[
International C. elegans Meeting,
1995]
Transposon mediated deletion mutagenesis is now a method of choice for reverse genetics. First a Tc1 insertion allele of the gene of interest is isolated. In case this insertion is not a loss-of-function allele, the Tc1 insertion can be used to select deletion derivatives. As reported previously, we constructed a library of Tc1 insertion mutants. We have screened this library on a routine basis for laboratories all over the world and have obtained now over seventy Tc1 insertion alleles of researcher's pet genes. In line with the genome sequencing project we have started sequencing Tc1 insertions in high copy number strains. Flanking sequences of these insertions are mapped by comparison with the genome sequence; all insertions will fall in place once the entire genome sequence is known. These polymorphic Tc1 insertions serve two functions: Insertions in or close to genes can be used to generate knock-outs of those genes. Second, the set of Tc1 insertions forms a dense grid of "PCR visible" genetic markers. We amplified and sequenced Tc1 flanks of the high copy number strains RW7000, CB4000 and KR1787 and obtained a set of at least 500 distinct Tc1 insertions. We mapped 35 of these insertions by alligning with the 10 Mbp of genome sequence. Strategies to use these insertions in mapping new mutant genes will be discussed.
[
International C. elegans Meeting,
1995]
Williams et al. [(1992) Genetics 131, 609] have described a genetic mapping system based on polymorphic sequence-tagged sites (STS). It is an alternative to classical two- or three-factor crosses, and can also be used to map polygenic traits [Ebert et al. (1993) Genetics 135,1003; Ayyudevara et al. poster]. One weakness has been that fewer than 80 C. elegans polymorphisms had been sequenced and mapped. This has changed. By shotgun sequencing, Korswagen et al. [WBG 13(5),90] have generated over 800 sequences adjoining Tc1 insertions in strains RW7000, KR1787, and CB4000. At this writing, 35 Tc1 alleles map to sequenced genome regions. This number will slowly increase as sequencing proceeds. We are mapping Tc1 alleles by YAC grid annealing to pooled DNA probes. Pooling is via the "matrix addressing" strategy [Zwaal et al. (1993) PNAS 90, 7431]; DNAs are arranged in 3-D arrays and pooled along each plane. An allele thus contributes to three orthogonal probe pools. A YAC or adjacent YACs labeled by only those three pools will in principle map the allele. In preliminary work, we identified and set aside alleles that align with repetitive DNA elements or anneal to total genomic DNA. We are evaluating probe mixtures of increasing complexity, to optimize 3-D array size.