Worm Breeder's Gazette 12(5): 83 (February 1, 1993)
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.
Our goal is to analyze the role that nematode P granules play in germline determination. Whitfield and Glover (Nature, 1989, 338:337-340) have demonstrated in Drosophila that cyclin B mRNA localizes to the germline precursor cells (pole cells) while cyclin A mRNA remains evenly distributed throughout the developing embryo. In order to determine if cyclin B mRNA is similarly localized in nematode embryos. we have cloned Caenorhabditis cyclin genes as reported earlier in the WBG (Richards, et al., January, 1991).
To date we have isolated genomic clones and cDNAs for Caenorhabditis cyclin A and B genes. Sequence analysis of the cDNAs has confirmed their identities, with SL1 found on the 5' end of two of the cyclin cDNAs. There appears to be two cyclin B genes and potentially a multigene family of cyclin A's. Multiple cyclin A's are unique to Caenorhabditis in that no other organism has multiple cyclin A's. Further analysis is required in order to determine if all of these genes are functional. Physical mapping localizes one cyclin B to LG IV near mec-3 and another to LG V near unc-76 .Two cyclin A's have been physically mapped, one to LG II near lin-31 and the other to LG III to the right of lin-12 .Further sequence analysis has revealed some interesting conserved elements in these genes. A conserved proline is present within the potential Caenorhabditis destruction box (required for cyclin protein degradation with each cell cycle). In the 3' untranslated region of the cyclin genes potential adenylation control elements (ACE) are present which have been shown in mice and Xenopus to specify the adenylation state (and thus the translation state) of maternal RNAs in oocytes (Cyclin RNAs have been shown to be abundant maternal messages in clams, Xenopus, and mice.)(Huarte, et al., Cell, 1992, 69:1021-1030).
We have also chosen to isolate the cyclin genes from Ascaris, a parasitic nematode whose embryogenic lineages are much like those of Caenorhabditis. We initially chose to use Ascaris for our localization studies by in situ hybridization because it provides the advantage of producing synchronous embryos, permitting easier interpretation of hybridization results. We have used the Caenorhabditis cyclin cDNAs to probe an Ascaris oocyte cDNA library, after first demonstrating cross hybridization to Ascaris genomic DNA, with slightly reduced stringency of 58° and washed to only 0.5XSSPE. We have isolated putative cyclin A and two B cDNAs from Ascaris. Sequence analysis of these cDNAs should provide confirmation.
Upon sequence confirmation we plan to use sectioned synchronous Ascaris embryos to demonstrate whether cyclin B RNA localizes to the nematode germline precursor cells while cyclin A RNA remains evenly distributed throughout the embryos. If we detect localization with Ascaris, we intend to repeat the in situ experiments using Caenorhabditis sectioned embryos.