The manner in which transposable element insertions affect the expression of the white gene of Drosophila was examined by analyzing polyadenylated RNA of flies with each of 9 insertions in or near the gene. In 5 mutants having insertions in the transcribed sequences of white, transcripts initiating at the white promoter are truncated within the insertions. Two insertions in the 3 kb [kilobase] intron of white alter neither the amount nor the structure of the mature white RNA. An insertion near the 5'' end of the gene blocks the accumulation of any white transcripts. Another insertion, located 1.2 kb upstream from the transcribed region of the gene, causes a mutant phenotype, yet has no obvious effect on the structure or abundance of the major white RNA. A mutation at each of 2 other loci that modulate the phenotype of the white-apricot insertion mutant are correlated with small but significant changes in the pattern of white transcripts.

The DNA sequence of the white locus of D. melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. The sequence of a complementary DNA clone which established the position of the 3'' end of the white RNA on this genomic sequence is reported. The probable exon-intron structure of the gene was predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with 7 insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wsm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11), 1 deletion mutant (white-spotted 4 (wsp4)) and 1 internal duplication mutant (white-ivory (wi)) were determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with 6 other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene Structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila,genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

The TART, HeT-A, and TAHRE families of Drosophila non-LTR retrotransposons specifically retrotranspose to telomeres to maintain telomeric DNA. Recent evidence indicates that an RNA interference mechanism is likely to regulate TART, HeT-A, and TAHRE retrotransposition, but the developmental and tissue-specific expression of telomeric retrotransposon proteins has not previously been investigated. We have generated antisera against TART ORF1 protein (ORF1p) and used these antisera to examine the pattern of TART ORF1p expression in Drosophila melanogaster. We detected TART ORF1p throughout most of development and observed particularly high levels of protein in late larval and pupal stages. In late-stage larvae, ORF1p accumulates in brain and imaginal discs tissues, rather than in terminally differentiated larval tissues. Accumulation of ORF1p in imaginal discs is intriguing, since TART antisense RNA has previously been detected in imaginal discs, and we discuss the implications of these findings for TART regulation. (C) 2008 Elsevier B.V. All rights reserved.

The Drosophila Gene Disruption Project (GDP) has created a public collection of mutant strains containing single transposon insertions associated with different genes. These strains often disrupt gene function directly, allow production of new alleles, and have many other applications for analyzing gene function. Here we describe the addition of similar to 7600 new strains, which were selected from >140,000 additional P or piggyBac element integrations and 12,500 newly generated insertions of the Minos transposon. These additions nearly double the size of the collection and increase the number of tagged genes to at least 9440, approximately two-thirds of all annotated protein-coding genes. We also compare the site specificity of the three major transposons used in the project. All three elements insert only rarely within many Polycomb-regulated regions, a property that may contribute to the origin of "transposon-free regions" (TFRs) in metazoan genomes. Within other genomic regions, Minos transposes essentially at random, whereas P or piggyBac elements display distinctive hotspots and coldspots. P elements, as previously shown, have a strong preference for promoters. In contrast, piggyBac site selectivity suggests that it has evolved to reduce deleterious and increase adaptive changes in host gene expression. The propensity of Minos to integrate broadly makes possible a hybrid finishing strategy for the project that will bring >95% of Drosophila genes under experimental control within their native genomic contexts.

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow(+) marker flanked by two inverted bacteriophage Phi C31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (> 500 bps) to promote precise integration of large constructs (> 5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

The Antlia dwarf galaxy, recently found to be a possible local Group member, is analyzed using VI photometry. The galaxy is resolved into a large number of stars and although the galaxy is intrinsically faint and of low surface brightness, its stellar populations reveal characteristics more typical of faint star-forming dIrs rather than dEs. Significant star formation is currently going on in the central part of Antlia although little or no tar formation is taking place in the outer regions. This indicates a two-component (core-halo) morphology which appears to be common, lot only in large spirals (disk-halo), but in dwarf galaxies as well. The SFR averaged over the lifetime of the galaxy is estimated to be <(psi)over bar>/A similar to 2-4 x 10(-10) M-. yr(-1) pc(-2) while the more recent star formation, averaged over the last 1 Gyr is much higher (<(psi)over bar>(1Gyr)/A similar to 3 - 9 x 10(-10) M-. yr(-1) pc(-2) for the central region). The total mass locked into stars and stellar remnants is estimated to be M(star)similar to 2-4 x 10(6) M-.. Its distance, estimated from the TRGB, is 1.32+/-0.06 Mpc, which places Antlia just beyond the Local Group, and makes it a close companion of the dwarf galaxy NGC 3109 (Delta r greater than or similar to 30 kpc), although it is not clear whether they are gravitationally bound. (C) 1997 American Astronomical Society.

The Antlia dwarf galaxy, recently found to be a possible local Group member, is analyzed using VI photometry. The galaxy is resolved into a large number of stars and although the galaxy is intrinsically faint and of low surface brightness, its stellar populations reveal characteristics more typical of faint star-forming dIrs rather than dEs. Significant star formation is currently going on in the central part of Antlia although little or no tar formation is taking place in the outer regions. This indicates a two-component (core-halo) morphology which appears to be common, lot only in large spirals (disk-halo), but in dwarf galaxies as well. The SFR averaged over the lifetime of the galaxy is estimated to be <(psi)over bar>/A similar to 2-4 x 10(-10) M-. yr(-1) pc(-2) while the more recent star formation, averaged over the last 1 Gyr is much higher (<(psi)over bar>(1Gyr)/A similar to 3 - 9 x 10(-10) M-. yr(-1) pc(-2) for the central region). The total mass locked into stars and stellar remnants is estimated to be M(star)similar to 2-4 x 10(6) M-.. Its distance, estimated from the TRGB, is 1.32+/-0.06 Mpc, which places Antlia just beyond the Local Group, and makes it a close companion of the dwarf galaxy NGC 3109 (Delta r greater than or similar to 30 kpc), although it is not clear whether they are gravitationally bound. (C) 1997 American Astronomical Society.