Synaptojanin is a phosphotidyl inositol-5-phosphatase, which is highly enriched at nerve terminals and binds proteins implicated in synaptic vesicle endocytosis. Four overlapping ESTs isolated by Yuji Kohara are highly homologous to rat synaptojanin. These ESTs hybridize to YAC Y67H2 which spans the cosmid gap between cosmids JC8 and W02A2 on LG IV. Using the ESTs as a launch pad into the gap, we have been able to reconstruct the entire synaptojanin genomic region by screening phage libraries and by PCR crawling into regions not represented in any library. The genomic region comprising synaptojanin extends greater than 25kb. cDNA sequence reveals a protein conserved for each of the four synaptojanin domains, that is, the Sac1, the OCRL, the phosphatase, and the proline-rich domains. We have also identified cDNAs lacking both the phosphatase and proline-rich domains, and splice variants that add or remove a single 20bp exon in the OCRL domain. Within the JC8/W02A2 cosmid gap lies
unc-26.
unc-26 mutants are resistant to acetylcholinesterase inhibitors, have behavioral phenotypes similar to the endocytic mutants
dyn-1, snt!1,
unc-11, and
unc-41, and are severely depleted for synaptic vesicles. These data suggest that UNC-26 may be required for synaptic vesicle endocytosis and thus is a likely candidate for the gene encoding synaptojanin. We have now demonstrated that
unc-26 encodes synaptojanin by sequencing mutant alleles:
s1710 (kindly provided by D. Baillie) is a frameshift in the fifth exon, and
n1307 and
n702 contain 600bp insertions in the first intron. Strikingly, the sequence of these insertions do not resemble Tc1-Tc5, and may represent a novel class of transposons active in the wild-type background. Given the large size of the locus and our current lack of upstream regulatory regions we have been unable to rescue
unc-26. However, Stephanie Chissoe has isolated fosmids of this region using 5' regions of the gene as a probe, and we are currently determining the ability of these fosmids to rescue
unc-26.