Fig 7. UNO cleavage is sufficient for elimination of homolog conjunction.
(A) UNO contains a separase cleavage site preceded by a threonine (T128 in D. melanogaster) that is highly conserved among drosophilids [22], as illustrated with the sequence alignment. Positions essential for UNO cleavage are indicated by arrows. In the consensus sequence of separase cleavage sites [6,49], Φ indicates hydrophobic, X any amino acid and ζ hydrophilic residues. Separase cleaves C-terminally after R. The UNOTEV-EGFP mutant contains three repeats of the recognition sequence cleaved by TEV protease (grey shading), replacing the separase cleavage site. (B) Squash preparations uno null mutant testes with bam>unoTEV-EGFP and either no TEV transgene, exumP-TEV or betaTub85DP-TEV (bTub85DP-TEV) as indicated were labeled with a DNA stain and analyzed microscopically to reveal presence and localization of UNOTEV-EGFP during spermatocytes maturation. As indicated (dashed line) in the images with the apical testes regions on the left, a premature UNOTEV-EGFP disappearance was induced by the TEV transgenes. This disappearance occurred after the S3 stage, which still displayed a normal chromosome territory organization and UNOTEV-EGFP signals of normal level and localization in sub-nucleolar foci even in the presence of betaTub85DP-TEV (right panel). (C-E) Progression through M I in spermatocytes homozygous for the unocc1 null mutation with His2Av-mRFP and bam>unoTEV-EGFP was analyzed by time-lapse imaging. The spermatocytes expressed either no TEV transgene (C) or betaTub85DP-TEV (bTub85DP-TEV) (D). Still frames of representative spermatocytes are displayed with time indicated (min:sec) with t = 0 at the onset of NEBD I. Chromosome organization at NEBD I (number of major chromosome territories), during prometaphase I (presence of univalents) and telophase I (presence of chromosome bridges) was scored. The results are displayed in the bar diagram (E) with numbers of analyzed spermatocytes (n) indicated. (F) Summary of the protein-protein and protein-DNA interactions of the AHC proteins SNM, UNO, MNM and TEF. See discussion for the implications for the mechanism of alternative homolog conjunction. Scale bars = 20 (B, left), 5 (B, right), and 3 (C,D) μm.