Worm Breeder's Gazette 10(3): 51

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 of the unc-104 Gene by Transposon (Tc1) Tagging

Ayyamperumal Jeyaprakash, Toinette Hartshorne, Rodrigo Franco, Edward Hedgecock and Anthony Otsuka

Figure 1

Mutations in the unc-104 gene result in lack of electron dense 
material at presynaptic nerve endings and absence of neuromuscular 
junctions.  We have cloned unc-104 by transposon (Tc1) tagging as a 
first step towards understanding the molecular mechanism of this 
defect.  Three alleles of the unc-104 gene (e2184, rh1016, rh1017) 
isolated from transposon high copy strains have been tested.  Southern 
blots of DNA from wild type N2, mutant (e2184*7).  and several 
spontaneous revertant strains were probed with Tc1.  A unique 3kb 
EcoRI fragment always cosegregated with the mutant phenotype, but was 
absent in revertant strains.  The 3kb fragment was cloned separately 
into bacteriophage lambda Charon 21A and pUC8 plasmid vectors.  The 
relationship between the transposon (Tc1) containing clone and unc-104 
was established by linkage analysis, excision of transposon Tc1 in 
several spontaneous revertants, and localization of Tc1 insertions in 
the alleles rh1016 and rh1017 to a nearby restriction fragment.  The 
unc-104 gene has been isolated on 6 genomic clones containing 20 kb of 
contiguous DNA from a bacteriophage lambda N2 library (kindly provided 
by N.  Nishiwaki and J.  Miwa, NEC, Japan ).  The genomic clones were 
mapped to a contig containing unc-104 and tra-2 and 4 cosmid clones 
containing 80 kb of contiguous DNA have been isolated by Alan Coulson 
and John Sulston ( MRC, England ).  Three cDNA fragments have been 
isolated from an N2 cDNA library on bacteriophage lambda gt10 ( kindly 
provided by J.  Arhinger and J.  Kimble, Univ.  of Wisconsin, Madison )
.  A 0.75 kb cDNA is the 5' most fragment thus far isolated and 
corresponds to the region where Tc1 insertion occurs in the e2184 
allele.  A 1.1 kb cDNA fragment is derived from the downstream 1.4 kb 
EcoRI genomic fragment containing the sites of Tc1 insertions in the 
rh1016 and rh1017 alleles.  The third cloned cDNA fragment hopefully 
contains sequences corresponding to the remaining 3' portion of the 
approximately 5.6 kb message detected by Northern blotting.  Only weak 
homologies have been found to genes and proteins in the Genbank and 
PIR data banks.
[See Figure 1]

Figure 1