The Drosophila gene disruption project: progress using transposons with distinctive site specificities

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 ～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.     

A P-insertion screen identifying novel X-linked essential genes in Drosophila.

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13 600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)_TPL and l(1)_TPG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.     

The carnegie protein trap library: a versatile tool for Drosophila developmental studies

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.     

A P-element insertion screen identified mutations in 455 novel essential genes in Drosophila

With the completion of the nucleotide sequences of several complex eukaryotic genomes, tens of thousands of genes have been predicted. However, this information has to be correlated with the functions of those genes to enhance our understanding of biology and to improve human health care. The Drosophila transposon P-element-induced mutations are very useful for directly connecting gene products to their biological function. We designed an efficient transposon P-element-mediated gene disruption procedure and performed genetic screening for single P-element insertion mutations, enabling us to recover 2500 lethal mutations. Among these, 2355 are second chromosome mutations. Sequences flanking >2300 insertions that identify 850 different genes or ESTs (783 genes on the second chromosome and 67 genes on the third chromosome) have been determined. Among these, 455 correspond to genes for which no lethal mutation has yet been reported. The Drosophila genome is thought to contain approximately 3600 vital genes; 1400 are localized on the second chromosome. Our mutation collection represents approximately 56% of the second chromosome vital genes and approximately 24% of the total vital Drosophila genes.     

FlyBase: a Drosophila database. The FlyBase consortium.

FlyBase is a database of genetic and molecular data concerning Drosophila. FlyBase is maintained as a relational database (in Sybase) and is made available as html documents and flat files. The scope of FlyBase includes: genes, alleles (and phenotypes), aberrations, transposons, pointers to sequence data, clones, stock lists, Drosophila workers and bibliographic references. The Encyclopedia of Drosophila is a joint effort between FlyBase and the Berkeley Drosophila Genome Project which integrates FlyBase data with those from the BDGP.     

Generating lineage-specific markers to study Drosophila development.

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.     

Competition may determine the diversity of transposable elements

Transposable elements are genomic parasites that replicate independently from their hosts. They harm their hosts by causing mutations or genomic rearrangements, and most organisms have evolved various mechanisms to suppress their activity. The evolutionary dynamics of transposons in insects, fish, birds and mammals are dramatically different. Mammalian genomes contain few, very abundant but relatively inactive transposon strains, while Drosophila and fish species harbour diverse strains, which typically have low abundance but are much more virulent. We hypothesise that the variation in the diversity and activity of transposable elements between various animal genomes is caused by the differences in the host defence mechanisms against transposon activity. In recent years RNAi, a mechanism capable of gene, virus and transposon silencing has been discovered. We model RNAi as a density dependant mechanism of defence, which can cause competition among transposons depending on its specificity, and test its predictions using the complete Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, chicken, mouse, rat and human genome sequences.     

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non‐essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.     

Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars [1, 2]. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor [3, 4], and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor [5-7]. We carried out molecular analyses of mutations in Tre to elucidate their involvement in the gustatory phenotype. Here, we show that Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression was not always associated with mutations in Tre. In flies with the spontaneous mutation Tre(01), both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre(01). Taken together, the mutation analyses support that Gr5a is allelic to Tre.     

Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila.

Drosophila melanogaster has been a premier genetic model system for nearly 100 years, yet lacks a simple method to disrupt gene expression. Here, we show genomic cDNA fusions predicted to form double-stranded RNA (dsRNA) following splicing, effectively silencing expression of target genes in adult transgenic animals. We targeted three Drosophila genes: lush, white, and dGq(alpha). In each case, target gene expression is dramatically reduced, and the white RNAi phenotype is indistinguishable from a deletion mutant. This technique efficiently targets genes expressed in neurons, a tissue refractory to RNAi in C. elegans. These results demonstrate a simple strategy to knock out gene function in specific cells in living adult Drosophila that can be applied to define the biological function of hundreds of orphan genes and open reading frames.     

An Inverse Pcr Screen for the Detection of P Element Insertions in Cloned Genomic Intervals in Drosophila Melanogaster

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of ``local' P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of ~10(-4) are distributed every 40-50 kb.     

A low copy number, copia-like transposon in maize.

Bs1, a transposable element that moved into the maize Adh1 gene following barley stripe mosaic virus infection, is shown to be present in 1-5 copies in all maize and teosinte lines tested. Bs1 sequences do not hybridize with the genome of barley stripe mosaic virus. The insertion of Bs1 is bounded by 304-bp perfect direct repeats, similar in structure to Ty1 in yeast, copia and related elements in Drosophila, and vertebrate pro-retroviruses, but different from all other known plant transposons. No free copies of the terminal sequences or large internal deletions of Bs elements could be detected. Bs1 is apparently not related to several transposons which moved into the Shrunken gene in lines made genetically unstable by barley stripe mosaic virus infection, suggesting that this virus may cause genome shock, resulting in a generalized liberation of transposons in response to environmental stress.     

Genome-wide patterns of large-size presence/absence variants in sorghum

The presence/absence variants （PAVs） are a major source of genome structural variation and have profound effects on phenotypic and genomic variation in animals and humans. However, little is understood about PAVs in plant genomes. Our previous resequencing effort on three sorghum （Sorghum bicolour L.） genomes, each 12？ coverage, uncovered 5 364 PAVs. Here, we report a detailed characterization of 51 large-size （＆gt;30 kb） PAVs. These PAVs spanned a total size of 2.92 Mb of the sorghum genome containing 202 known and predicted genes, including 38 genes annotated to encode celldeath and stress response genes. The PAVs varied considerably for repeat sequences and mobile elements with DNA trans-posons as the major components. The frequency and distribution of these PAVs differed substantial y across 96＆amp;nbsp;sorghum inbred lines, and the low-and high frequency PAVs differed in their gene categories. This report shed new light on the occurrence and diversity of PAVs in sorghum genomes. Our research exemplifies a new perspective to explore genome structural variation for genetic improvement in plant breeding.     

Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.     

Heterochromatic Trans-Inactivation of Drosophila White Transgenes

Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene, is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.