Worm Breeder's Gazette 12(3): 59 (June 15, 1992)

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.

Toward Cloning of smg-2

Brian Carr[1], Rock Pulak[2], Phil Anderson[1]

[1]Department of Genetics, University of Wisconsin, Madison WI, 53706.
[2]Department of Genetics, University of Wisconsin, Madison WI 53706.

mRNAs that contain premature translational termination codons are unstable in most eukaryotic cells. For example, the steady-state level of unc-54 nonsense-containing mRNA is only 5-2096 of that found in wild-type unc-54 mRNA (Pulak, 1991 C. elegans abstracts, pg. 134). The six smg genes are necessary for the instability of nonsense mutation-containing mRNAs. Mutations in any of the smg genes result in increased accumulation of nonsense-containing mRNAs. We think that the smg genes constitute all or part of a system for the removal of potentially harmful mRNAs. Such mRNAs may arise via errors of mRNA synthesis and/or processing. We are cloning smg-2 in order to understand the nature and action of this system.

We are attempting to clone the smg-2 gene by transposon tagging. Suppression of unc-54 ( r293 )is a strong screen for smg(-) mutations. Consequently, we have isolated many spontaneous (TR679-derived) and EMS-induced alleles of smg-2 .One particular TR679 -derivedallele, smg-2 ( r920 ),reverts spontaneously to wild-type at high frequency. smg-2 ( r920 )was backcrossed and recombined extensively with Bristol strains to yield multiple, independently derived, smg-2 ( r920 )lines. We hybridized Southern blots of these r920 mutant lines, three independent revertants of r920 ,and wild-type (N2) with probes for the known C. elegans transposable elements. The copy numbers of all transposons, including Tc1 ,are very low in the backcrossed r920 lines. One particular Tc4 element is present in all r920 -containinglines, but is absent from N2 and from three independent revertants of r920 .We believe that this Tc4 element is responsible for the r920 mutation.

The Tc4 element believed to be responsible for the r920 mutation is contained within a 4.0 kb Sac I fragment. We have clone size-selected (3.8 4.3 kb) DNA from a Sac I digest of r920 DNA into a plasmid vector. Only two Tc4 elements should be represented in this mini-library; the putative r920:: Tc4 and an additional Tc4 that was contained in the size-selected range. We have identified 13 colonies that hybridize to Tc4 ,at least two of which appear to correspond to the putative r920:: Tc4 insert. We are currently testing these Tc4 -containingclones to see if they contain sequences flanking Tc4 that are altered by other smg-2 mutations. We will then use this flanking sequence to isolate cosmid or lambda clones from the region, and attempt and rescue the smg-2 mutant phenotype by microinjection.

Literature Cited:

Pulak, 1991 C. elegans abstracts, pg. 134