Worm Breeder's Gazette 11(4): 24

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.

Cloning ced-1, a Gene Required for the Engulfment of Cell Corpses

Tony Gerber, Ronald Ellis and Bob Horvitz

In wild-type C.  elegans hermaphrodites, 131 cells undergo 
programmed cell death.  Each resulting cell corpse is quickly engulfed 
and degraded by a neighboring cell.  Ed Hedgecock showed that 
mutations in the genes ced-1 and ced-2 prevent the engulfment of cell 
corpses.  Since then, we have identified five additional genes -- ced-
5, ced-7, 
required for the engulfment step. 
In animals with mutations in any of these genes, corpses resulting 
from programmed cell death can persist for hours.  Genetic studies 
suggest that these genes fall into two groups that might have 
partially redundant functions in the engulfment process: ced-1, 
ced-7 and ced-8 constitute one group, while ced-2, 
te the other.  To understand 
better how the engulfment process works at the molecular level and why 
there appear to be two classes of genes involved in this process, we 
have undertaken a molecular analysis of ced-1.The ced-1(n1506) I 
mutation was isolated in a mut-2 genetic background, in which a number 
of C.  elegans transposons are active.  Southern analysis revealed a 
novel 2.9 kb EcoRI band in DNA from ced-1(n1506) animals, using the 
transposon Tc3 as a probe.  This polymorphic band is within 0.1 map 
units of ced-1.We cloned this Tc3 polymorphism from a size-
fractionated library made with DNA from ced-1(n1506) animals.  This 
clone, skT1, contains 2.0 kb of non-Tc3 sequence in addition to a 0.9 
kb EcoRI end fragment of Tc3.  We used parts of the non-Tc3 sequence 
of skT1 to probe a copy of the YAC 'polytene' filters generously 
provided by John Sulston and Alan Coulson.  Three overlapping YACs, 
Y5D9, Y53C10 and Y47H9, which form part of a previously unpositioned 
700 kb contig, were identified.  None of the cosmids that overlap all 
three of these YACs hybridizes to our probe, so we constructed a phage 
library from the YAC Y5D9 by ligating size-selected SauIIIA partial 
digests of Y5D9 into BamHI-digested Lambda Dash arms.  We obtained 
approximately 125,000 recombinant phage.  We screened 50,000 of these 
with the probe derived from skT1 and identified several thousand 
positive plaques.  We are currently analyzing these phage, with the 
goal of obtaining 30 to 40 kb of overlapping phage covering the region 
of the Tc3 polymorphism.  We plan to use these phage in microinjection 
experiments to attempt to rescue the ced-1 phenotype.