Targeted Gene Replacement in Drosophila Via P Element-Induced Gap Repair

Abstract

Transposable elements of the P family in Drosophila are thought to transpose by a cut-and-paste process that leaves a double-strand gap. The repair of such gaps resulted in the transfer of up to several kilobase pairs of information from a homologous template sequence to the site of P element excision by a process similar to gene conversion. The template was an in vitro-modified sequence that was tested at various genomic positions. Characterization of 123 conversion tracts provided a detailed description of their length and distribution. Most events were continuous conversion tracts that overlapped the P insertion site without concomitant conversion of the template. The average conversion tract was 1379 base pairs, and the distribution of tract lengths fit a simple model of gap enlargement. The conversion events occurred at sufficiently high frequencies to form the basis of an efficient means of directed gene replacement.

Targeted gene alteration in Caenorhabditis elegans by gene conversion

“…Heterologous transposon insertions will probably have similar or greater difficulties with regard to transposition and the generation of imprecise excision alleles. The gene conversion technique reported here should also be effective in this heterologous system, because the process of gene conversion, as shown by ourselves and others 20 , seems merely to require the presence of dsDNA breaks and the homologous template DNA. Similarly, the technique should work for generating targeted gene alterations in other systems, such as zebrafish and mouse, where heterologous insertion systems are also being developed [32][33][34] .…”

“…Last, deletions and insertions might be converted more easily than point mutations. This last possibility seems less likely, considering that, first, evidence from P elementinduced gene conversion in D. melanogaster suggests that deletions are converted at a lower rate than are point mutations and insertions 27,28 ; and second, point mutations in a conversion template should contain more contiguous homologous sequence near the transposon insertion site than should deletions, and the nearer a polymorphism in the homologous sequence is to the strand break site, the more likely it is to be converted 20 .…”

“…First, we constructed a plasmid containing an engineered deletion of a specific size in the genomic DNA corresponding to the area of the Tc1 insertion site. One endpoint of the deletion was placed near the Tc1 insertion site to facilitate the homology search at the DNA ends during dsDNA break repair 20 . We introduced the plasmid as an extrachromosomal transgene 2 into a mutator strain containing the gene-specific Tc1 insertion.…”

Repairing a double–strand chromosome break by homologous recombination: revisiting Robin Holliday's model

“…From studies of budding yeast MAT gene switching (Nasmyth 1982;McGill et al 1989), transposable element recombination in Drosophila (Gloor et al 1991) and from transformation experiments in mammalian cells (Belmaaza & Chartrand 1994), an alternative set of recombination models emerged, known as SDSA mechanisms (figure 2bϪd). Here, strand invasion was regarded as rate limiting so that one end would succeed while the second end remained unengaged.…”

The effects of trans modifiers and tandem duplications on meiotic recombination in maize

“…In contrast, the average tract lengths associated with Drosophila P element excision were somewhat larger, i.e., -1400 bp (GLOOR et al 1991 ;PRESTON and ENGELS 1996). In yeast, the average meiotic conversion tract lengths range from 0.4 to 1.6 kb in a 9-kb interval (BORTS and HABER 1989).…”