Worm Breeder's Gazette 11(4): 20
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.
We have been trying to clone the gene unc-80 by the method of transposon tagging, using advice from David Miller, Tim Schedl, Susan Bektesh and Alan Coulson. We searched for spontaneously arising alleles in the mutator strain, RW7097. We obtained 11 such strains, eight of which had spontaneous revertants. We isolated a 3.4 kb BglII fragment which contains a Tc1 and correlates with the unc-80 phenotype 1.)after 14 outcrosses, 2.)after crosses with the linked marker dpy-21, and 3.)after a late occurring (after eight outcrosses) revertant. This fragment was ligated into the plasmid pBS (BamHI site) and labeled pCW1 (we're a real big lab) and we were happy. Restriction mapping showed that the fragment contained a 300 bp region, the Tc1, and a 1500 bp region. We subcloned the 300 bp fragment, the 1500 bp fragment and the 1800 bp fragment resulting from clipping out the Tc1 with EcoRV (pCW2, pCW3 and pCW4, respectively--this is really embarrassing, maybe we should have started with pCW500). [See Figure 1] Alan Coulson sent us his neat YAC library which we screened with pCW4 and (roll of drums) it lit up one clone. We were very happy. Then we identified the YAC as Y24H1 which contains part of chromosome II (unc-80 is on V). We were very, very unhappy. Then it turned out that Y24H1 contained more than just one YAC. We were slightly less unhappy. So we sent pCW4 to Bob Waterston, while we were probing our genomics with this plasmid. Both results showed that pCW4 contained repetitive DNA. About 10 YACs lit up, and the genomics showed about 15-20 bands. Very, very unhappy again. However, using pCW2 and pCW3 separately showed that pCW2 (300bp fragment) contains the repetitive stuff and pCW3 is a unique sequence. We don't know at present if the repetitive sequences are local or widely distributed. We are now sequencing both pieces and extending the cloned DNA by walking. The initial sequence (only a few hundred bp) shows no homology to anything thus far, but is about 50% GC. At present we are no longer suicidal.