Worm Breeder's Gazette 12(1): 27 (September 1, 1991)

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.

The cloning of smg-5 ,a gene involved in the degradation of aberrant mRNAs

Kirk Anders, Rock Pulak, Phil Anderson

Laboratory of Genetics, University of Wisconsin, Madison, WI

The informational suppressor genes smg-1 through smg-6 are likely to play a role in the identification and degradation of aberrant mRNAs. The smg genes prevent the accumulation of unc-54 mRNAs containing nonsense and frameshift mutations. Mutations in any one of the six smg genes allow aberrant unc-54 mRNAs to accumulate to near wild-type levels (see Pulak and Anderson, 1991 Meeting Abstracts). We have cloned smg-5 to further understand its role in aberrant mRNA turnover.

We utilized the correspondence between the genetic and physical maps to identify cosmid clones likely to contain smg-5 .Using three-factor mapping, we localized smg-5 to a 0.07 m.u. interval that also contains unc-87 ,whose physical map position is known (see Goetinck and Waterston, WBG 11(4):30). The 95% confidence interval for the location of smg-5 extends 0.05 m.u. to the left and 0.02 m.u. to the right of unc-87 .In addition, we have isolated a spontaneous smg-5 allele, r919 .This smg-5 allele was isolated in a strain containing mut-2 ( r459 ),which exhibits a high frequency of transposon-induced mutations. smg-5 ( r919 )is unstable and we have isolated four independent smg-5 (+)revertants.

Using cosmid clones from the region surrounding unc-87 we searched for DNA alterations that cosegregated with the mutator-induced smg-5 ( r919 )allele. Southern blots probed with cosmid W02D3 (kindly supplied by J. Sulston and A. Coulson) detect an apparent insertion of 1.6 kb that is present in smg-5 ( r919 )but is absent in wild-type and all of the revertant strains.

To prove that we have identified DNA containing the wild-type smg-5 gene, we tested the ability of cosmid W02D3 to rescue a smg-5 mutation upon microinjection. The recipient worms were smg-5 ( r860 ) unc-54 ( r293 )double mutants. These worms are fully motile because the unc-54 ( r293 )allele is suppressed in a smg(-) background. Injection of a wild-type copy of smg-5 is expected to eliminate suppression of unc-54 ( r293 ),and transformed progeny should be paralyzed. We co-injected smg-5 ( r860 ) unc-54 ( r293 )double mutants with cosmid W02D3 and the marker plasmid pRF4 ( rol-6 (dm)).Paralyzed worms were observed among the F1 progeny. When prodded, these worms attempt to roll. This indicates that cosmid W02D3 indeed rescues a smg-5 mutation and contains the wild-type smg-5 gene. We are in the process of identifying smaller regions of W02D3 which will rescue smg-5 .Further molecular analysis will include identification of the smg-5 transcription unit and determination of its DNA sequence.