Worm Breeder's Gazette 11(5): 25

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.

A Cloned PCR Product Amplified from C. elegans Genomic DNA Shares Amino Acid Homology with Germline-Specific Helicases, Vasa and PL10

Deborah Lynn Roussell and Karen Bennett

We are interested in those genes (and their protein products) that 
establish the germline in the developing embryo of nematodes.  
Potential candidates for germline determinants include the protein 
component of the P-granules.  Mutants that lack or improperly 
segregate these maternally supplied granules do not develop a germline,
are sterile, and are phenotypically grouped into a class of mutants 
appropriately called grandchildless (Schupbach and Wieschaus, Dev.  
Biol.  195:302-317; Martin et al., C. elegans Meeting Report, CSH, pp.
197).  The first gene to be cloned that corresponds to a known 
grandchildless locus is the vasa gene from Drosophila melanogaster (
Lasko and Ashburner, Nature 335:611-617; Hay et al., Cell 55:577-587). 
We have cloned a potential vasa-equivalent from Caenorhabditis 
ethod of polymerase chain reaction (PCR).
Degenerate primers were designed from the predicted vasa amino acid 
sequence.  The PCR-amplified C.  elegans DNA was purified, cloned and 
sequenced.  Amino acid sequence comparisons show 48% perfect amino 
acid homology (and 66% homology with conserved amino acid changes) 
between the Drosophila vasa and the Caenorhabditis PCR clone that we 
call NC1.  In addition, this clone shares 48% perfect amino acid 
homology with the mouse gene PL10, a putative male germ cell-specific 
helicase specific (Leroy et al., Cell 57:549-559).  As members of the 
so-called DEAD family of helicases (a subclass of proteins with 
putative helicase activity upon nucleic acids), both vasa and PL10 
contain the five conserved helicase motifs that distinguish this 
family of proteins (Nature 337:121-122; Gorbalenya et al., NAR 17:4713-
4730).  In the portion of C.  elegans DNA that we amplified and 
sequenced there are also conserved helicase motifs and other short 
stretches of amino acids that are conserved among helicases.  However, 
most interesting to us is another region of perfect amino acid 
identity that NC1 shares only with the two germline-specific putative 
helicases, vasa and PL10.
The NC1 insert DNA hybridizes to a 6.4 Kb and 4.3 Kb DNA fragment on 
genomic Southerns of EcoRI digested C.  elegans genomic DNA.  We do 
not yet know if this represents one or multiple genes.  On Northern 
analyses of poly A+ RNA from mixed stage C.  elegans, NC1 hybridizes 
to an approximately 2.6 Kb RNA and also to a larger, relatively less 
abundant RNA.
The PCR clone, NC1, has also been used as a hybridization probe to 
isolate a C.  elegans genomic clone, CEA1, from a genomic library in 
lambda EMBL4 that was previously constructed in this laboratory.  
Sequence analysis of CEA1 should determine if the C.  elegans clone is 
vasa-like, PL10-like, or a new potential germline-specific helicase.  
Additionally, placement of this cloned gene on the physical map will 
determine if it corresponds to any known Caenorhabditis mutants.