The Mr 30,000-33,000 major protein components of the lateral elements of synaptonemal complexes of the rat

Synaptonemal complexes (SCs) are intranuclear structures which are formed during meiotic prophase between homologous chromosomes. The SC consists of two protein-rich axes, either of which is found at the basis of one of the homologous chromosomes. These axes, called lateral elements (LEs), are connected along their entire length by so-called transverse filaments. Between and parallel to the LEs runs a third element, called central element. The assembly and disassembly of SCs take place in a period during which a number of important events takes place at the chromosomal level: condensation, pairing, recombination and segregation of homologous chromosomes. The possible involvement of the SC in these events is an importnat topic in the research program of our section. This thesis focusses on the possible function of the LE and its components (Chapter 1). The experimental work described in this thesis (Chapters 2-5) concerns the characterization of the LE-components with relative electrophoretic mobilities of 30,000 and 33,000 (the M r 30,000-33,000 components).The isolation of cDNAs encoding the M r 30,000-33,000 components is described in Chapter 2. The isolation took place by screening of an expression cDNA-library with an affinity-purified polyclonal antiserum. The largest cDNA, 2A4, encodes a protein with a predicted molecular mass of 29.7 kDa, which we have termed s ynaptonemal c omplex p rotein 3 (SCP3). A polyclonal antiserum raised against SCP3 recognizes only the M r 30,000-33,000 components on a westernblot of SC-protein and exhibits a similar immunological localisation as monoclonal antibodies and a polyclonal antiserum raised against the M r 30,000-33,000 components. The deduced amino acid sequence shows that SCP3 is a potential ATP-binding protein and that the C-terminal half of the protein is capable of forming an amphipaticα-helix. Moreover, part of the amino acid sequence exhibits considerable homology to the predicted protein products of two members of a gene familiy of X-linked lymphocyte-regulated ( Xlr ) genes.We conclude that cDNA 2A4 encodes at least one of the M r 30,000-33,000 components and that SCP3 is a major component of the LEs of the rat. We speculate that the homology between SCP3 and two products of Xlr genes might be connected to a function of both types of protein in processes which share the common purpose of blocking certain recombination events.The M r 30,000- and the 33,000 component are closely related according to their almost identical peptide maps and the fact that all isolated antibodies always recognize both proteins. A first step to elucidate the difference between these two components and the level at which such a difference arises is described in the addendum to Chapter 2. We conclude that the M r 30,000-33,000 components are probably encoded by a single gene and a single messenger RNA and that the difference between the two components arises during or after translation. Mechanisms by which this could be achieved are discussed. A practical problem which arises here is that N-terminal sequencing of the two components by means of standard techniques is not possible.When SC-protein is separated by means of two-dimensional gel electrophoresis (2D-electrophoresis) and the gel is subsequently blotted and the blot used for immunological detection of the M r 30,000-33,000 components, a large number of variants, differing in isoelectric point and relative electrophoretic mobility can be discerned. In Chapter 3 experiments are described which were performed to determine (i) the nature of the differences between the variants and (ii) possible changes in the observed pattern on 2D-gels during subsequent stages of meiotic prophase. We conclude that differences in the number of attached phosphate groups are largely responsible for the existence of different variants of the M r 30,000 as well as the M r 33,000 component. Moreover, we observed a change in the phosphorylation pattern between early- and midpachytene, probably because of the addition of one phosphate group to all variants. We speculate that the phosphorylation pattern observed as early as zygotene is the result of the action of a kinase which responds to DNA-damage.The presence of the LEs at the bases of the chromatin of the homologous chromosomes prompted us to investigate whether points of contact exist between LEs and the chromatin, and if so, which LE-components and DNA-sequences are involved (Chapter 4). Based on crosslinking experiments in vivo of chromatin from mouse spermatocytes, we assumed that the M r 30,000-33,000 components are bound to DNA or at least associated with DNA. By immunoprecipitation of protein-DNA complexes, obtained after crosslinking in vivo of mouse spermatocytes, with antibodies raised against the M r 30,000-33,000 components as well as against isolated SCs, a region was identified within a 120 kbp gene cluster as a SC - a ssociated r egion (SCAR). This SCAR colocalizes with a so-called 'matrix attachment region' (MAR). We also developed a method by which specific binding of DNA-fragments to the SC can be tested in vitro . By means of this method we show that MARs from different organisms bind specifically to rat SCs. We conclude that the organisation of the chromatin during meiotic prophase shows a certain similarity to those during mitosis and interphase.After it was determined that the M r 30,000-33,000 components are probably bound to DNA in vivo , we have further analysed the possible DNA-binding of these proteins as well as SCP3 in vitro (Chapter 5). Because of the insolubility of SCP3 in aqueous solutions, these studies were carried out by means of the Southwestern blotting technique. We conclude that SCP3 binds to DNA in vitro and shows a preference for single-stranded DNA. From a comparison of different single-stranded DNA substrates we conclude that SCP3 probably favours binding to single-stranded DNA which adopts a spatial conformation caused by intra- or intermolecular interactions. In similar experiments the M r 30,000-33,000 components also exhibit a preference for single-stranded DNA, although the variants which caary the most phosphate residues show no detectable DNA-binding at all. Based on the assumption that in the situation in vivo the M r 30,000-33,000 components would exhibit a similar type of DNA-binding as in vitro , we speculate on the (temporal) existence of single-stranded DNA during meiotic prophase.Finally, I present a summary on what is known about the M r 30,000-33,000 components at the beginning of the general discussion (Chapter 6). Then I discuss a model which describes the chromatin organization during meiotic prophase, in which I particularly point at the modifications which, starting from a general organization pattern, are necessary for the proper progress of the processes of pairing, recombination and segregation of homologous chromosomes. Within this model I discuss the possible involvement of components of the SC. After that I focuss on the function of the M r 30,000-33,000 components in this model. I propose that the M r 30,000-33,000 components play a role in the structural organization of the chromosomes, in such a manner that recombination between sister chromatids is inhibited temporarily and cohesion between sister chromatids is maintained as long as this is needed.

Bibliographic Details

Main Author:	Lammers, H.
Other Authors:	Heyting, C.
Format:	Doctoral thesis biblioteca
Language:	English
Published:	Landbouwuniversiteit Wageningen
Subjects:	meiosis, protein composition, eiwitsamenstelling, meiose,
Online Access:	https://research.wur.nl/en/publications/the-mr-30000-33000-major-protein-components-of-the-lateral-elemen
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