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.

Transposon insertion display; a rapid method to identify mutated genes in a forward genetic screen.

Henri G.A.M van Luenen, Ronald H.A. Plasterk

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.