and a polyA tail. Northern hybridization analysis using COR1 cDNA asprobe, identifies an approximately 1150 nucleotide polyadeny-lated transcript in both hamster and rat. The predicted proteinis glutamine-rich (12% Q), most of the Q residues being in thecarboxyl-terminal half of the protein. Overall, the protein ishydrophobic, with 34% hydrophobic residues, 18% basic and15% acidic amino acids. A search of the PROSITE data base(Bairock, 1992) indicates the Cor1 protein has a potentialATP/GTP-binding site motif A (P-loop), potential cAMP- andcGMP-dependent protein kinase C and casein kinase II phos-phorylation sites, as well as potential N-myristoylation andamidation sites. Significant similarity exists in both nucleotideand amino acid sequence (40% identity) with a cDNAencoding a 208 amino acid protein called pM1, a member ofan X-linked, lymphocyte-regulated family (XLR) of the mouse(Siegel et al., 1987). The Cor1 protein also shows 16% identityover a 203 amino acid stretch with skeletal muscle myosinheavy chain, while the XLR family of proteins has similar

Table 1. Names and lengths of the Syn1 fusion proteinsLength

Name Nucleotides (bp) Amino acids Old name

Syn1e 292-927 653 98-309Syn1d 403-1678 1275 135-560 HSC16Syn1f 643-1678 1035 215-560 HSC12Syn1c 1003-2235 1232 336-745 HSC17Syn1b 1298-2235 937 434-745 HSC14Syn1a 1513-2286 773 505-762 HSC15Syn1g 2200-(A)n 734-end

2752

identity with nuclear structural proteins lamins A and C, andthe structural protein mouse epidermal keratin II, members ofthe intermediate filament family of proteins. Cor1 protein, likemyosin heavy chains, intermediate filament proteins and thepM1 protein, is predicted to have regions of heptad repeats andis therefore likely to form coiled-coil structures.

Syn1 and Cor1 fusion proteinsSyn1We constructed translational fusion proteins in pGEX-2T fromfour overlapping partial cDNA clones derived from the SYN1gene. Analysis of the soluble, insoluble and total proteins fromthe E. coli (DH5aF′) transformants by SDS-PAGE and westernblotting, shows that for all four, SC-antigenic material isproduced mainly in the insoluble fraction. Shown in Fig. 3Ais the total Coomassie-stained E. coli protein from strainscarrying the SYN1 fusion constructs: a (lane 1), d (lane 2), c(lane 3); and from a strain carryingthe parental pGEX-2T vector (lane4). The predicted full-length fusionproteins of 77 kDa (Syn1d, lane 2)and 75 kDa (Syn1c, lane 3); areclearly visible as are a truncated 32kDa Syn1a fusion protein (lane 1) andthe 27 kDa GST protein (lane 4).Lanes 5-8 are a western blot of lanes1-4 visualized with antibody againstthe fusion protein Syn1d produced inE. coli. Two E. coli proteins ofapproximately 73 and 37 kDa stainquite strongly with this and otherantibodies against fusion proteins, butthis antibody does not recognize theGST segment of the fusion proteins(Fig. 3A, lane 8). Other stronglystaining bands in lanes 5-7 representfull-length or truncated Syn1 fusionproteins. This is confirmed in thewestern blot shown in Fig. 3A, lanes9 (pGEX-2T), 10 (Syn1c), 11(Syn1d) and 12 (Syn1a), visualizedwith anti-GST antibody. In proteinproduced from an E. coli straincarrying pGEX-2T, only the 27 kDaGST protein and an approximately 73kDa E. coli protein are visualized(lane 9). Fusion proteins visualizedby the anti-GST antibody (Fig. 3A,lanes 12, 11, 10) are the same as thoserecognized by the anti-Syn1d fusionprotein antibody (Fig. 3A, lanes 5, 6,7), demonstrating that antibodiesagainst different epitopes of thefusion proteins recognize the samefusion proteins. Antibodies againstthe Syn1 fusion proteins alsorecognize predominantly the 125 kDaSyn1 protein in western blots ofhamster testicular extracts (Fig. 3B,lanes 2-5) while the original sera usedto screen the cDNA libraries

recognize a number of SC proteins including the 125 kDa Syn1and 30 kDa Cor1 proteins (Fig. 3B lanes 6 and 7).

To determine whether the Syn1 protein might display DNAbinding activity, a western blot suitable for Southwesternanalysis was prepared from an SDS-PAGE gel on whichinsoluble protein from E. coli expressing SYN1c, SYN1d andGST had been electrophoresed. The transfer was incubatedwith a 0.4 kb [32P]dATP-radiolabelled DNA fragmentexcised from SCB9, a rat DNA sequence originally cloned onthe basis of its co-purification with rat SCs (Pearlman et al.,1992) (Fig. 3C). The multiple bands in the Syn1c and -d lanescorrespond with immunoreactive labelled species in thesetwo extracts (Fig. 3A). GST and bands representing otherabundant E. coli proteins did not bind the radiolabelled DNAprobe. The data suggest that the SC Syn1 protein has DNAbinding capability. This binding capability, however, is notsequence-specific, since other DNA sequences such as the

M. J. Dobson and others

CG -80

CAAAGGCGCAGCCGGCTCAGAAGCGTCGAGGGAGCTGAGGCGTCGACCTCCGTCCCGGGCCGCTGAAGAAACTCTAAAG -1

ATG GTG CCT GGT GGA AGA AAG CAC TCT GGG AAA TCT GGG AAG CCA CCA CTG GTG GAT CAG 60M V P G G R K H S G K S G K P P L V D Q 20

GCT AAA ACA GCC TTT GAC TTT GAG AAA GAA GAT AAA GAA CTG AGT GGT TCA GAG GAG GAT 120A K T A F D F E K E D K E L S G S E E D 40

GTT GCT GAT GAA AAG ACT CCA GTA ATT GAT AAA CAT GGA AAG AAA AGA TCT GGG GGA CTA 180V A D E K T P V I D K H G K K R S G G L 60

GTT GAA GAT GTG GGA GGT GAA GTA CAG AAT ATG CTG GAA AAA TTT GGA GCT GAC ATT AAC 240V E D V G G E V Q N M L E K F G A D I N 80

AAA GCT CTT CTT GCC AAG AGA AAA AGA ATT GAA ATG TAT ACC AAA GCT TCT TTC AAA GCC 300K A L L A K R K R I E M Y T K A S F K A 100

AGT AAC CAG AAA ATT GAG CAA ATT TGG AAA ACA CAA CAA GAA GAA ATA CAG AAG CTT AAC 360S N Q K I E Q I W K T Q Q E E I Q K L N 120

AGT GAA TAT TCT CAG CAA TTT ATG AGT GTG TTG CAG CAG TGG GAA CTG GAT ATG CAG AAA 420S E Y S Q Q F M S V L Q Q W E L D M Q K 140

TTT GAG GAA CAA GGA GAA AAA CTA ACT AAT CTT TTT CGA CAA CAA CAG AAG ATT TTT CAG 480F E E Q G E K L T N L F R Q Q Q K I F Q 160

CAG TCT AGA ATT GTT CAG AGC CAG AGA CTG AAA GCA ATC AAA CAG CTA CAT GAG CAG TTC 540Q S R I V Q S Q R L K A I K Q L H E Q F 180

ATA AAG AAT TTG GAG GAT GTG GAG AAA AAT AAT GAT AAT CTA TTT ACT GGC ACA CAA AGT 600I K N L E D V E K N N D N L F T G T Q S 200

GAA CTT AAA AAA GAA ATG GCA ATG TTG CAA AAA AAA GTT ATG ATG GAA ACT CAG CAG CAA 660E L K K E M A M L Q K K V M M E T Q Q Q 220

GAG ATG GCA AAT GTT CGA AAG TCT CTT CAA TCC ATG TTA TTC TGA TGA GTCTTTGAAGAAAGA 723E M A N V R K S L Q S M L F * * 234

ACTTGAACCTATGTAATATATGATACAGTTAAAACATTATCTATGAGGCATGCCTATAGAAAGTATACTTTGAACTATA 802

ACATTCATAACCATAGCTTGTTTAAGTGGAAGACTTCTGTTCCTGTTAACTTTTAAATAAAACTTAACAGCTGTATAAG 881

TAGCAGCTATTTCAGTGTATCAAGCTTTCAACTCTTATAATAGTGAATTGTTTGCTACTATTGTGTCAATAAAAATGAT 960

TTAAATTTA(A)n 969

Fig. 2. Nucleotide sequence of the cDNA encoding the hamster Cor1 (30/33 kDa) protein and thederived amino acid sequence (single letter designation). The cDNA sequence (upper line) is 1050nucleotides up to the first A in the poly A sequence. The derived amino acid sequence (lower line)begins at the first M in the longest open reading frame and terminates with adjacent TGA codons(*). A perfect match to a nucleotide (ATP/GTP) binding site motif A (P-loop) is underlined. Theaccession number for this sequence is X77371.

2753SC proteins

375 bp EcoRI/BamHI fragment from pBR322 are also boundby Syn1 (not shown).

Cor1Six independent cDNA fragments of this gene, all initiated atthe 3′ poly A tail, were isolated in multiple screening of thehamster cDNA library and excised as Bluescript plasmids. Ofthese, the longest fragments were apparently unclonable as in-frame translational fusions in the pGEX-2T vector. Insertswere recovered in the reverse orientation, however, suggestingthat the longer Cor1 fusion constructs may be toxic to the E.

coli host. The only cDNA to be subcloned in the correct ori-entation was COR1a, which encodes the carboxyl half of theprotein. Electrophoresis of protein extracts from this transfor-mant on SDS-PAGE gels showed that a fusion protein of theexpected size was produced predominantly in the insolublefraction (Fig. 3D).

Antigen occurrence and localizationThe polyclonal antibodies against the Syn1 and Cor1 fusionproteins are specific for meiotic chromosome antigens (cores,lateral domains, centromere regions and synapsed regions).

Fig. 3. (A) SDS-PAGE (Coomassie Blue,lanes 1-4) and western blots (lanes 5-12) fromtotal protein from E. coli expressing Syn1-GST fusion proteins or GST protein. Equalamounts of protein from IPTG-induced E.coli were loaded on an SDS-polyacrylamidegel as follows: lane 1, construct carrying theSyn1a fusion (major stained induced protein,32 kDa); lane 2, construct carrying the Syn1dfusion (major stained induced protein, 77kDa, 50 + 27 GST); lane 3, construct carryingthe Syn1c fusion (major stained inducedprotein, 75 kDa, 48 + 27 GST); and lane 4,construct carrying parental vector pGEX-2T,which expresses GST (27 kDa) but no Syn1protein. Lanes 5-8 are a western blot of thegel probed with a 1/3000 dilution of serumfrom a mouse inoculated with Syn1d-GSTfusion protein (inclusion bodies) produced inE. coli. An additional gel with the sameamounts of protein as on lanes 1-4 (lane 9,pGEX-2T vector; lane 10, Syn1c-GST fusion;lane 11, Syn1d-GST fusion; and lane 12,Syn1a-GST fusion), was electrophoresed,blotted to nitrocellulose and probed with anti-GST antibody prepared in goat (Pharmacia).Lane M between lanes 4 and 5 contains lowmolecular mass markers (Bio-Rad); and laneM between lanes 8 and 9 contains rainbowmarkers (Amersham). Sizes in kDa areindicated. (B) Western blot of proteins fromhamster testicular nuclei separated by SDS-polyacrylamide gel electrophoresis(Coomassie Blue, lane 1) and probed withantibodies generated in mice against Syn1-GST fusion proteins expressed in E. coli orwith antibodies produced in rabbits againstpurified hamster synaptonemal complexes.Lane 2, mouse anti-Syn1b antibody; lane 3,mouse anti-Syn1a antibody; lane 4, mouseanti-Syn1d antibody; lane 5, mouse anti-Syn1c antibody; lane 6, rabbit B anti-SCantibody; and lane 7, rabbit D anti-SCantibody (Moens et al., 1992). Sizes in kDaare indicated. (C) Southwestern analysis ofthe Syn1c (lane 1) and -d (lane 2) fusionprotein extracts. Lane 3, pGEX-2T extract.Probe is rat DNA, sequence SCB9, known tobe tightly associated with rat SCs (Pearlman

et al., 1992). Sizes of immunoreactive Syn1c (75 kDa) and Syn1d (77 kDa) proteins (B and C) are given (arrows). (D) SDS-polyacrylamide gel(Coomassie Blue, lanes 1 and 2) and western blot (lanes 3 and 4) from insoluble protein from E. coli expressing the Cor1a-GST fusion protein.Lane 1, 42 kDa Cor1a-GST fusion protein; lane 2, 27 kDa GST; lane 3, western blot of lane 1; lane 3, western blot of lane 2 using rabbit anti-SC antibody. Size markers (thin arrows), the 42 kDa Cor1a-GST fusion protein and the 27 kDa GST protein (thick arrows) are indicated.

A

B C

D

2754 M. J. Dobson and others

Fig. 4. Progression of chromosome synapsis at meiotic prophase in hamster spermatocytes is visualized with differential immunolabelling ofthe core and synaptic proteins. For A, C and E the primary antibody is mouse anti-Cor1a and the secondary antibody is rhodamine-conjugatedgoat anti-mouse IgG. For B, D and F the primary antibody is rabbit anti-Syn1c and the secondary antibody is FITC-conjugated goat anti-rabbitIgG. Bar, 10 µm. (A,B) The earliest stage of chromosome synapsis, zygotene. The cores in A are mostly single except for a few synapsedregions shown in B. Three synapsed regions are already quite lengthy and it appears that the extension of synapsis is rapid relative to theestablishment of new initiation sites. (C,D) Midway through the zygotene stage there is extensive synapsis. (E,F) The cores are completelysynapsed and the image of the Cor1 protein now coincide with the image of the Syn1 protein. Aggregates of Cor1 protein, separate from theSCs, are present at early pachytene, E (arrow), but not at later stages. At the resolution of the epifluorescence microscope, the antibodies to theseveral Syn1 fusion proteins give the same image.

2755SC proteins

Cryosections of hamster body tissues are negative, with theexception of testes where the spermatocyte nuclei are positive(Fig. 5). Details of the occurrence and location of meiotic chro-mosome antigens was obtained from surface-spread spermato-cyte preparations.

The Cor1 antigen is first detected when the cores are stilllargely unpaired at the leptotene-zygotene stages of meioticprophase (Fig. 4A). In those cells the Syn1 antigen is presentwhere cores from homologous chromosomes have synapsed(Fig. 4B). Although there are few such regions at earlyzygotene, they are already quite long, indicating that synapsisis rapid relative to the establishment of new pairing sites. Assynapsis progresses, the number of single cores is reduced (Fig.4C, anti-Cor1), while the synapsed regions are longer and morenumerous (Fig. 4D, anti Syn1). The images of the core andsynaptic antigens are identical when all chromosomes are fullysynapsed (Fig. 4E and F), with the exception of some aggre-gates of core antigen at early pachytene (Fig. 4E, arrow). Atdiplotene, the cores separate and the Syn1 antigen is presentonly at the remaining points of contact (Fig. 6A; Cor1 antigenis red, the Syn1 antigen is green but appears yellow when over-lapped with red). The presence of one or two small pairingregions for each of the 20 bivalents gives a noticeably differentstaining pattern from the early zygotene configuration wherethere are relatively few but long pairing regions. Therefore thestaging of these cell types is unambiguous.

At metaphase I substantial amounts of Cor1 antigen co-localize with each of the 40 double centromeres identified byCREST immunostaining and small amounts of Cor1 antigenare present along the long axis of the chromosomes. In Fig. 6Bthe metaphase I bivalents are stained fluorescent blue withDAPI and the Cor1 antigen is fluorescent red. The samenucleus in Fig. 6C demonstrates the centromere positions thatcoincide with the Cor1 positive regions in Fig. 6B. One of thedoubled centromeres is indicated by a double bar. At anaphaseII the 20 double centromeres separate leaving the Cor1 antigenbehind (Fig. 6D; centromeres in green and Cor1 antigen inred). Cor1 antigen can be detected in later stage spermatids butthey are no longer associated with the kinetochores.

Epitope mappingSyn1a (aa 505-762) and Syn1b (aa 434-745)Polyclonal antibodies are against the carboxyl region of theSYN1 gene product (Table 1). The mode and mean of the goldgrain distribution are at the inner edge of the lateral element asdefined by osmium tetroxide staining (Fig. 7). The distributionof grains to either side of the mean can be attributed, in part,to the size of the antibodies, the size of the gold grain, and theunbiased error of each measurement. About 50% of the grainsare over the central region (Table 2). Syn1b is detected mar-ginally more towards the centre of the SC, concomitant withan additional 71 amino acids towards the amino terminus ofSyn1. Both these fusions lack the Syn1 leucine zipper domain.

Syn1c (aa 336-745) and Syn1d (aa 135-560)The carboxyl end of Syn1c is identical to Syn1b but there are100 additional amino acids towards the amino end, includingeight heptad repeats of the leucine zipper motif. Relative toSyn1a and -b, the mode and mean of the gold grain distribu-tion are shifted more than 10 nm towards the centre of the SCand some 70% of the grains are over the central region (Table

2). This portion of the Syn1 protein appears to occupy aposition in the central region of the SC, about 20-25 nm fromthe middle of the SC (Fig. 7). Compared to Syn1c, Syn1d lacks185 amino acids at the carboxyl end and has an additional 201amino acids at the amino end. It includes the complete leucinezipper motif. The mode and mean of the immunogold graindistribution are similar to that of Syn1c, shifted slightly moretowards the centre of the SC. Approximately 80% of the grainsare over the central region of the SC (Table 2).

Cor1aThe Cor1a fusion protein contains a 130 amino acid carboxyl-terminal fragment of the Cor1 protein. The immunogolddetects the Cor1 antigen in a broad lateral domain of the SC(Figs 7, 8B). This distribution is much broader than the con-ventional lateral element as visualized with electron-densestains (Fig. 8A,B). The distribution coincides with the dimen-sions of the shadow-cast image of the lateral domain (Fig.8C,D).

SC dimensionsElectron micrographs of sectioned or surface-spread meioticprophase cells stained with electron-dense stains (uranylacetate, lead citrate, osmium tetroxide or phosphotungstic acid)reveal lateral elements of about 47±6 nm width and a centralregion of 100±25 nm, for a total width of about 194±13 nm(60 OsO4-stained SCs measured, ± standard error) (Fig. 8A,B).Different dimensions are apparent in electron micrographs ofshadow-cast SC preparations (Fig. 8C, D). The lateral domainsare considerably wider, on average about 110 nm (range 80-170 nm), and the central region is narrower, on average only50 nm, and the total width is approximately 260 nm. The coresand the lateral elements that react with metal ions appear to bean internal component of the lateral domain. The broad lateraldomain as seen in shadow-cast images corresponds to thestructure detected by antibodies to the chromosome coreprotein, Cor1a, where the major portion of the immuno-goldgrains cover a lateral domain of about 100 nm in width (Table2, Figs 7, 8B,D).

DISCUSSION

SC dimensionsThe substructure of the SC as it is disclosed by antibodyepitope mapping and shadow-casting shows noticeable differ-

Table 2. Numerical values of the gold grain distributionsshown in Fig. 7

Percentage of grainsMode Mean

Antigen (nm) (nm) Outside LE* Central

Syn1a 35-40 44.67 11.14 38.39 50.47Syn1b 30-40 44.56 10.57 40.38 49.06Syn1c 20-25 31.45 3.46 22.63 73.90Syn1d 20-30 28.55 2.75 16.28 80.97

Cor1a 80-90 71.89 45.72 39.12 15.16

For each of the anti-fusion protein antibodies the positions of 400-500grains were digitized and computationally accumulated (Moens et al., 1987).

*Lateral element.

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ences from the standard descriptions of SCs, which are basedon electron microscopy of SCs stained with metal ions(tungsten, lead, uranium, osmium). For the rat, mouse andhamster, the electron-dense lateral elements are about 50 nm

wide, separated by a central region of about 80-100 nm. Thisimage leads to the expectation that the structural componentsof the central region abut the distinctive lateral elements. Anti-bodies against the carboxyl end of hamster chromosome-core

M. J. Dobson and others

66

6

2757SC proteins

protein 1, Cor1, indicate a much broader lateral elementdomain than is apparent in the traditional electron-dense lateralelement of rodents (Figs 7, 8, Table 2). The gold grain distri-bution extends some 40 nm to the outside of the dense lateralelement. That lateral element domains are indeed broader thanis apparent from electron microscopy of metal-ion-stainedlateral elements is further evident from shadow-cast prepara-tions, which show that the chromosome cores and the lateraldomains are about twice as broad as the metal-ion-stainedelectron-dense lateral elements (Fig. 8). The broaderdimension is in part at the expense of the central region, whichbecomes narrower, and in part due to a widening of the SCfrom approximately 200 nm to 260 nm or more. A mode of thegold grain distribution to the outside of the lateral element hasalso been observed for the 190 kDa lateral element protein (C.Heyting and E. Hartsuiker, personal communication). Appar-

ently, traditional EM staining with metal ions has visualizedonly a specific portion of the lateral domain.

The implication of these new dimensions is that the proteinsthat comprise the synaptic domain have a greater degree ofoverlap with the protein constituents of the lateral domains ofthe SC than was apparent from earlier observations. Thisarrangement suggests that direct interactions might beexpected between some of the SC proteins. The availability ofSC protein-encoding genes will allow in vitro assessment ofpotential protein-protein interactions.

Epitope mappingThe characteristics of SCP1, the rat homologue of the hamstersynaptic protein Syn1, have been reported by Meuwissen et al.(1992). It is predicted to consist of 946 amino acids and haslong regions that show sequence similarity to the coiled-coilregion of the myosin heavy chain. In addition, there is a leucinezipper, DNA binding motifs, and potential target sites forP34cdc2 protein kinase (Meuwissen et al., 1992). The publishednumbering of the SCP1 amino acid sequence is used here toidentify the hamster fusion proteins Syn1a to -d in Table 1, andin Fig. 7. It is clear from Fig. 7 that each extension of the fusionprotein in the direction of the amino end of the Syn1 proteincoincides with a displacement of the peak of the gold grain dis-tribution towards the centre of the SC. The polyclonal anti-bodies against the fusion protein products of these four cDNAfragments demonstrate that the Syn1 protein is oriented withthe amino end towards the centre of the SC and with thecarboxyl end extending into the lateral domain. The orienta-tion agrees with the more general observation of Meuwissenet al. (1992) that a monoclonal antibody against an epitope atthe carboxyl end of rat synaptic protein SCP1 produces a goldgrain distribution that is closer to the lateral element than thatof a polyclonal antibody against SCP1 as a whole. A differentsynaptic protein, Zip1, has been identified in the yeast, S. cere-visiae. Sym et al. (1993) calculated that the 875 amino acidZip1 is sufficiently long to span the distance between thelateral elements.

The in vivo significance of the apparent DNA binding char-acteristics of the Syn1 protein, indicated by the DNA bindingmotifs (Meuwissen et al., 1992) and the non-specific DNAbinding in Southwestern blots of the Syn1 fusion proteinsreported here (Fig. 3D), remains to be determined. Tradition-ally, the chromosome cores are thought to be the binding sitesfor the chromatin loops because the meiotic prophase chro-mosomes usually acquire their cores before synapsis. At a laterstage, when the homologous cores are being aligned, thesynaptic protein becomes part of the SC. The deduced aminoacid sequence of Cor1 (Fig. 2) does not have a recognizableDNA binding motif. DNA binding may be the function of othercore proteins. A nucleotide (ATP/GTP) binding site motif maybe of functional significance as might putative cAMP- andcGMP-dependent, protein kinase C and casein kinase II phos-phorylation sites. The rat homologue of the COR1 gene, SCP3,has also recently been cloned (C. Heyting, personal communi-cation).

Cor1 antigens in rodent spermatocyte nuclei can occuroutside the context of the chromosome core or SC. At the timethat chromosome synapsis is being completed there are smallaggregates of antigen among the SCs but not necessarilyconnected to the SCs (Fig. 4E). Such extra-SC material has

Fig. 5. A cryosection of hamster testicular tubules immunostainedwith antibody to Syn1c fusion protein and stained with the DNAbinding stain propidium iodide. The FITC-conjugated secondaryantibody marks the SCs of the spermatocytes in green. Otherspermatogenic and non-spermatogenic nuclei are red and lack theSyn1 antigen.Fig. 6. Chromosome disjunction. (A) Cor1 antigen in red (rhodaminefluorescence), Syn1 antigen in green (FITC fluorescence), andoverlapping areas in yellow. Surface-spread mouse spermatocyte. Atdiplotene the chromosomes separate but remain attached at a fewpoints, the chiasmata, presumably the sites of crossovers. The Syn1antigen gradually disappears, lastly from the chiasmata sites. TheCor1 antigen remains present axial to each chromosome. (B) Cor1antigen in red (rhodamine fluorescence), chromatin in blue (DAPIfluorescence). At metaphase I most of the Cor1 antigen co-locateswith the 40 doubled centromeres while small amounts are presentalong the chromosome axes. The X-chromosome (x) retains moreCor1 antigen than the other chromosomes. Bar, 10 µm. (C) The samenucleus as in B immunostained with human anti-centromere CRESTserum and FITC-conjugated secondary antibody. The correspondingcentromeres in B and C are marked (c). A doubled centromere ismarked with two short bars. The superimposed FITC and DAPIimages are slightly offset. (D) Cor1 antigen red, centromeres green(FITC fluorescence). The association between centromeres and Cor1antigen persists till anaphase II at which time the sister centromeresseparate and the Cor1 antigen appears to lag between the separatingcentromeres.Fig. 7. Distributions of 5 nm immune gold grains over hamster SCsdetected by polyclonal antibodies to Syn1 and Cor1 fusion proteins.Since the SC is bilaterally symmetrical, only one half of the SC isshown, with position 0 nm in the centre and position 140 nm on theoutside. The positions of the electron-dense lateral element and thebroader lateral domain are marked as such. Computation adjusts allmeasurements to an arbitrary standard SC (central region 80 nm,lateral element 40 nm). The Syn1 fusion proteins to which theantibodies were made are defined by the amino acid numbers startingat the amino end of the protein (Meuwissen et al., 1992) and areshown diagrammatically at the top of the graph. The region of theleucine zipper in 1c and 1d is an open rectangle. The graphdemonstrates that the peaks of the gold grain distributions shift froma central position to a lateral position as the fusion protein fragmentsshift from the amino to the carboxyl end of the protein. It isconcluded that Syn1 is oriented with the amino end towards thecentre and the carboxy end extends into the lateral domain. Thegraph also demonstrates that the method is sensitive to shifts of a fewnanometres in the antigen location. Polyclonal antibodies to thecarboxy end of Cor1 detect a broad lateral domain of the SC (purplecurve, Cor 1a).

2758

been observed in several organisms and has been associatedwith the self-assembly of excess SC material; for example, inpachytene-arrested yeast mutants (Moens and Kundu, 1982).

The structural aspects of the SC are summarized in Fig. 9,an elaboration of Fig. 1. The Syn protein is shown extendingfrom the middle of the SC out into the lateral domain. Alter-native arrangements, such as a Syn1 protein that bridges theentire distance between the lateral elements or a bidirectionalorientation of the Syn1 protein, are not supported by theevidence shown in Fig. 7. The binding of DNA to the Syn1

protein is not addressed in the diagram. It is clear, however,from a separate study that combines SC immunofluorescencewith in situ hybrization of probe that is specific for a 2 Mbphage DNA insert on mouse chromosome no. 4, that the DNAloops attach to the SC (Heng et al., 1994), but the details ofthe attachment are not clear as yet. Since the chromatinbecomes attached to the cores before Syn1 is associated withthe cores, the diagram shows chromatin attached to the lateraldomain proteins. The known lateral domain proteins of rodentssuch as SCP2 (190 kDa) (E. Hartsuiker and C. Heyting,

M. J. Dobson and others

Fig. 8. SC ultrastructural dimensions revealed by different techniques. Bar, 200 nm. (A) A thin section stained with uranyl acetate and leadcitrate. Typically, the lateral elements are rather narrow, about 40 nm, while there is a wide central region of about 80-100 nm. (B) In surface-spread preparations, the uranyl/lead-stained lateral elements are similar in width and spacing to the sectioned material in (A), but thedistribution of the Cor1 antigen detected by the 5 nm gold grains betrays a wider lateral domain, particularly to the outside of the lateralelement. (C) The greater width of the lateral domain is also evident in shadow-cast preparations of surface-spread SCs. As a result, the SC as awhole is wider and the space between the lateral domains is reduced, leaving room only for the central element. (D) The coincidence of theimmuno-stained (5 nm gold grains) lateral domains and the shadow-cast image is apparent in this diplotene SC where the chromosome coresare starting to separate. The implication is that there is a considerable overlap between locations of the core and the synaptic proteins.

Fig. 9. A summary diagram of the positions ofcore and synaptic proteins in the SC. No details ofthe protein structures, their interactions andassociation with chromatin are known as yet.

2759SC proteins

personal communication) and Cor1 all have a broad distribu-tion that exceeds the more narrow lateral element as seen withelectron-dense stains. The implication is that synaptic and coreproteins have a broad region of overlap. No antibodies thatrecognize the central element have been reported to date, norany antibodies against the SC-associated dense nodes, some ofwhich may be implicated in reciprocal crossovers (Carpenter,1975; Albini and Jones, 1988).

Chromosome synapsisUsing antibodies raised in different hosts against variousmeiotic chromosome components, the temporal and spacialrelationships of cores, lateral domain proteins and synapticproteins can be visualized simultaneously with two-colourimmuno-labelling for epifluorescence microscopy or by dif-ferential immunogold labelling for electron microscopy. At theonset of chromosome synapsis (Fig. 4A), there are predomi-nantly unpaired chromosome cores (Cor1) and a few synapsedregions (Fig. 4B, Syn1). There are relatively few synapsedregions but the ones that are there are already fairly lengthy,about 5 µm, indicating that the progression of synapsis is rapidrelative to the rate with which new initiation sites are estab-lished. The same conclusion holds for later zygotene (Fig.4C,D) where synapsis is more advanced. It has been arguedthat the initial sites of pairing require homology recognitionand may represent potential sites of genetic recombination(Radman and Wagner, 1993; review, Moens, 1994). Theextension of synapsis may be less dependent on homology thaninitiation of synapsis. The rapid, possibly non-homologous,extension of SCs observed here may be the mechanism thatforestalls additional homology searches near an initiation siteand thereby provides a mechanism for positive recombinationinterference. In support of such a mechanism is the observa-tion that in organisms with limited SC formation, such asSchizosaccharomyces pombe, or with faulty SC formation asin asynaptic meiosis, positive genetic interference tends to beabsent or reduced (Bahler et al., 1992; Havekes, 1992; Moens,1969). The use of two-colour immune-staining permits anefficient quantification of the process of chromosome synapsisat meiotic prophase but is beyond the scope of the presentreport.

Chromosome disjunctionAt anaphase of mitosis sister chromatids separate, but atanaphase I of meiosis the sister chromatids remain associatedat their centromeres and chromosomes rather than chromatidssegregate. Regular chromosome disjunction at meiosis I ofmost sexually reproducing organisms is thought to depend onat least two mechanisms: chiasma maintenance and sister kine-tochore cohesion. Observations on the orientation of acentricfragments that result from a crossover in an inversion in maizemeiosis has lead Maguire (1993) to a preference for sisterchromatid cohesion as one of the main factors responsible forthe maintenance of chiasmata (reciprocal cross-over). Sheproposes that SC components are likely candidates for sisterchromatid cohesion at metaphase I. The presence of SC com-ponents axial to the sister chromatids was demonstrated byelectron microscopy of serially sectioned grasshoppermetaphase I chromosomes (Moens and Church, 1979). Herewe report the presence of Cor1 protein in the metaphase chro-mosome I axis (Fig. 6B). Because the Cor1 protein is associ-

ated with sister chromatids throughout prophase we assumethat Cor1 continues in that role during metaphase I, therebygiving support to the suggested function for SC components insister chromatid cohesion and chiasma maintenance. The axisof the X chromosome has more Cor1 antigen than theautosomes (Fig. 6B).

Most of the Cor1 antigen at metaphase I occurs in associa-tion with the pairs of sister kinetochores (Fig. 6B, C). Here, too,the Cor1 protein may function as a cohesive element betweenthe sister kinetochores. As such, it can contribute to themechanism for chromosome, rather than chromatid, segregationat anaphase I. The finding that the Cor1 protein lags betweenthe separating sister kinetochores at anaphase II (Fig. 6D) andthat it has not been observed in association with mitoticmetaphase centromeres, further supports the suggested functionof Cor1 in sister kinetochore cohesion at meiotic metaphaseI/anaphase I. The loss of a kinetochore-associated protein atanaphase is not uncommon (for reviews see Earnshaw andBernat, 1991; Rattner, 1992). The observed presence of a silver-stained centromeric filament at metaphase I/anaphase I in anumber of mammalian species also led Solari and Tandler(1991) to the conclusion that the sister kinetochores remainjoined together and co-oriented by this SC remnant.

The research was supported by grants from NSERC to R.E.P. andP.B.M. We gratefully acknowledge the technical assistance of NoraTsao and Anita Samardzic with the molecular biology, and thankMary Lou Ashton for help with the confocal and electron microscopy.

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(Received 25 January 1994 - Accepted, in revised form,6 June 1994)

M. J. Dobson and others