The shrinker phenotype of
unc-49 suggests a defect in the system of cross-inhibitor GABAergic motorneurons, the DDs and VDs. Immunocytochemical staining with antibodies against GABA reveals normal DD and VD morphology and axonal trajectories, and suggests normal levels of GABA (Mclntyre, pers. comm. and our own observations). GABA receptor activity, however, is impaired. Binding studies reveal a 3 fold reduction in GABA binding in
unc-49 as compared to N2, confirming observations by Silver and Way of altered GABA binding ( pers. comm.). Muscimol [8mM] inhibits motility, measured either as rate of locomotion on an agar plate as well as frequency of swimming motions. We observed a 10 fold increased delay in this response in
unc49, consistent with reports by Mclntyre of
unc-49 displaying resistance to GABA ligands in behavioral assays. Analysis of muscle responses to GABA and Muscimol in cut worm assays also reveals altered responses in
unc-49. Polyclonal antibodies against the a-subunit of the mammalian GABAA receptor reveal that a clear pattern of immunoreactivity in N2 musculature is absent in
unc-49. Preliminary results using in situ hybridization show the presence of a putative
unc-49 message in the body wall musculature.We are currently testing various GABA receptor ligands with known binding sites and establishing dose-response relationships in behavioral assays to try to determine the degree of functional homology of the
unc-49 product with the mammalian GABAA receptor and to localize the defects in
unc-49 to a specific sub- unit. Unc-49 has been cloned by transposon tagging. Using 82 recombinant strains of
unc-49 (
n1324) and two flanking markers, a 4.5 kb TC1 band was identified that consistently segregates with the unc- 49 phenotype. The 2.9 kb of flanking genomic sequences were cloned and used to isolate three cDNAs (1.5 kb, 2.8 kb and 3.5 kb) and to identify two YACs, Y40C2 and Y52C5, as putatively containing the unc- 49 coding region. These two YACs overlap and are located in a region of the physical map predicted to correspond to the position of
unc-49 in the genetic map. Identification of the
unc-49 message and analysis of its temporal and spatial pattern of expression, by Northern blot hybridization of staged mRNA and in situ hybridization, is currently in progress.