Worm Breeder's Gazette 16(4): 24 (October 1, 2000)

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.

Locating spe-6 by mapping the overlap of two LGIII deficiencies, tDf7 and ctDf3

Paul J. Muhlrad1, Samuel Ward2

1 Department of Molecular and Cellular Biology, University of Arizona, Tucson 85704 pmuhlrad@u.arizona.edu
2 Department of Molecular and Cellular Biology, University of Arizona, Tucson 85704 samward@u.arizona.edu

In the later half of the second millennium, when we first set out to clone the spe-6 gene, the physical map in its region of the right arm of chromosome III was tattered and moth-eaten. spe-6 lay in a large hole to the right of tra-1. When nob-1 was cloned, the hole appeared patched, so we were able to gain a foothold to move in on spe-6. spe-6 is the only reported gene whose mutations fail to complement both of the overlapping deficiencies, tDf7 and ctDf3. We therefore decided to locate this overlap region on the physical map. We used PCR on individual dead homozygous ctDf3 or tDf7 mutant embryos, targeting regions of the genome that we suspected either closely flanked or fell within one or both of the deficiencies. With each tester primer set, we also multiplexed a set of primers whose target we knew fell under the deficiency and one primer set whose target was definitely outside of the deficiency. These controls proved essential, because we frequently encountered false positives and false negatives in our PCR reactions. Starting with targets in cosmid T28D6 and YAC Y41C4, we honed in on the overlap region until we had narrowed it down to…oblivion. We could not identify a region on the physical map that was deleted in both deficiencies. Further molecular analysis of the region (represented in YAC Y66A7, accession # AL161712) revealed that its structure was not as depicted on the physical map. Following a series of increasingly pathetic emails to the Sanger Centre, Alan Coulson pulled out a new YAC, Y66D12, that bridged the newly discovered gap, and John Sulston (himself!) constructed a library and sequenced the YAC. The new sequence, about 166 kb, showed numerous extended repeats, which proved difficult to sequence, and were probably responsible for the problems with cloning this region. Continuing with our PCR strategy, we narrowed the tDf7/ctDf3 overlap to within about 12 kb, a region containing three tightly packed genes, represented by Kohara EST clones. EST clones from the leftmost gene are from a hermaphrodite embryonic library, suggesting that this gene was probably not spe-6, a gene involved in spermatogenesis. The other two genes both had clones from a him-8 library. Most enlightening, the middle gene was also was also represented by a clone isolated recently by Harold Smith in our laboratory from a normalized and subtracted cDNA library highly enriched for spermatogenesis genes (i.e. expressed in fem-3 (q23gf) mutant hermaphrodites, which make only sperm, but not in fem-1(hc17ts) mutants, which make only oocytes). Further analysis of this gene (described in the accompanying article) confirmed that it is in fact spe-6.