Worm Breeder's Gazette 14(5): 28 (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.
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We want here to share our experience with Tc1 mutagenesis. We described at the last Worm Meeting the existence of a gene related to lin-26 immediately upstream of lin-26, which we called lir-1 for lin-26 related gene. To determine the function of lir-1, we set out to isolate a mutation using the Tc1 insertion strategy designed by Ronald Plasterk and his collaborators (1). The Plasterk lab was able to identify a Tc1 within the first lir-1 intron, lir-1(pk96), which was inserted approximately 1.7 kb from the first lir-1 exon. Not surprisingly, we found that lir-1(pk96) animals are wild-type. Starting from lir-1(pk96), we decided to try and get a jump-out event that would generate a concomitant excision of lir-1 sequences. Using a 5' oligonucleotide located approximately 1 kb upstream of the first lir-1 exon and a 3' oligonucleotide located 0.4 kb downstream of the Tc1 (about 3.3 kb downstream of the 5' oligo in a wild-type strain), we screened pools of animals to find deletion events. We first screened the progeny of 10,000 F0 lir-1(pk96) animals in pools of three to ten animals per plate and did not obtain any deletion. While we were conducting that screen, Piali Sengupta and her colleagues published a paper in which they mentioned that to get odr-10(ky255) they treated animals with UV prior to screening (2). Apparently, they had done so because in initial screens they were unsuccessful using a standard protocol (Cori Bargmann, personal communication). We decided to use a somewhat similar strategy, except that we subjected animals to mutagenesis with trimethylpsoralen and UV (3). We screened 2500 pools of F0 lir-1(pk96) animals that had first been mutagenized with TMP/UV and obtained a 779 nucleotide deletion that takes out only intronic sequences, lir-1(pk96 mc32) (the phenotype induced by mc32 is still under investigation). The deletion does not break at the Tc1 insertion point but rather about 380 pb from the original insertion point of pk96; it starts or finishes with an AT dinucleotide. We do not know what was the actual effect of psoralen mutagenesis: it could have increased the frequency of Tc1 mobilization and excision, or it could have induced the deletion by itself. Still, mutagenizing with TMP/UV and Tc1 can be an alternative strategy in certain difficult cases. Our thanks to Ronald Plasterk for isolating lir-1(pk96). (1) Zwaal R. R, Broeks A., van Meurs J., Groenen J. T. M. and Plasterk R. H. A. (1993). Proc. Natl. Acad. Sci. USA 90: 7431-7435. (2) Sengupta P., Chou J. H. and Bargmann C. I. (1996). Cell 84: 899-909 1996. (3) Yandell M. D., Edgar L. G. and Wood W. B. (1994). Proc. Natl. Acad. Sci. USA 91: 1381-1385.