Worm Breeder's Gazette 14(5): 26 (February 1, 1997)
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.
|1||Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Md. 21210 USA and Notre Dame College of Maryland|
|2||Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Md. 21210 USA|
At the 1995 international C. elegans meeting, Kevin Fitzgerald and Rock Pulak suggested to us a scheme for engineering conditional expression from virtually any promoter. This strategy takes advantage of the Smg RNA surveillance system in C. elegans . Similar uses of conditional Smg activity had also been proposed by Papp  and Ransom et al.  The proposed scheme is as follows: I. Isolate conditional mutations in the smg system that are unable to degrade aberrant mRNAs at a non-permissive temperature, but are able to do so at a permissive temperature. II. Construct a fusion DNA that has three components a.A promoter with a defined activity pattern b.A coding region to be expressed conditionally c.A 3'UTR which is arbitrarily extended by irrelevant "junk" sequence. III. Transform the fusion DNA into the smg-conditional host. IV. Use temperature shifts to control activity of the transgene. The expectation is that the extended 3' untranslated region will destabilize the mRNA in a wild type (Smg+) genetic background, while in a Smg- background, the mRNA surveillance will fail and the mRNA will be translated. Thus the transcript is under dual control of the promoter and the state of Smg activity. As one example, a tissue specific promoter could be used to limit expression to one cell type, while a conditional smg background could be used to limit expression a single stage in the life cycle. Previously, Papp had reported some smg mutants with partial cold sensitivity. Also smg-7 alleles  show some degree of temperature sensitivity . Since both of these show incomplete temperature dependence, we carried out additional screens. smg mutations can readily be isolated following mutagenesis of the smg-suppressible unc-54 allele, r293 . Performing this screen at 25C, we isolated 75 putative smg mutations . Of these, three (cc543, cc545, and cc546) showed temperature sensitivity. Two of these have been shown to be allelic with smg-1, the third has not been analyzed further. We have used a variety of reporter constructs to test the feasibility of using smg-1(cc546ts) to generate conditional expression. Previous studies [2,6] had shown that (as with endogenous genes) unusual 3' UTR regions in transgenes could cause smg-dependent decay. We find that a myo-3::gfp fusion transgene with an artificially long 3' UTR (consisting of lacZ sequences) showed temperature-dependent activity in a smg-1(cc546) background. We would like to extend the technical usefulness of this by finding a "junk" 3' UTR which would not interfere with other aspects of experimental design (and which in particular would be lacking any reporter sequences or cis acting transcriptional signals). We are in the process of trying out different candidates. Table: Temperature sensitive smg revertants from r293 smg-?(cc543ts);unc-54(r293)- Movement: moderately Unc at 16, Almost wild type at 25 smg-1(cc545ts)unc-54(r293)- Movement: very Unc at 16, Slightly Unc at 25 smg-1(cc546ts)unc-54(r293)- Movement: very Unc at 16, Almost wild type at 25 References and Notes 1. Cali, B., and P. Anderson (1995) C. elegans meeting abstract 142. 2. Okkema, P., Harrison, S.W., Plunger, V., Aryana, A., and Fire, A. (1993) Genetics 135:385-404. 3. Papp, A. (1991) C. elegans meeting abstract 229. 4. Pulak, R., and Anderson, P. (1993) Genes and Development 7:1885-1897. 5. Ransom, D., Sharp, S., Alberico, C., and B. De Stasio (1995) C. elegans meeting abstract 431. 6. Wilkinson, H., Fitzgerald, K., and Greenwald, I. (1994) Cell 79:1187-1198. 7. B. Cali, S. Kuchma, and P. Anderson, personal communication. 8. These mutations were isolated in the F2 following mutagenesis and growth at 25C. Since alleles of some smg mutations show a maternal rescue effect , it is possible that the screen was biased toward genes, such as smg-1, that do not show maternal rescue. 9. Thanks to R. Pulak, K. Fitzgerald, B. Cali, P. Anderson, S. Kuchma, I. Greenwald, and T. Dill for their help and suggestions.