P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.We used a transposable P-element insertion allele of the white gene to study the repair of a double-stranded DNA break in Drosophila melanogaster. Unrepaired double-stranded DNA breaks can be cell lethal after mitosis. Consequently, organisms have developed efficient methods for their repair. Double strand break repair is thought to occur by a process in which the broken ends search for a homologous sequence, invade it, and serve as primers for DNA synthesis to reconstitute the broken chromosome (29, 32, 39).P elements are thought to transpose by a cut-and-paste process in which excision of the element breaks both DNA strands of the chromosome (6,12,19). Experiments have shown that the break is usually repaired by copying the corresponding sequences from the sister strand (6), thus restoring a copy of the P element to the break site. Alternatively, sequence may be copied from a template on the homolog (18,25) or one inserted in the genome at an ectopic site (12). The DNA sequence flanking the P excision site is usually converted as a continuous block with an average tract length of about 1,400 nucleotides. Recent results have also shown that small insertions and deletions from the homolog can be converted into the excision site (18).These observations suggested to us that the gap repair process could be adapted for use as an efficient gene targeting method in D. melanogaster (Fig. 1). We tested this possibility by using a P-element insertion in the white gene, whd (whd80KJ7), which is known to excise at high frequencies in the presence of P transposase. We then recovered gap repair products that occurred in the presence of one of five ectopi- cally located templates. In four of these templates, the white gene had been altered by the addition of unrelated sequences of lengths up to approximately 7,970 bp. We found that each of the nonhomologous sequences could be copied into the white locus almo...

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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.

SUMMARYHybrid dysgenesis has been described as a syndrome of aberrant traits including sterility, male recombination, and mutation, which occurs in some inter-strain hybrids of Drosophila, but only from one of the two reciprocal crosses. In a series of experiments in which hybrids of various pedigrees were tested for sterility, it was found that a case of hybrid dysgenesis could be most easily interpreted as the interaction of two components. One component was found to be a polygenic Mendelian factor linked to each of the major chromosomes of π2, the paternally contributing strain (‘P strain’). These chromosomes were capable of causing sterility when inherited from either parent, provided the appropriate maternal component was also inherited. The ability to transmit this maternal component was designated ‘cytotype’ to indicate that it is a property of the entire cell. It was possible to classify nearly all hybrid females as either P or M cytotype on the basis of their ability to produce sterile daughters. All daughters of the M-cytotype mothers were susceptible to the sterilizing effects of the π2 chromosome, whereas all, or nearly all daughters of P-cytotype mothers were immune. When more than one of the π2 chromosomes were received by daughters of M-cytotype females, chromosomal interactions could be detected statistically, but the model of independent action remained a useful approximation. Cytotype was shown to be determined by chromosomal factors, but with limited cytoplasmic transmission. This unusual mode of inheritance can be compared with other cases of hybrid dysgenesis where the behaviour resembles that of self-replicating cytoplasmic particles which are dependent on certain chromosomes. The lack of sterility from intra-strain crosses can be explained by the fact that chromosomes capable of causing sterility also induce the P cytotype, and thus prevent sterility in the next generation.

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