Materials and Methods

PCR analysis of Trip13 cDNA.
Trip13 was amplified from samples of Clontech's Mouse Multiple Tissue cDNA Panel I (http://www.clontech.com), using the following primers: 5′-GCACCATTGCACTTCACATC-3′ (TRP3-6F) and 5′-TGACCATCAGACTGTCGAGC-3′ (TRP3-6R). These primers correspond to exons 3 and 6, respectively, and amplify a 330-bp cDNA product. The cDNAs in this panel are equalized to allow quantitative analysis by RT-PCR.

Generation of Trip13-deficient mice.
The mouse embryonic stem cell line RRB047 (strain 129/Ola) containing a gene trap insertion in Trip13 was obtained from BayGenomics (http://www.baygenomics.ucsf.edu/). The gene-trapping vector used to create this line, pGT1lxf, was designed to create an in-frame fusion between the 5′ exons of the trapped gene and a reporter, βgeo (a fusion of β-galactosidase and neomycin phosphotransferase II). The gene-trapped locus creates a fusion transcript containing exons 1–3 of Trip13 and βgeo. To identify the exact insertion site within intron 3, PCR was performed using one primer within the gene trap vector, and the other primer at various positions in intron 3 pointing towards the 3′ end of the gene. Product from a productive reaction was sequenced, revealing that the insertion site was 445 bp into intron 3.

Genotyping of mice.
Three primers were used to distinguish wild-type and mutant alleles of Trip13: primer 1, 5′-CGTCGCTCCATTGCTTTGTGC-3′; primer 2, 5′-AGTAGTGGTACACTGTATTTTTGCTTTCATTGA-3′; and primer 3, 5′-GTAGATCCCGGCGCTCTTACCAA-3′. Primers 1 and 2 are located upstream and downstream, respectively, of the gene trap insertion within the intron 3. Primer 3 corresponds to pGTlxf sequence. Primers 1 and 2 amplify a 700-bp band from a wild-type allele; primers 1 and 3 amplify a 540-bp fragment from a mutant allele. Separate reactions were used to assay the presence or absence of each amplicon from a DNA sample. The cycling conditions were: 94 °C 2 min; 35 cycles of 94 °C 30 s, 57 °C 45 s, and 72°C 50 s; and 72 °C 2 min.

RT-PCR.
Total RNA was isolated from adult testes with the RNeasy Mini Kit (Qiagen, http://www.qiagen.com), and 4.0 μg was oligo dT–primed and reverse-transcribed with Superscript II (Stratagene, http://www.stratagene.com). The entire Trip13 protein-coding sequence was amplified with primers 5′-ATGGACGAGGCGGTG-3′ and 5′-TCAAACATAAGCTGAAAGTT-3′. The cycling conditions were: 94 °C 2 min; 94 °C 30s, 55 °C 45 s, and 72 °C 80 s for 35 cycles; and 72 °C 2 min. The primers for amplifying the Med31 coding sequence as control were : 5′-ATGGCCGCGGCCGTCGCTATGG-3′ and 5′-TCATTTCCCTGCTGTGTTATTCTGCTGCTGCTGC-3′. The cycling conditions were: 94 °C 2 min; 94 °C 30 s, 55 °C 30 s, and 72 °C 35 s for 35 cycles; and 72 °C 2 min.

Development and purification of chicken antibodies.
A peptide corresponding to amino acids 25–40 of TRIP13, VLQRSGSTAKKEDIK, was conjugated to KLH and used to immunize chickens (done by Sigma Genosys, http://www.sigmaaldrich.com). Polyclonal IgY was isolated from eggs with the Eggcellent Chicken IgY Purification kit (Pierce, http://www.piercenet.com). IgY antibodies were then affinity purified using the immunizing synthetic peptide.

Western blotting.
50 μg of testis extract in RIPA buffer was separated by 8% SDS-PAGE and electrotransferred onto a Pure Nitrocellulose membrane (Bio-Rad, http://www.biorad.com). The membrane was incubated with a polyclonal rabbit anti-human TRIP13 antibody (18-003-42687; Genway, http://www.genwaybio.com). According to the manufacturer, the immunogen was a synthetic peptide embedded in sequence we deduced to correspond to exon 3. Binding was detected by chemiluminescence ECL kit (Pierce) using a rabbit anti-chicken IgG horseradish peroxidase conjugate (Pierce).

Histological analyses.
Testes or ovaries were fixed in Bouin's, embedded in paraffin, sectioned at 6 μm, and stained by hematoxylin and eosin. Antigen retrieval for immunohistochemistry of testis sections was as described [60]. Oocyte and follicle numbers were counted as described [61]. Only follicles containing an oocyte with a clearly visible nucleus were scored.

Immunocytochemistry.
Immunolabeling of surface-spread spermatocytes and oocytes was performed as described [39,62]. To reach conclusions on the pattern of staining for various proteins, 30 (unless otherwise indicated) well-spread nuclei of particular meiotic stages were first identified under the fluorescent microscope on the basis of SYCP3 or STAG3 staining, then imaged at both appropriate wavelengths to determine the pattern of second proteins with focal patterns such as RAD51 or RPA. Unless otherwise indicated, the panels shown in the figures were the exclusive or predominant patterns seen. The exception for this approach was in the case of staining for MLH1 or MLH3 plus RAD51 (in which case SYCP3 or STAG3 was not available to find chromosome cores). Nuclei in this situation were identified first by MLH1/3 foci clustering, then imaged for both fluorescent wavelengths.
Primary antibodies used in this study were as follows: mouse anti-SCP3 (1:500; Abcam, http://www.abcam.com); rabbit anti-SYCP1 (1:1,000; a gift from C. Heyting) [63]; rabbit anti-REC8 (1:100; a gift from C. Heyting); rabbit anti-RAD51 (1:250, this polyclonal antibody recognizes both RAD51 and DMC1; Oncogene Research Products, http://www.merckbiosciences.co.uk); rabbit anti-γH2AX (1:500; Upstate Biotechnology, http://www.upstate.com/); rabbit anti-STAG3 (1:1,000; a gift from R. Jessberger); rabbit anti-MLH3 (1:400; a gift from P. Cohen); mouse-anti-human MLH1 (1:50; BD Biosciences, http://www.bdbiosciences.com); rabbit-anti-TopBP1 (1:100; a gift from J. Chen) [22]; mouse-anti-ubiquityl-histone H2A (1:200; Upstate Biotechnology); rabbit-anti-TRF2 (1:500; a gift from T. de Lange); and rabbit-anti-BLM (1:50; a gift from R. Freire). All secondary antibodies conjugated with either Alexa Fluor 488 or 594 (Molecular Probes, http://probes.invitrogen.com/) were used at a dilution of 1:1,000. All images were taken with a 100× objective lens under immersion oil.

Metaphase I spermatocyte spreads and OA treatment.
Metaphase fixed spermatocytes from 8-mo-old Trip13RRB047 homozygotes, using 23-d-old wild-type mice as control, were prepared and stained with Giemsa as described [64].
For OA treatment, cells were exposed to 5 μM OA (Calbiochem, http://www.emdbiosciences.com) for 6 h at 32 °C in a humidified environment of 5% CO2 before spreading [65]. These preparations were stained with DAPI to visualize metaphase nuclei and chromosomes.

Phylogenetic analyses.
TRIP13 orthologs were identified by BLASTP searches of Genbank and other sources providing gene models such as Ensembl. The selected orthologs can be found in Table S1. Amino acid alignments were done with Clustal W, using the default settings with and without removing the regions outside of the AAA-ATPase central domain. The trees were constructed by using the neighbor-joining method with Poisson correction. The reliability of internal branches was assessed by using 500 bootstrap replicates, and sites with gaps were ignored in this analysis. Neighbor-joining searches were conducted by using the computer program MEGA3 [66].