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.

Shotgun sequence analysis of transposon Tc1 alleles in high copy number strains

Hendrik C. Korswagen, Miriam T. Smits, Richard M. Durbin*, Ronald, H.A. Plasterk

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.