Worm Breeder's Gazette 10(1): 71
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.
An increasingly popular method to identify and study gene function is anti-sense mediated inhibition of gene expression. Inhibition by anti-sense RNA presumably results from the annealing of a complementary RNA to its target message. Anti-sense mediated inhibition has been used successfully in cultured cell lines, Dictyostelium, Drosophila and Xenopus oocytes. Surprisingly, attempts to inhibit gene expression in Xenopus embryos were not successful. It has since been shown that there is a developmentally regulated activity that denatures RNA:RNA duplexes in Xenopus (Bass and Weintraub, Cell 48, 1986). The denaturing activity in Xenopus is found in embryos but not oocytes and therefore could account for the difference in anti-sense inhibition at these developmental stages. However, it should be noted that there is no direct evidence that this denaturase is relevant to previous failures of anti-sense inhibition in Xenopus embryos. Because of our interest in employing anti-sense methodologies to the study of early development in C. elegans, we were curious to see if C. elegans also had a RNA:RNA duplex denaturase activity. Following the protocols utilized for Xenopus, we made S-100 extracts from worms and assayed them for RNA duplex unwinding activity. We find that C. elegans has such a denaturase and that it appears to be developmentally regulated. S-100 extracts from either embryos, L1s or L2-L3s show the denaturase is abundant in embryos and L1s and relatively absent from L2s and L3s. These developmental differences hold whether comparing microgram equivalent, cell equivalent or nuclear equivalent amounts of extract protein. As with Xenopus, it is unclear if the worm RNA duplex denaturase is relevant to anti-sense experiments in C. elegans. If the denaturase becomes problematic, alternative approaches may circumvent the unwinding activity. For example, it is not known if the unwinding activity is localized in the cell (eg. nuclear vs. cytoplasmic). Preliminary evidence from Xenopus embryos suggests a nuclear localization for the denaturase. If the worm denaturase is nuclear, then cytoplasmic injections of anti-sense RNA could conceivably inhibit expression at the translational level. In any event, it would be prudent to demonstrate that presumptive sense and anti-sense duplexes are stable and persistent before evaluating the success or failure of anti-sense RNAs in C. elegans.As a footnote, a recent variation to the anti-sense theme has emerged in which modified oligodeoxynucleotides are used as the antisense substrate. The modified oligos have been successful in inhibiting gene expression in Xenopus embryos and trypanosames. The numerous modifications available are designed to either increase the intracellular half life of the oligo or cause functionally irreversible duplexes to form. Modified oligos appear to be the best available method for transient anti-sense mediated inhibition.