Worm Breeder's Gazette 13(2): 93 (February 1, 1994)
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.
Messenger RNAs are rapidly degraded in wild type cells if they contain upstream nonsense codons or are incorrectly processed. The six smg genes in Caenorhabditis elegans (1) are involved in this specialized decay of aberrant messages, which may protect the cell from errors in transcription and processing (2). Multiple alleles of five of the smg genes have been isolated, but only one allele of smg-4 has been found so far, suggesting that this gene may be in some way unusual.
In order to investigate the molecular mechanism of smg gene activity, I have begun to clone smg-4 .Because initial attempts to rescue the smg-4 mutant with cosmids from the region of the physical map predicted to contain the gene (to the right of her-1 )failed, I mapped the locus more precisely. Three factor mapping showed that smg-4 is located just to the right of the gene unc-42 ,within 0.05 map units [ unc-42 (1) smg-4 (24) sma-1 ].Physical mapping of smg-4 relied upon strains deleted for this region. By Southern blot analysis of DNA from strains carrying sDf29 and eDf1 ,I identified cosmids which hybridize to polymorphic bands in deficiency strain DNA and thus define two of these deficiency breakpoints, denoted as * or @ below. As can be seen from the figure below, these data led to the conclusion that both unc-42 and smg-4 physically lie under a YAC bridge. The cosmid at the right end of the bridge, T07C12 ,was also tested in germline transformants and does not rescue the smg-4 or unc-42 mutants tested.
I have now shown that two overlapping YACs, Y42 FSand Y1D1 ,each containing approximately 230 Kb of C. elegans genomic DNA, will not only rescue smg-4 ; unc-54 ( r293 )mutant animals (resulting in paralysis), but also a mutant in the neighboring unc-42 gene (allowing wild type movement). Interestingly, rescue is not apparent in F1 transformants in either case, but only in later generations. Furthermore, both the Smg male tail phenotype and hermaphrodite protruding vulva are fully rescued in a large proportion of the animals.
I am pursuing two strategies to identify a minimal rescuing fragment and ultimately the smg-4 gene. Both rescuing YACs have now been moved into the appropriate yeast genetic background to begin making deletion derivatives by homologous recombination with a YAC fragmentation vector (reagents kindly provided by Stuart Kim). I am also planning to utilize clones from phage mini-libraries carrying S au3 AI partial fragments of the rescuing YACs in further rescue attempts.
(I) J. Hodgkin et al. 1989. Genetics 123(2):189-198.
(2) R. Pulak and P. Anderson. 1993. Genes and Dev. 7:1885-1897.