Worm Breeder's Gazette 13(5): 90 (February 1, 1995)
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.
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; *The Sanger Centre, Hinxton Hall, Hinxton, Cambridgeshire CB10 1RQ, United Kingdom We devised a new approach to map large numbers of Tc1 transposon insertions in the genome of C.elegans. Strains have been described that contain up to 500 polymorphic Tc1 insertions; most of these are between genes or in introns, therefore the animals exhibit no clear phenotypes. We amplified the termini plus flanking sequences of Tc1 elements in the genome of high Tc1 copy number strains RW7000, CB4000 and KR1787 by an anchored PCR based method. The PCR products were cloned directly into M13 sequencing vectors, and over two thousand of these clones were shotgun sequenced. Clustering of identical sequences resulted in a set of 821 distinct Tc1 flanks. Since more right than left flanks were sequenced, this set represents at least 500 Tc1 insertion alleles; about two thirds of the total Tc1 number present in these strains. Alignment of these sequences reveals a weak Tc1 insertion site consensus sequence that is symmetric around the invariant TA target site and reads A C/T (notT) T A (notA) A/G T. This is in reasonable accordance with results reported previously on the basis of much smaller datasets. The tracks of Tc1 flanking sequences were compared with the 5 Mbp of C.elegans genome sequence that is known today. We found 16 insertions within the sequenced area, approximately one per 300 kbp. In addition, three insertions map within cloned cDNA tags and four insertions are within the rDNA cluster. With every new release of genome sequence information, remaining Tc1 alleles will fall into place. These Tc1 insertions can serve three functions: 1. Insertions in or near genes can be used to isolate deletion derivatives that have that gene mutated. 2. They also represent a dense collection of polymorphic markers to fine map new mutations. Tc1 insertions can be visualized by PCR and do not have the inherent disadvantages of visible mutations. 3. In addition, these insertions can be used to connect the genetic map to the genome sequence of C.elegans. The approach lends itself to a scale-up aimed at getting transposon insertion alleles of all genes in the C.elegans genome. The current limitation lies in getting new strains with independent high Tc1 copy numbers. The set of Tc1 alleles with a search routine to screen for insertions in your own sequence of interest will be made available through ACeDB.