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.

Detection of Coding Regions in C. elegans

T.P. Snutch, D.L. Baillie

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 
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.