JCS031799 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1. Tetrahymena (Tt) proteins with a potential function in meiosis. Predictions are based on their affiliation with orthologous clusters of mouse, Mus musculus (Mm), budding yeast (Sc), fission yeast (Sp) and Arabidopsis (At) proteins with known meiotic members. Orthology relationships are derived from OrthoMCL and InParanoid (http://inparanoid.sbc.su.se/cgi-bin/index.cgi) databases, and reciprocal proteome BLASTs (Chen et al., 2007). The criteria supporting the homology of a Tetrahymena protein to other members within the cluster are indicated in brackets using the following codes: O, OrthoMCL; I, Inparanoid ortholog (high confidence); i, Inparanoid inparalog; R, reciprocal BLAST hits. R2 and R3 indicate the rank sums 2 or 3 of reciprocal hits (1). Also shown is the representation of Tetrahymena proteins by expressed sequence tags (ESTs) in conjugating (meiotic) cultures, the common name of the protein family, and function (where known) of meiotic members of the family. For Tetrahymena identifiers the common TTHERM_-key is omitted, as is the ENSMUS key in mouse proteins.

• Supplemental Table S2 -

  Table S2. Homologous pairing of an intercalary (A) and a distal (B) chromosomal region (see Fig. 5) as determined by FISH. If a FISH probe to a chromosomal region produces two separate dots within a MIC, the chromosomes are considered not to be paired in this region. Pairing is indicated by two closely associated (double dot) FISH signals or by the fusion of FISH signals (single dot). It can be seen that in wild type the frequency of nuclei with pairing (double dots + single dots) increases throughout meiotic prophase for both regions. In the hop2AΔ mutant, the maximal level of pairing achieved in stage IV is slightly lower. In the spo11Δ mutant, the maximal pairing (at aberrant stage III) achieves a level comparable to the one found in stage-III MICs of the wild type.

• Supplemental Figure S1 -

  Fig. S1. Multiple sequence alignments of Mnd1- and Hop2-family proteins. Protein-sequence identifiers are composed of two-letter species codes (Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Mm, Mus musculus; At, Arabidopsis thaliana; Tt, Tetrahymena thermophila) and common protein names; for Tetrahymena proteins the TTHERM_ identifier is provided. (A) Multiple sequence alignment of the predicted winged helix-like domain in Mnd1 proteins, which also represents the region with highest conservation within the Mnd1 family. Shading according to conservation and BLOSUM62 scores (Clamp et al., 2004). Predicted α-helices (white bars) and β-strands (gray arrows) are indicated below the alignment. (B) Multiple sequence alignment of the predicted winged helix-like domain of Hop2- and Mnd1-family proteins. Shading according to conservation and BLOSUM62 scores.

• Supplemental Figure S2 -

  Fig. S2. (A) Design of the knockout constructs for the disruption of HOP2A, SPO11 and HOP2B, and sizes of the restriction fragments for Southern testing of the wild-type (WT) and the disrupted locus. (B) Southern blots of several knockout (KO) isolates of HOP2A and SPO11 demonstrating the (almost) complete loss of wild-type copies of these genes. The (untranscribed) micronuclear copies are retained. For HOP2B, Southern blotting produced significant wild-type bands in all isolates. Relative densities suggest that the cells contained between 30 and 50% of the wild-type locus. Therefore, only a partial knockdown (KD) of HOP2B was achieved, as it is probably essential for vegetative growth.