Worm Breeder's Gazette 12(5): 95 (February 1, 1993)
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.
Tandem repeats of the sequence TTAGGC are not only located at the ends of the C. elegans chromosomes, but also at internal chromosomal sites as shown by B al31 digestion experiments. Different cloning approaches (e.g. YAC deficiency cloning) failed to discriminate between telomeres and internally repeated TTAGGC sequences.
To solve this problem, we have constructed a lambda Zap library from specially prepared chromosomal long C. elegans DNA. Worms were embedded in 0.5 % low melting agarose and lysed according to Waterston (WBG 9: 2, p. 108. 1986). The resulting chromosomal DNA was then treated with the Klenow polymerase and digested with the restriction enzyme XbaI in the agarose. After extraction from the agarose, the different populations of fragments were ligated to a lambda Zap vector containing a XbaI compatible end and a blunt end. This cloning procedure specifically tags sequences that are localized at the ends of the chromosomal DNA. The resulting telomeric library was screened for recombinants containing TTAGGC repeats. About 70 positive clones were found. So far, 13 isolated clones were partially sequenced and three of them satisfy all criteria required for telomeric clones. The TTAGGC tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. Furthermore, the G-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. Finally, preliminary hybridization data with one of these putative telomeric clones show that a subtelomeric fragment hybridizes to B al31 -sensitivefragments on a Southern blot with C. elegans DNA, indicating that this clone represents a C. elegans telomere. The same probe does not hybridize to a YAC grid and thus might contain sequences, which are not yet physically mapped. Besides the fact that telomeres represent specialized structures important for chromosome stability, the particular significance of cloned C. elegans telomeres is that they will contribute to achieve the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial C. elegans chromosomes.