Worm Breeder's Gazette 14(4): 23 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5427
Pairing and crossing over between homologous chromosomes are required to direct proper orientation of homologs on the meiosis I spindle, thereby ensuring their faithful disjunction. By screening directly for mutants with high levels of meiotic nondisjunction, we identified mutations in at least twelve genes required for normal levels of crossing over between homologs. One of these mutations is an allele of him-14, a gene initially identified in a screen for maternal-effect lethals (Kemphues et al., Genetics 120:977-86). him-14 mutants are defective for the segregation of all chromosomes, and thus produce broods consisting mainly of dead aneuploid embryos plus a few healthy, fertile adult survivors, many of which are male, that by chance received a euploid (or near euploid) chromosome complement. him-14 is required for the formation of crossovers between homologs (Duffy, Basl, and Kemphues, WBG 10(1):104; A.V.). Cytological examination reveals a high frequency of achiasmate chromosomes in him-14 oocyte nuclei at the diakinesis stage of meiotic prophase. Whereas wild-type oocytes at this stage contain six bivalents, indicative of each pair of homologs having undergone a crossover, him-14 oocytes contain up to 12 univalents, and few or no bivalents. him-14 mutants also exhibit a severe reduction in genetic recombination frequencies, indicating that the absence of chiasmata is due to failure to form crossovers rather than to a defect in chiasma maintenance. Temperature-shift experiments suggest that the him-14 gene product functions during the pachytene stage of meiotic prophase, when chromosomes are fully synapsed and recombination is taking place. We shifted young gravid him-14 (it44ts) hermaphrodites from permissive to restrictive temperature (and vice-versa), fixed batches of worms at various times following the shift, and examined DAPI-stained oocytes for the presence of achiasmate chromosomes. By estimating the rate of progression of nuclei through the gonad (based on egg-laying rates and estimates that 1/3 to 1/2 of nuclei undergo programmed cell death), we inferred the stage at the time of the shift for the oocyte being scored. These experiments show that the requirement for him-14 begins during the pachytene stage, and is complete before the end of pachytene. A pachytene requirement indicates that him-14 is not required to establish pairing or synapsis of homologous chromosomes, and suggests a more direct involvement in crossover formation. Our molecular analysis strongly suggests that him-14 encodes a member of the MutS family of mismatch repair proteins. We mapped him-14 between unc-104 and lin-26, immediately to the right of the right breakpoint of mnDf30, which we mapped to cosmid ZK1127 (between sequence coordinates 6743 and 30841) by PCR from mnDf30/mnDf30 dead eggs. By SSCP analysis and/or sequencing, we have now identified mutations in four of six him-14 alleles in the candidate gene ZK1127.11, which encodes a MutS family member most closely related to the MSH4 protein from S. cerevisiae . MutS proteins function in mismatch repair by binding directly to mismatched base pairs or insertion loops and directing the mismatch repair machinery to the site of the mismatch. In humans, homologs of MutS and other mismatch repair proteins are mutated in several types of familial cancer syndromes. Members of the MutS gene family, including MSH4, have recently been shown to play a role in meiotic recombination in yeast. Although the role of MutS proteins in mismatch repair has been well studied, how members of this family might interact with DNA to promote the formation of crossovers is not at all understood. We are attracted by the hypothesis that they may function by binding to Holliday junction recombination intermediates to influence resolution in the direction of crossovers. Our laboratory is currently testing whether HIM-14 can in fact bind Holliday junctions in vitro. We are also testing whether him-14 mutants are defective for mismatch repair.