SCP1,a majo rprotei n componento f synaptonemal complexes ofth era t SCPl, eenbelangrijk e eiwit-component van synaptonemale complexenva nd era t Promotor: dr.C .Heytin g hoogleraar ind emoleculair ee n celgenetica Ralph Leo Johan Meuwissen SCP1,a majo rprotei n componento f synaptonemal complexes ofth era t Proefschrift terverkrijgin g van degraa d van doctor op gezagva n derecto r magnificus van deLandbouwuniversitei t Wageningen, dr.CM . Karssen, inhe t openbaar te verdedigen opdinsda g4 februari 1997 desnamiddag s tehal ftwe e ind eAula . CIP-DATAKONINKLIJK E BIBLIOTHEEK,DE NHAA G Meuwissen, Ralph LeoJoha n SCP1, amajo r protein component of synaptonemal complexes ofth era t/ R.L.J.Meuwissen . - [S.l. :s.n.] .- 111. Thesis Wageningen. -Wit href . With summary inDutc h and Spanish. ISBN 90-5485-649-1 Subject headings:meiosi s / SCP1/ synaptonemal complex/ Rattus rattus norvegicus Theresearc h described inthi sthesi swa sfunde d byth eMedica l Science Section (GMW)whic h issubsidise d byth eNetherland s Organization for Scientific Research (NWO). NMoS^Ol.fc^S Stellingen 1. SCPl isee nhoofdcomponen t vand etransversal e filamenten vanhe t synaptonemale complex. dit proefschrift 2. Onderzoek waarbij polyklonale antisera,opgewek ttege noverlappend eS C eiwitfragmenten, wordengebruik tvoo rimmunogou d labeling experimenten metS C preparaten van gespreide spermatocyten, leverenonbetrouwbar eresultate n opdi enie t zonder meergebruik t kunnen wordenvoo rd elokalisati e bepaling vandi teiwi t inhe tSC . Dobsone tal .J. Cell Sci. 107 :2749-276 0 dit proefschrift 3. Deafzonderlijk e SCeiwitte n vanS. cerevisaee nzoogdiere nkunne nee ngroter e structurelee nfunctionel e overeenkomstvertone nda nme no pgron dva nnu ngelijkeni s ind e primaire aminozuurvolgorde zouverwachten . 4. Devraa g ofee nintact e SCstructuu r verantwoordelijk isvoo rhe tverschijnse l van "crossover interferentie" kanbete ri nee nredl danee nziplredl achtergrond inS. cerevisae worden bestudeerd. Storlazzi etal .Proc. Natl. Acad. Sci.USA 93: 9043-9048 5. Hetafdwinge n van sluitingstijden doorhe t gebruikva n semi-professionele bewakingsapparatuur inee nuniversitai r laboratorium leidta fva nd eprimair edoelstellin g als faciliteit voorhe t optimaal kunnenuitvoere nva nwetenschappelij k onderzoek. Deze maatregel werkthierdoo r volledigcontra-productief . 6. Machtswellust isvaa kgebaseer d oppur eangst . 7. Nietallee nJezu sdroe gzij n eigenkruis . 8. Deopmerkin gva nwesters esociaal-historic i datAfrik a een "verlorencontinent "zo uzij n methe too g opstabiel ewelvaartsgroei ,gaa tvoorbi j aanhe t feit datd edeplorabel e economischetoestan d inmeni gA&ikaans estaa tjuis t ind ehan dword tgewerk tdoo rhe t actiefsteune n vanincorapetent ee ncorrupt eregime sdoo rwesters e regeringen. 9. Eent ehoo gaandee iva nspelletje s enloterij-programma' s inhe taanbo dva nd e commercieletelevisi eontaard ta lgau wi nplee-TV . 10. DNAhebje voor 't leven. 11. Eenslapp ehaa nkraai tnie tmeer . 12. Eendispuu t brengtweini gvrienden . 13. Intellectuelen zijn vaak tesli m voorhe topiosse n vaneenvoudig eproblemen . Steilingenbehoren d bijhe tproefschrif t 'SCP1,a majo r componento fsynaptonema l complexes ofth erat ' vanRalp hMeuwisse n Wageningen,4 februari 1997 Voormij n ouders Contents Chapter1 Introduction, 3 Chapter2 Acoiled-coi lrelate dprotei nspecifi c for synapsed regions of meioticprophas e chromosomes,2 5 Chapter3 Human synaptonemal complexprotei n 1 (SCP1): isolation and chracterization of the cDNAan d chromosomal localization of thegene ,3 7 Chapter4 Bindingo fDN Ab y SCP1,a majo r componento f synaptonemal complexes, 53 Chapter5 Organization of SCP1protei n moleculeswithi n synaptonemal complexes of therat , 79 Chapter6 GeneralDiscussion , 101 Summary 115 Resumen 119 Samenvatting 123 Dankwoord 127 CurriculumVita e 129 CHAPTER 1 Introduction INTRODUCTION Meiosis isa nessentia l process inth e sexual reproduction cycle ofeukaryotes . Itbring s about the transition from diploid to haploid generations of cells. At fertilization, the diploid state isrestore d whentw ohaploi d gametes fuse to form adiploi d zygote. Meiosis consists of two successive nuclear divisions, meiosis I and II, which follow a single round of DNA replication. After premeiotic S-phase, during the prophase of meiosis I, a series of characteristic chromatin rearrangements take place by which homologous chromosomes condense and pair, and the non-sister chromatids of homologous chromosomes (homologues) recombine with each other (von Wettstein et al, 1984; Loidl, 1994; Griffiths et al, 1996). These events are followed by segregation ofth ehomologue s (Figure 1). The segregation of the homologues causes the reduction of the chromosome complement from diploid to haploid: meiosis I is therefore called a reductional division. Meiosis II, in contrast, is an equational division because the ploidy level is maintained: the two sister chromatids of each chromosome are then separated, as in a mitotic division (Fig.l). The characteristic aspects of chromosome behaviour in meiosis are summarized in Table I, which shows that many meiosis-specific events take place during meiotic prophase. These events, namely the pairing and recombination of homologous chromosomes, are accompanied by the assembly and disassembly of meiosis-specific structures, the synaptonemal complexes (SCs) (Moses, 1969). TableI Chromosome behaviour during eukaryotic cell divisions Meiosis 1 Meiosis II Mitosis Pairing of homologous Usually no pairing between chromosomes homologous chromosomes High frequency of recombination, Low frequency of recombination, preferably between non-sister preferably between sister chromatids chromatids Sister chromatid cohesion until no cohesion cohesion until anaphase anaphaseI The two sister-centromeres act as a two sister-centromeres act two sister-centromeres act unit independently from each other and independently from each other and are separated are separated 1)n.a. , not applicable DNA replication DNA replication X pairingo f homologous chromosomes duplicated chromosomes line up individually on thespindl e homologous pairso f duplicatedchromosome s line upo nth espindl e ivision >^\. Figure 1. The process of meiosis compared to mitosis. Only one set of homologous chromosomes is shown. The pairing of the duplicated homologous chromosomes isuniqu e to meiosis. During meiosis, two nuclear divisions are needed to produce haploid gametes. Each diploid cell entering meiosis thus produces four haploid cells (from Alberts et al., 1989). Synaptonemal complexes (SCs) The SC is aproteinaceous , zipper-like structure which holds the homologues in close apposition along their entire length during meiotic prophase (von Wettstein et al, 1984) (Figure 2a).A t the beginning ofmeioti c prophase, after premeiotic S-phase, the two sister chromatids develop a single proteinaceous axis, called axial element. As meiotic prophase proceeds, these axial elements are connected along their entire length by numerous thin, transverse filaments (TFs). Between the axial elements resides a third longitudinal structure, referred to as the central element (CE). The two axial elements together with the CE make up the so-called tripartite SC structure. Within the context of the SC,th e axial elements are also called lateral elements (LEs) of the SC. Homologues are called synapsed if they are connected by the tripartite structure. The tripartite SC structure is conserved among sexually reproducing eukaryotes (Dresser and Giroux, 1988;Schmeke l etal, 1993;Schmeke l and Daneholt, 1995), although there is much variation in both CE structure and LE shape (von Wettstein etal, 1984). A schematic overview of the (dis)assembly of SCs and the concurrent chromosome rearrangements is shown in Figure 2a and 2b. The successive stages of meiotic prophase are defined by the stages of SC (dis)assembly (Figure 2a): during leptotene, a single axial element is formed between the sister chromatids; during zygotene, the axial elements of homologues are connected by TFs, and the CE appears in synapsed regions. During pachytene, the homologues are synapsed from telomere to telomere. In diplotene, the tripartite structure starts to disassemble; in mammals the TFs disappear first (von Wettstein et al, 1984). At the end of diplotene and the onset of diakinesis, the LEs have disappeared and the two scaffolds of the individual sister chromatids (shown in Figure 2b) become discernible as the now recombined chromosomes condense further. The chiasmata become visible as the results of reciprocal exchange between twonon-siste r chromatids ofhomologues . In metaphase I, the nuclear envelope disappears and the chromosomes condense further while the centromeres are attached to the spindle and chromosome pairs (bivalents) orientthemselve s inpreparatio n ofchromosom e disjunction. Recombination nodules During the assembly of SCs, electron-dense spherical or ellipsoidal structures with a diameter of 100-200n mar e formed, the so-called recombination nodules (RNs) a) Leptotene Pachytene I I I I 14 ^Mii, I Diplotene Interphase Zygotene followed by diakenesis > b) Leptotene/zygotene MetaphaseI Nuclear envelope Figure 2. a) A schematic figure showing the successive stages of meiotic prophase as defined on the basis of morphological changes of the SC: leptotene (proteinaceous axes start to form along the homologous chromosomes), zygotene (start of synapsis of the homologues), pachytene (synapsis is completed and the SC extends from telomere to telomere), diplotene (SC disassembles) and