We are studying the C. elegans member of a novel family of DNA polymerases also present in Drosophila, Arabidopsis and humans. This family is distantly related to the mitochondrial Pol-gamma, but is evolutionary closer the bacterial Pol-A family. With the exception of one human homologue, all other members of this family include a domain with helicase similarity 5 of the polymerase domain. These constitute a unique category of proteins with a helicase and a polymerase within the same gene. We have cloned the worm orthologue (chr. III,
w03a3.2 & .3) by RT-PCR and established its actual exon-intron (19 exons) organization. Consistent with the fly orthologue
mus-308, the helicase and the polymerase domains produce a single transcript in C. elegans. In addition, the gene is spliced in trans to SL-1 and encodes
ceh-10 within the fifth intron on the opposite strand.
mus-308 is involved in DNA repair of interstrand cross-links. To elucidate the function and developmental pattern of the new gene, we have targeted the two individual regions of the gene using RNAi by injection and ingestion (transformed bacteria producing dsRNA). With either method of dsRNA delivery, we have observed a set of various defects at low penetrance. These range from embryonic and larval arrest (L1-L2) with generalized malformations, to small morphological defects that do not seem to affect viability of these nematodes in lab conditions. Successive generations of (genotypically) N2 Bristol kept on plates of transformed bacteria expressing dsRNA for the two domains can be propagated, while the phenotypes mentioned above can simultaneously be observed in the same plate (as positive control in the RNAi feeding experiments, we have used
unc-22 and
npr-1, both giving striking phenotypes under identical conditions). Several questions have arisen. Is the knockdown of this gene an all or nothing event and why is it so infrequent? Do we need a challenging agent or condition to observe the authentic phenotype? To elucidate the expression pattern of this gene, we are also establishing lines with GFP fusions including 3, 1.6 kb and 250 bp upstream of the helicase gene. We are currently titrating the transgene to inject, as only F1s have been obtained so far (toxic gene?). In addition, polyclonal antibody production for immunolocalization studies is also in progress.