Worm Breeder's Gazette 14(2): 28 (February 1, 1996)
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.
Dept. of Biological Sciences, University of Alberta, Edmonton, Alberta CANADA T6G 2E9
The LGIII-derived aberrations eDf2 and eDp6 were obtained concomitantly after acetaldehyde mutagenesis(1). The left-hand eDf2 portion (approximately two-thirds of LGIII) behaves genetically like a terminal deletion, and the remaining portion of LGIII formed the free duplication eDp6, which may be circular (Hunter and Wood, WBG 10#2, p.150). eDf2 complements mutations to the left of unc-119, and eDp6 complements all those to the right. We have previously shown that animals homozygous for eDf2 and eDp6 display the same phenotype as unc-119 mutants, and that eDf2; eDp6 animals transgenic for unc-119 rescuing clones are indistinguishable from wild type, consistent with unc-119 being the only gene affected(2). It is of interest to understand the nature of these aberrations, perhaps as a model for understanding the healing of chromosome breaks in C. elegans. Our previous long-range restriction analysis has shown that there are at least 100 kbp of DNA that have been added beyond the eDf2 breakpoint (WBG 13#2, pp.64-65), suggesting that the healing¹ of the break involved the addition of a large amount of DNA, perhaps from the original right end of LGIII, either as a direct splice¹ or as a result of a recombination event. Using a size-selected minilibrary, and screening with a fragment just upstream of the unc-119 transcription initiation site, we have cloned a 5.6-kbp fragment of eDf2 (pDP#MM071). The insert of pDP#MM071 was used to probe genomic Southerns and to test against the polytene YAC grid. Confirmation of the uniqueness of the added DNA was obtained: In addition to expected bands from the unc-119 portion of the probe, an apparently unlinked, ~12 kbp SstI fragment was found in all strains examined, even eDf2; eDp6. We sequenced across the breakpoint junction and verified that this clone contains part of the presumptive unc-119 promoter followed by an abrupt change to eDf2-specific sequences. We searched the worm databases using ~700 bp of sequence of this extra¹ DNA but did not find any 100% matches, only strong similarities in repeated regions to those found on cosmids throughout the genome. Therefore, the source of the added sequences was pre-existing in the genome and is not solely telomeric in nature. The polytene grid results were unclear. The strongest positive was Y41E4 on LGIIL, near lin-31. However, none of the overlapping YACs Y2C8, Y14H12 and Y8A9 were positive, so this result is probably an artifact; the clone is probably just not represented in the grid. However, this would nicely explain why the original ~12 kbp fragment is present in eDf2; eDp6 - this strain was originally backcrossed many times and only selected for the unc phenotype. If the sequences added to eDf2 contain genes, it may be possible for eDf2 to complement mutations that do not map on LGIII to the left of unc-119. Indeed, Starich et al report such a gene, dyf-2, at the far right end of LGIII, near unc-64, but which is apparently complemented by both eDp6 and eDf2(3). [Figure accomanies original] 1.Hodgkin, Genetics 96:649-664. 2.Maduro and Pilgrim, Genetics 141:977-988. 3.Starich et al, Genetics 139: 171-188.