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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section VIIRepair After Tc1 Excision

It is important to distinguish transposon excision__the reaction by which the transposon is released from flanking DNA sequences by two double-strand DNA breaks__from transposon loss. The latter event consists of excision followed by repair of the double-strand break left behind in the donor DNA. Strictly speaking, the excision is probably always precise, and it is the subsequent repair that can result in precise or imprecise loss of the transposon. It is likely that the transposon does not “care” what happens to the break it leaves behind, in the sense that the repair is probably mechanistically identical to that of any double-strand DNA break in the worm genome and totally carried out by proteins that are not transposon-encoded. Emmons et al. (1983) showed that somatic excision of Tc1 could be monitored by Southern blot analysis, which reveals an empty donor restriction fragment, i.e., a fragment of which the size would suggest that the transposon is more or less precisely lost. Apparently, the cell puts the two flanks together again without much loss or addition of sequences. Analysis of the sequence at the junctions of the repaired somatic donor sites indicates that some microheterogeneity exists, resulting in “footprints” of only a few base pairs (Ruan and Emmons 1987; Eide and Anderson 1988; van Luenen et al. 1994). As shown in Figure 3, the excision leaves two nucleotides of each 5′transposon end behind at each end of the double-strand break, and indeed some of these are present in the footprint. The difference between excision and transposon loss has been visualized by synchronized induction of transposase expression. Expression leads to the appearance of a broken DNA donor fragment, which is subsequently chased into a repaired fragment (R.H.A. Plasterk and H.G.A.M. van Luenen, unpubl.).

Repair of the donor break in the germ line is discussed here in some detail because it is important for several applications of the transposon in genetic analysis. It has not yet been possible to detect the products of germ-line excision in a direct physical assay. Only repair products that result in reversion of a phenotype have been analyzed, and this implies a bias in product selection. Nevertheless, a common footprint is TAcaTA (Eide and Anderson 1988; Plasterk 1991), which would indicate that the direct ligation of the broken ends occurs in germ cells as well. The resulting 4-bp footprint would of course lead to a frameshift in a coding region and therefore usually goes unnoticed in a reversion screen.

Kiff et al. (1988) noted that among revertants of an unc–22 Tc1-insertion allele, there were rare alleles that contained a deletion. Zwaal et al. (1993) took a reverse genetic approach to show that such deletions occur among the progeny of most Tc1 insertion mutants. The endpoints of these deletions are often at short direct repeats, suggesting that the two broken DNA ends scan each other's flank until a match is found, after which the break is sealed at that point and the intervening sequence is lost. Anecdotal evidence suggests that the events occur at widely different frequencies for different insertion alleles, and thus the excision frequency may be different for each genomic position or the preferred repair products may be sequence-dependent.

A third class of repair products differs from direct ligation or deletion and results from a template-dependent repair process. Plasterk (1991) found that frequent precise loss of a Tc1 element depends on the presence of a wild-type sequence on the homologous chromosome. The wild-type sequence is used to repair the double-strand break, resulting in an apparent precise excision. Among rare revertants of a homozygous Tc1 allele are products that result from partial template-directed repair, and these contain short stretches of the transposon ends as a footprint. The same template-dependent repair process has been described by Engels for the Drosophila P element (Engels et al. 1990).

Mechanistically similar to this allelic template-directed repair is repair that uses an ectopic template. The main difference is the searching process that precedes the alignment of template and broken ends; the relative inefficiency of this process may explain the reduced frequency of ectopic template usage. Plasterk and Groenen (1992) showed that an extrachromosomal transgenic array could be used as template to repair the break after excision of Tc1 from a chromosomal unc–22 gene. The transgene contained unc–22 sequences marked with some polymorphisms, and these polymorphisms were found copied into the repaired unc–22 gene. This method can in principle be used to shuttle any point mutation into the chromosome, provided a nearby transposon insertion is present.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20157

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