Worm Breeder's Gazette 11(5): 66
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 tagging remains an efficient way to clone mutationally defined genes in C. elegans, whether by cloning out the flanking DNA or by looking for allele-specific rearrangements with wild-type probes. Often, multiple alleles of the gene of interest are obtained from mutator backgrounds, but the effort required to finely map or clone out more than just a type specimen does not usually warrant the exercise. It is useful to know the location of insertion sites relative to the established structure of the gene, because insertions in exons indicate probable null alleles, while insertions upstream or downstream of the transcript can identify potential cfs-acting regulatory sequences. Here I describe a novel strategy ( 'echolocation') to obtain quite precise (+ or - 10bp) estimates of the site of insertion by PCR (recently published in NAR 18: 6741 qv for more details). The ends of most transposons are defined by short sequences that are inverted with respect to each other. Thus a single oligonucleotide, oriented such that it would prime DNA synthesis away from the transposon, can do so from both ends of the transposon simultaneously ( see also Hill and Sternberg CSH '89 Abstracts p.124). The strategy involves pairwise combination of such an oligonucleotide with each of several oligonucleotides of either orientation derived from the genomic sequence throughout the region of suspected transposon insertion. Whenever the transposon primer has sufficient proximity to an appropriately oriented genomic primer, a product ('echo') will be formed, whose length can be determined by high-resolution agarose gel electrophoresis. For example, the x508 allele of the lev-1 gene of C. elegans (which encodes a muscle acetylcholine receptor sub unit) arose in TR679, and contains the transposon Tc1 (Barnes and Lewis, WBG 11(2):47). Nine different primers were derived from the wild-type genomic sequence of the lev-1 gene of C. elegans (Mike Squire, pers. comm), (Fig. A). The transposon primer was a 20mer derived from bp 1582 to 1601 of the Tc1 sequence (1), designed such that it avoided the exact end of Tc1 which is somewhat similar between different transposons (2). Each of the genomic primers was then paired with the Tc1 primer in a PCR reaction, using unbackcrossed lev-1(x508) genomic DNA as template. The products were resolved on a 2% agarose gel, allowing the size of the smallest fragment to be determined with high accuracy. Only four primers produced products, and from their sizes they indicate that the site of insertion lies near an intron/exon boundary (Fig. B). The two favorably oriented primers that failed to produce products (primers 1 and MS2) are probably too distant from the Tc1 primer to work with a 2 minute extension time. This method has also been applied to the two other Tc1 alleles, x504 and x562, which has located them near a different intron/exon boundary, and in an exon, respectively. The precise estimate can be converted into an exact guess by dint of the site preference for Tc1 (4). Each of the defined regions had just two TpA sequences, and one was clearly a better match to the consensus than the other in each case. Thus x504 is predicted to have occurred in a splice donor, and x508 in an exon. The reliability of the strategy is demonstrated by the fact that different genomic primers, from either side of the transposon, produce fragments which are consistent with the same single site (Fig. B). The technique is also fairly robust: the DNA used has ~600 priming sites for the Tc1 primer per genome (3), yet there is little problem with spurious bands; when evident, these are also produced by TR679 DNA. Finally, to determine the site of insertion exactly, the PCR products can be easily cloned and sequenced. [See Figure 1]