In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

A combined bioinformatic and genetic approach was used to conduct a systematic analysis of the relationship between ribosomal protein genes and Minute loci in Drosophila melanogaster, allowing the identification of 64 Minute loci corresponding to ribosomal genes.

Multiple stringent confinement strategies should be used whenever possible

BackgroundChromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome.ResultsA large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes.ConclusionsDrosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.

The conserved heterochromatic location of centromeres in higher eukaryotes suggests that intrinsic properties of heterochromatin are important for chromosome inheritance. Based on this hypothesis, mutations in Drosophila melanogaster that alter heterochromatin-induced gene silencing were tested for effects on chromosome inheritance. Here we describe the characterization of the Su(var)2-10 locus, initially identified as a Suppressor of Position-Effect Variegation. Su(var)2-10 is required for viability, and mutations cause both minichromosome and endogenous chromosome inheritance defects. Mitotic chromosomes are improperly condensed in mutants, and polytene chromosomes are structurally abnormal and disorganized in the nucleus. Su(var)2-10 encodes a member of the PIAS protein family, a group of highly conserved proteins that control diverse functions. SU(VAR)2-10 proteins colocalize with nuclear lamin in interphase, and little to no SU(VAR)2-10 is found on condensed mitotic chromosomes. SU(VAR)2-10 is present at some polytene chromosome telomeres, and FISH analyses in mutant polytene nuclei revealed defects in telomere clustering and telomere-nuclear-lamina associations. We propose that Su(var2-10 controls multiple aspects of chromosome structure and function by establishing/maintaining chromosome organization in interphase nuclei. Heterochromatin is an enigmatic component of higher eukaryotic genomes. The paucity of genes and the abundance of repetitive sequences in heterochromatin contribute to it being described as functionally inert. However, heterochromatin houses essential single copy genes (Liu et al. 1998) and the rDNA loci, the most highly transcribed genes in the genome (for review, see Williams and Robbins 1992). In addition, the centromerethe site of kinetochore formation, spindle attachment, and checkpoint control during mitosis and meiosis-is usually buried deep within heterochromatin. Elegant studies from a variety of organisms (Goday and Pimpinelli 1989;Bass et al. 1997) indicate that heterochromatin plays other important roles in chromosome inheritance. For example, heterochromatic homology is required for faithful homolog pairing and chromosome segregation during male and female meiosis in Drosophila (McKee and Karpen 1990; Dernburg et al. 1996b; Karpen et al. 1996).Heterochromatin can silence the expression of genes that are normally found in euchromatin, resulting in a phenomenon known as position-effect variegation (PEV). PEV manifests as the mosaic or variegated expression of an affected locus owing to its abnormal juxtaposition near centric heterochromatin or telomeres (for review, see Wallrath 1998). Changes in the chromatin structure surrounding a variegating gene motivated the hypothesis that PEV is caused by spreading of the heterochromatic state into neighboring regions of euchromatin (for review, see Wakimoto 1998). However, PEV also occurs when heterochromatic associations produce large-scale alterations in the nuclear organization of chromosomes. For example, a large block of heteroc...