Worm Breeder's Gazette 14(4): 20 (October 1, 1996)
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.
The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands telephone +31 20 512 2083, telefax: +31 20 669 1383, email: hluenen@nki.nl).
The reverse genetic analysis of C. elegans has become important as result of the fast amount of sequence data that come out of the genome project. Techniques have been developed to analyse the functions of newly identified genes (e.g. transposon induced gene inactivation). Although forward genetic screens are also facilitated by the genome project, there is no quick route yet from mutant phenotype to gene identification. We have developed a method that greatly reduces the time required to identify transposon insertion mutants from forward genetic screens. The method is based on the disruption of a gene by a known transposon sequence. It starts with a mutator strain of low Tc1 and Tc3 copy number from which transposon insertion mutants are isolated with a specific phenotype. Genomic DNA is digested with a frequently cutting enzyme, and an oligonucleotide-vectorette is ligated to the digested DNA (Riley, J., et al. (1990) Nucleic Acids Research 18:2887-2890). A PCR is performed using a primer corresponding to the transposon end and a primer for the vectorette. This will result in the amplification of only those restriction fragments that contain a transposon end. A second PCR is performed with nested primers, of which one is radiolabeled. The products from this reaction are separated on a denaturing polyacrylamide gel. The autoradiogram shows in one lane the transposon insertions present in the genome of one strain. The novel band (absent in the non-mutant starting strain) is excised from the dried polyacrylamide gel, further amplified, and sequenced. The sequence of the flanking DNA can be compared to the database and the gene that is responsible for the phenotype can be identified. We have used this transposon insertion display method to identify a gene that is involved in the perception of copper. Wild type animals have an aversion for copper; they will not cross a line of 0.25 M CuSO4 even if there is an attractant on the other side (isoamylacohol). We have used the mutator strain NL917 (mut-7) and isolated a mutant (NL953) that did cross the copper. The mutant was out- crossed with Bristol N2 and a panel of mutant and wild type animals was obtained. Using the display we identified a Tc1 insertion that was present in all mutant animals and absent in wild type animals. The flanking sequence of this insertion was determined and compared to the database. A match was found in the dataset of unfinished sequences released on September 8th 1996. We are further optimizing the protocol and we are analysing other copper aversion mutants as well as other classes of mutants. The current version of the protocol is available on request.