Worm Breeder's Gazette 12(5): 86 (February 1, 1993)

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.

Physical Mapping of Deficiency Breakpoints Near unc-3 by Fluorescent in situ Hybridization

Sean Eddy

MRC-LMB, Hills Road, Cambridge CB2 2QH, UK

I am interested in cloning the gene unc-3 .Motor neuron axons in the ventral cord of unc-3 animals are misplaced and disorganized (J. White). Mosaic analysis by Bob Herman has shown that the focus of unc-3 action is likely to be in the motor neurons themselves (Genetics 116:377-38, 1987). It is likely that the unc-3 product is involved in fasciculation (adherence) of motor neuron processes to neighboring processes in the cord.

Meneely and Herman generated a number of deficiencies covering unc-3 (Genetics 92:99-115, 1979). In order to pin unc-3 down on the physical map, I mapped five such deficiencies using fluorescent in situ hybridization (D. Albertson, EMB0 J. 3:1227-1234, 1984, and EMB0 J. 4:2493-2498, 1985) to probes derived from a set of overlapping YACs covering about 2 Mb to the left. Or unc.7.

Rhodamine-labeled probes were produced by degenerate oligonucleotide primed PCR from a bit of purified YAC gel slice, and hybridized to squashes of pachytene meiotic chromosomes of mnDp1 /+V; mnDf X young adult hermaphrodites. mnDp1 is a V-linked duplication of the right arm of X which balances the homozygous deficiencies. Its presence in all the nuclei provides a convenient internal hybridization control. Two hybridization spots indicates hybridization to both mnDp1 and the paired Df chromosomes and one spot indicates that the YAC probe does not hybridize to the Df chromosomes. YACs spanning- a deficiency breakpoint are usually recognizable by the ambiguous and variable hybridization signals their probes give.

Results are summarized in the figure below. The intervals in which the breakpoints fall are marked by dashed lines and brackets. The left breakpoint of mnDp1 is deduced from the observation that all five deficiencies show single hybridization spots for the YACs left of the Y46E3 / Y41C8 !overlap. unc-3 lies within or to the right of Y16B4 ,and within or to the left of Y70D2 - about a 300 kb interval.

There are some complications, possibly because neither the deficiencies nor mnDp1 ,all gamma-ray induced rearrangements, were created by simple single events. Both mnDf15 and mnDf18 appeared to contain similar extra small deficiencies, which makes me think mnDp1 is missing a chunk of DNA under Y49B6 ;but I haven't tested mnDp1 by itself to be sure. Also, the mnDf2 breakpoint is too far left to agree with the Meneely and Herman's genetic mapping of let-9 relative to these same Df breakpoints.

These data also narrow the physical intervals for a few of let loci in the region, notably let-7 which is in the same interval as unc-3 .

[See Figure]