Suppression of him-14(it44ts) by a transgene insertion expressing GFP::COSA-1

Abstract

Meiotic crossover formation requires the activity of multiple pro-crossover factors, including the MutSγ complex and the cyclin-related protein COSA-1, that become concentrated together at the sites of crossover recombination intermediates. Here we show that a transgene insertion expressing GFP::COSA-1 can suppress the crossover deficit caused by a partial reduction in MutSγ function. Our data, combined with previous findings, support a model in which COSA-1 promotes crossover formation, at least in part, through positive regulation of MutSγ function.

Figure 1. Suppression of the him-14(it44ts) crossover deficit at semi-permissive temperature by the GFP::COSA-1-expressing transgene insertion meIs8

A) Example oocyte karyotypes (modified from Zalevsky et al. 1999) illustrating a normal wild-type karyotype (top) with all 6 pairs of homologous chromosomes connected by chiasmata (6 bivalents), and a karyotype from a recombination-deficient mutant (bottom) with 12 achiasmate chromosomes (12 univalents). B and C) Graphs quantifying numbers of resolvable DAPI-stained bodies in diakinesis oocyte nuclei; each diamond represents a nucleus, and mean and standard deviation are indicated. him-14(it44ts) is a temperature-sensitive mutation affecting the HIM-14/MSH-4 subunit of the MutSγ complex; meIs8 is a transgene insertion expressing a GFP::COSA-1 fusion protein. Fixation and DAPI staining were conducted as in Bessler et al. 2007, and numbers of DAPI bodies were counted in oocyte nuclei in the -1 to -3 positions. For the 20°C data, worms raised at the semi-permissive temperature of 20°C were fixed at 24 h post L4; for the 24° and 25°C data, worms were raised at 20°C until the L4 stage, then were shifted to 24° or 25°C for 24 h before fixation. (Note that this assay tends to underrepresent the actual numbers of DAPI bodies present, as some chromosomes may be too close together to be resolved unambiguously.) Genotypes and numbers of nuclei scored: B) him-14(it44ts) unc-4 20°C, n = 62; meIs8 him-14(it44ts) unc-4 20°C, n = 65; meIs8 him-14(it44ts) unc-4 25°C, n = 40; C) meIs8 24C°, n = 63; him-14(it44ts) 24°C, n = 53; meIs8 him-14(it44ts) 24C°, n = 47. p-values are for two-sided Mann Whitney tests. Because of the temperature-sensitive nature of him-14(it44ts), all plotted data for each graph are derived from cohorts of worms raised, fixed and stained in parallel in the same experiment. The finding that meIs8 suppresses the him-14(it44ts) CO deficit at 20°C has been replicated in at least four independent experiments. The findings that meIs8 him-14(it44ts) unc-4 is severely defective in CO formation at 25°C and that suppression of him-14(it44ts) by meIs8 is abrogated at 24C° have each been replicated in at least two independent experiments.