recombination phenotype. Our observations confirm and extend the notion that CE assembly and CE-specific protein components are not necessary for recombination initiation but essential for recombination progression [27,30–32]. What is the physical link between the recombination machinery and the SC? In mammals, early recombination events and formation of ENs take place before SC central region assembly [27]. Early recombination events also appear to be independent of AE assembly during leptotene/zygotene as shown in mice lacking SYCP3. In these mice, AE assembly is impaired. However, they show normal levels of DMC1 foci formation and synapsis occurs between homologous chromosome regions [21,22]. Recombina- tion progression and crossover formation can also take place in the absence of AE assembly. As shown in female Sycp32/2 mice, oocytes reveal the presence of chiasmata but at a lower level than in the wild-type, which results in a reduction but not a complete loss of fertility [21]. In contrast, assembly of the SC central region is required for recombination progression and crossover formation as shown in mice lacking TF protein SYCP1 or any of the CE- specific proteins ([27,30–32]; this study). Previous electron microscopical studies revealed a close contact between RNs and SC components (e.g. [49,50]). RNs seem to coalesce with the CE and fibers were shown to connect RNs and LEs [50]. These and other observations lead to the proposal that components of RNs might play a role in SC assembly. On the other hand, the SC central region can be seen as a platform for attachment and organization of RN components required for proper recombination progression (see [50], [30] and references therein). Our knowledge about the protein-protein interactions between components of the recombination machinery and the SC of mammals is rather fragmentary. Interactions have been reported between RAD51 and TF protein SYCP1 as well as CE protein SYCE2 [30,51], and between TN component Tex11 (ZIP4H) and LE protein SYCP2 [52]. Although still preliminary, the emerging picture leads to the proposal that SCs and RNs are held together through a network of protein-protein interactions. Therefore, the recombination phenotype described here might be caused by the inability of RN components to bind SYCP1 and SYCE2 due to the defective localization and assembly of these proteins. However, a direct involvement in these interactions of additional CE element proteins (including SYCE3) cannot be excluded at present. Despite the progress in recent years, elucidation of the mutual dependence between SC assembly and meiotic recombination events would require additional experiments. Materials and Methods Ethics Statement All animal care and experiments were conducted in accordance with the guidelines provided by the German Animal Welfare Act (German Ministry of Agriculture, Health and Economic Cooper- ation). For the generation of Syce3 knockout mice we obtained approval from the Landesdirektion Dresden (24-9168.11-9/2005- 1). Animal housing and breeding was approved by the regulatory agency of the city of W u¨rzburg (Reference ABD/OA/Tr; according to 111/1 No. 1 of the German Animal Welfare Act). All aspects of the mouse work were carried out following strict guidelines to insure careful, consistent and ethical handling of mice. Primary Structure Analysis, Posttranslational Modifications, and Sequence Alignment Taking the mouse SYCE3 protein sequence (GenBank accession number: NP_001156354) as query we analyzed Novel Synaptonemal Complex Protein SYCE3 predicted coiled-coil domains using PSORTII (http://psort.hgc. jp/) [53] and predicted phosphorylation sites (http://www.cbs.dtu. dk/services/NetPhos/) [54]. The mouse Syce3 cDNA sequence also served as query for searching all GenBank sequences with the BlastN and TBlastN algorithm (http://blast.ncbi.nlm.nih. gov/Blast.cgi). Multiple sequence alignments were performed online with CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/ index.html) [55]. RNA Extraction, Reverse Transcription, PCR, and cloning of SYCE3 cDNA Whole RNA from mice testes from different ages and from various tissues of adult mice was extracted using peqGOLD TriFastTM (Peqlab, Erlangen, Germany) according to the manufacturer’s protocol. cDNA was synthesized from 1 mg of RNA by reverse transcription with Oligo(dT) primers and M- MLV reverse transcriptase (Promega, Mannheim, Germany). Reverse transcribed cDNA samples were stored at 220uC before they were used in a polymerase chain reaction. Specific primers used for RT-PCRs and respective PCR conditions are listed in Table S1. In order to clone SYCE3 cDNA (GenBank accession number: HQ130280), we amplified full-length SYCE3 from cDNA derived from a reverse transcription of total testis RNA, as described above and using the same oligonucleotides and PCR conditions described for SYCE3 RT-PCR (Table S1). Antibody Generation To raise SYCE3 specific antibodies, we generated and purified a GST-SYCE3 fusion protein using the vector pGEX-5X-1 (Amersham Pharmacia Biotech, Braunschweig, Germany) and the Bulk GST Purification Module (Amersham) according to the manufacturer’s protocol. Anti-SYCE3 antisera were raised by immunizing a rabbit and a guinea pig with the purified GST- SYCE3 fusion protein (Seqlab, Go¨ ttingen, Germany). Specificity of both affinity-purified antibodies was validated by testing them on separate Western blots with protein lysates from wild-type, Syce3+/2 and Syce32/2 littermates (generation of Syce32/2 mice is described below). The presence of the expected band of 12 kDa in wild-type and Syce3+/2 testis lysates and moreover the absence of the aforementioned band in Syce32/2 testis lysates confirmed the specificity of both antibodies (see Figure S1A). SDS-PAGE, Tricine-SDS-PAGE, and Immunoblot Analysis Protein samples derived from co-immunoprecipitation analysis were separated on 10%–15% polyacrylamide gels [56]. Separation of protein samples from testicular cells of adult mice was carried out by using tricine-SDS-PAGE (16% separating gel/6 M urea; [57]). Proteins were transferred to nitrocellulose membranes using the semi-dry Western blotting system described by Matsudaira [58]. The membranes were blocked overnight at 4uC in TBST buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% milk powder. Incubation with the respective primary antibody was carried out in blocking solution for 1 h at room temperature: guinea pig anti-SYCE3 (1:1000), rabbit anti- SYCE3 (1:1000), mouse anti-myc (1:2000; R950-25, Invitrogen, Darmstadt, Germany), mouse anti-GFP (1:200; sc-9996, Santa Cruz Biotechnology, Heidelberg, Germany), guinea pig anti- SYCE1 (1:1000) [29], guinea pig anti-SYCE2 (1:400) [29], mouse anti-actin (1:10000, A4700, Sigma-Aldrich, Munich, Germany). Peroxidase-conjugated secondary antibodies were applied as specified by the manufacturer (Dianova, Hamburg, Germany). Bound antibodies were detected with the enhanced chemilumi- nescence system (Amersham). Western blots were stripped by PLoS Genetics | www.plosgenetics.org 12 May 2011 | Volume 7 | Issue 5 | e1002088