Worm Breeder's Gazette 15(3): 25 (June 1, 1998)

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.

Cloning and characterization of mei-2.

Martin Srayko, Paul E. Mains

University of Calgary, Calgary, Alberta, CANADA T2N 4N1

    The mei-1 gene product is required for assembly and/or maintenance of the meiotic spindle.  mei-2 acts genetically as an activator of mei-1.  Recently, mei-2 was identified by the RNA-mediated interference (RNAi) approach.  We chose a clone within the genetically defined mei-2 region because it contained a predicted gene with very weak similarity to a synaptonemal complex (SC) protein.  Initially, we injected RNA transcribed from a genomic region that contained a portion of this putative SC gene.  This gave no phenotype.  However, a second clone (a 4.8kb XbaI genomic fragment derived from cosmid F57B10) did give the mei-2(ct102) phenotype.  This clone contained the SC gene and another small gene (genefinder prediction), transcribed in the opposite direction.  A 1.5kb XbaI-SpeI subclone that just contained the other gene, gave the mei-2 RNAi phenotype (the SC gene turned out to be a red herring, but it thankfully led us to the right gene!).  Chris Shelton and Bruce Bowerman independently found mei-2 in a large-scale survey for maternal-effect RNAi-induced phenotypes (Thanks to Chris and Bruce for communicating their data).
    RT-PCR -derived cDNA for mei-2 was used to establish the gene structure, shown in Figure 1.  mei-2 is trans-spliced to SL1 and encodes 280 amino acids.  All four mei-2mutations have been sequenced (Fig 1.).  Each of the alleles contain G/C-A/T transitions, resulting in missense mutations.  sb31 has an additional 35bp deletion that removes a splice donor sequence within the third intron.  Based on this molecular data and complementation data, we believe sb31 represents the null phenotype of mei-2.
Figure 1.  The molecular structure of mei-2 and the location of mutations.

    Northern analysis of mei-2 (probed against gravid N2, L1, L2/L3, L4, glp-1(lf), fem-1(lf), fem-3(gf) indicates strong expression in both female and male germlines.  This is somewhat surprising, given that mei-2 mutations exhibit strict maternal-effect lethality.
    BLAST searches with the predicted MEI-2 sequence suggest it is a pioneer protein.  However, another C. elegans gene does have high similarity and we are currently testing to see if this is mel-45, a gene that genetically interacts with mei-2 and mei-1.
    MEI-1 protein shares sequence similarity to katanin p60 subunit, a sea urchin ATPase implicated in microtubule severing activity at the centrosome.  Although it is unlikely that MEI-1 is an orthologue (MEI-1 is localized to the meiotic spindle, not the centrosome), it is possible that MEI-1's role is to produce very short meiotic spindles by severing microtubules.  Surprisingly, MEI-2 sequence has a short region of similarity (albeit at a low level) to katanin's p80 subunit.  This subunit localizes the complex to the centrosome in sea urchin.  Perhaps MEI-2 localizes MEI-1 to the meiotic spindle in an analogous fashion.  Antibody production is currently underway to determine the localization pattern of MEI-2.