Worm Breeder's Gazette 10(1): 34
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.
Unc-5 is one of three identified genes that act in combination to guide mesodermal cell and axon migrations along the dorsal/ventral coordinate of the body wall. We isolated 11 spontaneous unc-5 mutations, two in TR679 and nine in RW7097. Both TR679 isolates were made congenic with N2 Bristol by ten cycles of outcrossing. The unc-5( rh1034) congenic strain did not contain a Tc1 that mapped to unc-5. The unc-5(ev435) congenic strain contained nearly 20 additional Tc1's not present in N2 DNA. Using flanking dpy-13 and unc-44 markers we mapped these Tc1's. One Tc1 of about 2.2 Kb was not separated from unc-5(ev435) by nine recombination events between unc-5 and unc-44. This Tc1 was not present in either of two independent spontaneous revertants of unc-5(ev435), so we believe it is responsible for the unc-5 mutation. We cloned the 2.2 Kb EcoRI fragment containing this Tc1 in lambda gt10, subcloned to puc-19, then removed the Tc1 by cutting with EcoRV and religating. The remaining 0.6 Kb of flanking sequence (containing 38 bp's of Tc1's inverted repeats) should contain at least part of the unc-5 gene or regulatory sequence. A 2.2 Kb EcoRI fragment was labelled on Southern blots of DNA from unc-5(ev435) probed with the 0. 6 Kb fragment, whereas a 0.6 Kb EcoRI fragment was labelled in blots of DNA from the following strains: N2, the two spontaneous revertants of unc-5(ev435) and three unc-5 mutations independently derived from Don Moerman's mutator strains RW7097. No detectable hybridization was seen on blots of DNA from two other spontaneous unc-5 mutations ( rh1036 and rh1037 isolated in RW7097), suggesting that these may have deleted the 0.6 Kb fragment. We used the 0.6 Kb fragment to probe Chris Link's N2 genomic EMBL-4 library and pulled out several positive plaques. One of these contains the expected 0.6 Kb EcoRI fragment which hybridizes to the 0. 6 Kb probe. We will use this genomic clone to examine whether several other unc-5 mutations isolated in mutator strains have DNA polymorphisms in the cloned region. We hope that this clone will allow the identification of cosmids (A. Coulson and J. Sulston) that cover unc-5 and perhaps neighboring genes.