No 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.

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.

A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable. Its transposase activity is higher than that of an entire P strain, but it rarely undergoes internal deletion, excision or transposition. This element was constructed by F. Laski, D. Rio and G. Rubin for other purposes, but we have found it to be useful for experiments involving P elements. We demonstrate that together with a chromosome bearing numerous nonautonomous elements it can be used for P element mutagenesis. It can also substitute efficiently for "helper" plasmids in P element mediated transformation, and can be used to move transformed elements around the genome.

The Drosophila Dmblm locus is a homolog of the human Bloom syndrome gene, which encodes a helicase of the RECQ family. We show that Dmblm is identical to mus309, a locus originally identified in a mutagen-sensitivity screen. One mus309 allele, which carries a stop codon between two of the helicase motifs, causes partial male sterility and complete female sterility. Mutant males produce an excess of XY sperm and nullo sperm, consistent with a high frequency of nondisjunction and/or chromosome loss. These phenotypes of mus309 suggest that Dmblm functions in DNA double-strand break repair. The mutant Dmblm phenotypes were partially rescued by an extra copy of the DNA repair gene Ku70, indicating that the two genes functionally interact in vivo.