Genetic instability in Drosophila melanogaster: P-element mutagenesis by gene conversion.

Abstract

We report the molecular characterization of several P element-induced mutations and their revertants at the yellow (y) locus ofDrosophila melanogaster. One of the mutants analyzed, y7W8, results from the insertion of a P element into the 5'-transcribed, untranslated portion of the y gene. Sequence analysis of several revertants of y76d28 shows that P excision occurs imprecisely. These events result in insertion of additional ATG codons in the y locus mRNA but are without phenotypic effect. In addition, we describe the molecular structure ofP-associated mutations induced in a near wild-type revertant of y76W08 that carries an internally deleted 0.4-kilobase P element in the 5' noncoding region. Sequence analysis of two of these mutants demonstrates that they arose as a result of the integration of a larger P element at the exact location as in the parental stock without the 8-base-pair additional duplication associated with P insertions. The phenotype of these y alleles is dependent on the size and orientation of the integrated P element. We infer that P-element replacement in these mutants has occurred by a recombination/gene conversion mechanism.Several classes oftransposable elements have been identified in Drosophila melanogaster that collectively account for 10o of the genome (1). Molecular analyses of mutations at a number of loci have indicated that the major cause of spontaneous mutations in D. melanogaster is insertion of a transposable element into a gene altering its expression (2)(3)(4)(5). Mechanisms by which these elements transpose and exert their mutagenic effects depend on the properties of a given element.One of the best characterized elements in D. melanogaster is the P element. Complete P elements are 2.9 kilobases (kb) long and contain short 31-base-pair (bp) terminal inverted repeats flanking four open reading frames that encode a transposase function (6). P-element transposition is restricted to germ-line cells as a result of a tissue-specific splicing event required for the production of transposase (7). In addition, P-element movement is influenced by the presence of a repressor protein, thought to be produced from the P mRNA in which the germ-line-specific splice has failed to occur (8). P elements form a heterogeneous family of elements originating from the autonomous 2.9-kb P factor by internal deletions (6).Insertion sites of P elements have been found to be clustered in target genes (9-12). In several cases, these sites are located in the 5' end, either upstream of the transcribed region or in the transcribed, untranslated portion of the gene (9-12). Mechanisms by which these P insertions affect gene expression are not well established. In at least one case, transcriptional interference has been implicated (13).We have used the yellow (y) locus as a model system to study the mobilization and mutagenic effects of P elements. The y gene is required for production ofthe normal brownishblack pigmentation of larval and adult cuticle structures. This gene encodes a single 1.9-kb RN...

Efficient Gap Repair in Drosophila melanogaster Requires a Maximum of 31 Nucleotides of Homologous Sequence at the Searching Ends

“…(iii) Internal conversion events are not dependent on sequence homology between DNA sequences internal to the template and external to the P-w hd element. This is in accordance with observations by other groups (18,21,22). The template, P{walLy}, used for the majority of the experiments contained an altered mini-white gene.…”

“…This result demonstrates that internal conversions form by a symmetrical repair process. The orientation preference observed by other investigators is probably a consequence of the method used to select for internal conversion events (18,21). (iii) Internal conversion events are not dependent on sequence homology between DNA sequences internal to the template and external to the P-w hd element.…”

“…One explanation for their rapid spread is that a P-element excision is repaired typically by copying from the sister strand (10,11). However, as this and other work demonstrates that a P element located anywhere in the genome can be used as a template to repair the DSB remaining after P-element excision (18,21,22), it is probable that internal conversion of P elements was another factor contributing to the rapid spread of P elements throughout the species.…”

“…A significant proportion (Ͼ20%) of the remaining flies had the P-w hd element replaced exactly by the template P{walLy} element. P-element replacements at the yellow, vestigial, and Broad loci have been observed by a number of groups (18,21,22). However, none of these experiments provided sufficient data to determine how the events were formed.…”

“…Therefore, one might expect that the orientation of the original insertion would influence the orientation of the internal conversion events. Nonrandom orientation of the newly inserted template has been observed at other loci (18,21). However, in these previous cases, the primary screen for the internal conversion FIG.…”