Worm Breeder's Gazette 8(3): 66
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 number of C. elegans workers have demonstrated the feasibility of utilizing RFLDs as molecular genetic probes for cloning and mapping genomic DNA sequences (Emmons et al, 1979; Rose et al, 1982). In addition, Sulston et al (Newsletter vol. 8, no. 2) have begun the herculean task of mapping the entire C. elegans genome. A question that arises from these efforts is how to identify the cloned sequences which are of interest - ie. coding sequences. The standard techniques of S-1 mapping, R-looping, Northern blotting and screening of cDNA libraries are labor intensive and too time consuming for the initial screening of large regions of cloned DNA. We believe that this problem can be simplified by utilizing the hybridization of cloned C. elegans probes to genomic blots of C. briggsae DNA. Evidence reported by Emmons et al (1979) and our own both indicate that while some portions of the C. briggsae and C. elegans genomes hybridize to each other, the majority of sequences have diverged significantly and do not cross-hybridize under moderate stringency conditions. Since C. elegans and C. briggsae are nearly identical, and in fact, are considered twin species (Dougherty, 1953), it is reasonable to assume that the divergent regions include mostly noncoding DNA sequences while the highly conserved regions code for the proteins necessary for nematode growth, development and reproduction. Our own results seem to support this idea. In the course of our work on C. elegans heat shock genes we have found that essentially only the cloned regions which we have found to react positively with RNA also show conservation to sequences within the C. briggsae genome. The condition that we use are probes nicktranslated to a specific activity of 1-2x10+E8 cmp/ g hybridized at 68 C in 5 x SSPE, 0.2% SDS and 2.5 x Denharts for 24 hours and then washed 4 times 20 minutes in 0.2 x SSPE, 0.2% SDS at 68 C. We have tested about 60 kb of cloned DNA around three different C. elegans regions which show homology to the Drosophila hsp70 gene. Two regions about 3 kb in size each are conserved between C. elegans and C. briggsae under these stringency conditions. From both Northern blots and the screening of B. Myers cDNA library we have found that these regions are coding. The third region of hsp70 homology does not react positively with RNA blots nor can we find any corresponding cDNAs in the cDNA library. In addition, none of the flanking regions of any of these regions reacts with RNA, shows positives from the cDNA library or cross-reacts with the C. briggsae genome. We believe that the initial blotting of cloned DNA to C. briggsae will give a good indication of whether the DNA is coding or not. We normally expose the filters for 24-36 hours with a screen. On longer exposures minor homologies between the two species appear but these are easily distinguishable from the coding regions which give good, strong signals.