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.

Desperate about getting the Tc1 to jump out from your favourite gene? Try first to mutagenize your Tc1 insertion strain with trimethylpsoralen/UV

Satis Sookhareea, Pascale Dufourcq, Michel Labouesse

IGBMC, BP163, 67404 Illkirch Cedex, France.

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 
(3) Yandell M. D., Edgar L. G. and Wood W. B. (1994). Proc. Natl. Acad.
Sci. USA 91: 1381-1385.