Robert Levis

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.

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.

Transformation of white locus DNA in Drosophila: Dosage compensation, zeste interaction, and position effects

P-element-mediated DNA transformation was used to generate transformants carrying segments of DNA from the white locus of D. melanogaster. The vast majority of transduced copies of an 11.7 or a 14.3 kb segment of DNA from white successfully rescued the white- eye-color phenotype when inserted in many different chromosomal locations. However, two transformants with abnormal eye pigmentation--apparently a consequence of the genomic positions of the transduced white gene--were also recovered. In all seven cases tested, autosomal insertions of white, which is dosage-compensated in its normal location on the X chromosome, retained the property of dosage compensation. In contrast to the relative insensitivity of eye-color pigmentation and dosage compensation to genomic position, the transduced white DNA segments differed widely in their interactions with the zeste mutation, ranging from greater than normal repression by zeste to insensitivity to the presence of zeste.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

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 ΦC31 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.

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 library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001

Terminal repeats of the drosophila transposable element copia: Nucleotide sequence and genomic organization

We have determined the nucleotide sequence of the terminal regions of two members of the copia sequence family of D. melanogaster. The first 276 bp at one end of a copia element are repeated in direct orientation at its other end. The direct repeats on a single copia element are identical to each other, but they differ by two nucleotide substitutions between the two elements which were examined; this suggests that during transposition only one direct repeat of the parent element is used as a template for both direct repeats of the transposed element. Each direct repeat itself contain a 17 bp imperfectly matched inveted terminal repetition. The ends of copia show significant sequence homology both to the yeast Ty1 element and to the integrated provirus of avian spleen necrosis virus, two other eucaryotic elements known to insert at many different chromosomal locations. Analysis of the genomic organization of the direct repeat sequence demonstrates that it seldom, if ever, occurs unlinked to an entire copia element.

Separable cis-acting control elements for expression of the white gene of Drosophila.

The white gene of Drosophila is required for the pigmentation of the eyes, ocelli, Malpighian tubules and testis sheath. We have examined the regulation of white expression in germ line transformants that carry the mRNA‐coding sequences of white flanked by varying amounts of the sequences normally located adjacent to them. A segment with as little as 1.9 kb of 5′‐ and 2.1 kb of 3′‐flanking DNA was expressed to give essentially wild‐type pigmentation in all of these tissues and interacted in trans with the zeste‐1 allele. In contrast, transformants having segments with 1.1 kb of 5′‐flanking DNA had reduced levels of pigment in their eyes, no detectable pigment in their testes and did not interact with zeste‐1. The eyes, Malpighian tubules and ocelli of transformants with as little as 0.4 kb of 5′‐flanking DNA were pigmented and the eye pigmentation exhibited dosage compensation. We conclude that there are sequence elements present in the region from 1.1 to 1.9 kb upstream from the 5′ end of the white transcript which are required for expression in the testes and interaction of white and zeste‐1. The same or other elements in this region appear to augment the pigmentation of the eye.

FB elements are the common basis for the instability of the wDZL and wc Drosophila mutations

The DNA insertions that cause the highly unstable mutations wC and wDZL share extensive homology with the FB family of transposable elements. FB elements carry long, internally repetitious, inverted terminal repeats and thus differ in structure from other transposable elements. Our results suggest that FB elements may excise and cause chromosomal rearrangements at unusually high frequencies. The wC insertion is a single FB element. The wDZL insertion differs in that it contains two FB elements, one at each terminus. The wC and wDZL insertions contain 4.0 and 6.5 kilobase nonhomologous segments between their terminal repeats. In contrast to the middle repetitive FB elements, the central segment of the wDZL insertion is single-copy and present at a fixed location in the wild-type genome. It has apparently been transposed by the action of flanking FB elements, causing the wDZL mutation at its new location.

The unstable wdzl mutation of Drosophila is caused by a 13 kilobase insertion that is imprecisely excised in phenotypic revertants

We have analyzed the lesion in wDZL, a genetically unstable mutant allele of the eye color locus, white, of Drosophila melanogaster. We have cloned the DNA of the white locus region of flies carrying the wDZL allele and find a 13 kilobase insertion not present in the wild-type at the corresponding location. In 12 independent cases examined, reversion to a wild-type eye color phenotype correlates with the excision of a portion of this 13 kilobase insertion, indicating that the insertion is the cause of the mutation. The portion of the insertion that is excised in these eye color revertants is heterogeneous in size but appears to include the central 6 kilobases of the insertion in all cases. Many of these eye color revertants continue to undergo mutation at the white locus, indicating that the residual portion of the insertion in these revertants is sufficient to promote mutations.