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]