SCP1, a major protein component of synaptonemal complexes of the rat

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes during meiotic prophase. SCs consist of two proteinaceous axes, one along each homologue, that are connected along their length by numerous transverse filaments (TFs). The assembly and disassembly of SCs closely correlates with the successive events at the chromosomal level: the condensation, pairing, recombination and segregation of homologous chromosomes, In Chapter 1, recent advances in the analysis of the structure and composition of SCs are described, and possible roles of SCs in meiotic chromosome pairing and recombination are considered. The regulation of meiotic recombination and the formation and maintenance of stable chiasmata are now considered as possibly the principal functions of SCs.The experimental work described in this thesis is focused on the structure and function of SCP 1, a major protein component of rat SCs (Chapters 2-5).Chapter 2 describes how cDNA clones encoding SCP1 of the rat were isolated by screening a cDNA library with monoclonal antibodies that recognize a 125,000 M r SC component. A polyclonal antiserum raised against the translation product of one of the isolated cDNA clones recognizes a single protein on Western blots of proteins of isolated SCs with identical electrophoretic mobility as the protein recognized by the monoclonal antibody that was used for screening. The protein encoded by the cDNAs (called SCP 1: lynaptonemal -complex protein 1) has a predicted molecular weight of 117 kDa. 'Me central part of SCP1 is capable of forming an amphipathic (alfa-helix, whereas the C-terminal domain carries motifs that are characteristic of DNA-binding proteins (Ser[]Fhr-Pro motifs). In immunogold labeling experiments, the monoclonal antibody that was used for screening and the polyclonal antiserurn that was elicited against the translation product of the cDNA both labeled the central region of the SC. We conclude that SCP I is most probably a major component of the transverse filaments of SCs, and speculate that SCP I has evolved by specialization of a nuclear matrix protein.In order to identify conserved features of SCPI, we isolated and characterized cDNAs encoding human SCP 1 (Chapter 3). Human SCP 1 and rat SCP 1 have 75% amino acid identity. Most of the prominent structural features of rat SCP1 are conserved in human SCP1. The human SCP1 gene was localized on human chromosome 1p12-p13. We conclude that human SCP 1 is most probably the functional homologue of rat SCP 1, and that SCP 1 is not a very conserved protein.Because SCP1 contains amino acid sequence motifs which are characteristic of DNAbinding proteins, we analysed the DNA binding capacity of SCP1, by means of a quantitative south-western blot assay (Chapter 4). The DNA binding capacity was confined to the C-terminal domain. This domain has about the same affinity for total rat genomic DNA as for a Drosophila SAR DNA probe, but its affinity for E. coli DNA is significantly lower. Similar results were obtained for full-length SCP 1.Analysis of the kinetics of DNA binding revealed that there is probably one type of DNA binding site on SCP I. Distamycin A could completely inhibit binding of DNA by the C- terminal domain. We therefore concluded that the C-terminal domain, and thus also full- length SCP1, binds to DNA through interaction with the minor groove, The binding of SCP I to DNA in vivo was shown by paraformaldehyde crosslinking of living spermatocytes. The DNA-binding capacity of the C-terminal domain of SCP1 is in agreement with the localization of this domain in the LEs of SCs, where most of the DNA is situated. We speculate about the DNA sequences to which SCP1 binds in vivo and about the implications of this for the role of SCP I in the assembly and function(s) of SCs.Chapter 5 describes the analysis of the organization of SCP1 molecules within SCs. Using polyclonal antibodies elicited against non-overlapping fragments of SCP1, we performed immunogold of SCs. Two types of SC preparations were used for this purpose, namely surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. The distribution of immunogold label on surfacespread spermatocytes differed significantly from the distribution of label on sections. This difference is probably due to masking of SCP1 epitopes within the SCs in surfacespread preparations and/or the surface morphology of the surface spreads. The results obtained on sections were therefore used for the localization of subdomains of SCP1 within the SC structure. We present the following model for the organization of SCP1 molecules within SCs: the C- terminus of SCP1 lies in the inner half of the lateral element (LE), and the molecules protrude from the LE through the central region into the central element (CE), so that N-termini of SCP1 molecules from opposite LEs overlap. The implications of this model for the assembly of SCs and for the possible functions of SCP 1 are discussed.In Chapter 6, the cytological and biochemical features of SCP 1 are compared with data about TF components in other organisms. Especially the possible function(s) of a putative TF component of yeast, Zip1p, are discussed, and 1 consider the relevance of the Zip1p data for the possible function(s) of SCP1. Finally, 1 present a model for the function(s) of SCP 1 during meiotic prophase.