Figure 1  The Mouse PCH2 Ortholog TRIP13 and Expression in Wild Type and Mutant
(A) Phylogenetic tree of presumed PCH2/TRIP13 orthologs. The database sequence accession number of each protein is presented in Table S1. Numbers shown are bootstrap values (see Materials and Methods). The clustering was the same regardless of whether the whole entire AA sequence or trimmed sequences (where regions showing little conservation were removed) were used for the analysis. Major eukaryotic groups are indicated in color, with deuterostomia in blue, plants in green, protostomia in purple, and fungi in maroon.
(B) Amplification products of cDNA from the following tissues: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis; 9, E7 embryo; 10, E11 embryo; 11, E15 embryo; and 12, E17 embryo.
(C) Intron–exon structure of TRIP13 and insertion site of gene-trap vector. See Materials and Methods for details on how the precise insertion site was identified.
(D) RT-PCR of Trip13 and a control gene Med31 from testis RNA. The Trip13 primers are situated in the first and last exons (see Materials and Methods).
(E) Western blot analysis of testis protein with anti-TRIP13 antibody. The blot was later probed with anti-alpha tubulin actin as a loading control. The expected TRIP13 protein is ∼48 KDa.
(F) Localization of TRIP13 in testes. Wild-type (top) and mutant (bottom) testis sections were probed with chicken anti-TRIP13, and detected with HRP-conjugated anti-chicken IgG (brown/red staining). Expression in WT was most prominent in the nuclei of Type B spermatogonia (Sg), leptotene spermatocytes (LS), and early pachytene spermatocytes (PS), but not late pachytene spermatocytes (LP). No nuclear staining was seen in mutant testis sections, although reddish cytoplasmic background is present. Identification of cell types was judged in part by estimating the epithelial stage of the tubules as described [67].
(G) TRIP13 localization in surface-spread spermatocytes. Preparations were immunolabeled with anti-SYCP3 (S) and TRIP13 (T). Both individual and merged images are shown for leptotene (Lep), zygotene (Zyg), and pachytene (Pac) spermatocytes. Nuclear staining was absent in the mutant.