We are using a positional cloning approach to clone
smg-3, one of 7 smg genes required in C. elegans for the degradation of nonsense mutant mRNAs.
smg-3 was localized to LGIV between
unc-44 and
deb-1 using an RFLP mapping approach.
smg-3 maps between the cosmids T13A10 and F15B10. Until recently, the T13A10/F15B10 region represented a cosmid gap bridged by a single 600 kb YAC, Y73B6. Positioning of
smg-3 to the T13A10/F15B10 gap was confirmed by transformation rescue experiments. Y73B6, which spans and extends to the right of the T13A10/F15B10 region has
smg-3(+) activity. A circularized derivative of Y73B6 containing only the C. elegans genomic DNA from T13A10 to F15B10 was isolated. This derivative, TR#242, has
smg-3(+) activity. Y48F8, which extends into but not across the cosmid gap, does not rescue
smg-3 mutants. Recently, the physical map of the T13A10/F15B10 region was refined. A cosmid contig and a lambda contig were positioned within the T13A10/F15B10 gap. The cosmid contig overlaps with and extends approximately 88 kb to the left of F15B10 (toward T13A10). The lambda contig extends from T13A10 rightward for approximately 55 kb (8 kb beyond the right end of Y48F8). Both
gut-4 and
bli-6 are contained on this 55 kb lamda contig (A. Davies, pers. comm.). Three-factor mapping data suggests that
smg-3 is tightly linked to
bli-6. These data suggest that
smg-3 is close to the right end of Y48F8. Transformation rescue experiments using the lambda clones isolated from the walk are in progress. The leftmost cosmid in the contig, C01G5, does not contain
smg-3(+) activity in rescue experiments. The circular YAC derivative, TR#242, was linearized by homologous recombination in yeast. The resulting linear YAC is approximately 350 kb and contains the region between T13A10 and F15B10. Using the estimated size of this linear YAC, the distance between the end of Y48F8 and C01G5 is approximately 215 kb. We are currently purifying the linear YAC DNA for transformation rescue experiments, sequencing and construction of a BAC library.