The isolation of a cloned DNA segment carrying unique sequences from the white locus of D. melanogaster is described. Sequences within the cloned segment hybridized in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements in which breakpoints bracket the white locus. 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 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. A simple, rapid method is described 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 its application to the cloning of the DNA sequences of other genes in Drosophila is discussed, including those identified only by genetic analysis and for which no RNA product is known.

The white locus of D. melanogaster is a genetically well-characterized locus, mutations in which alter the degree or pattern of pigmentation of the eyes. Using a previously cloned DNA segment containing a portion of the white locus of a mutant allele, the DNA of a 48-kilobase chromosomal region of the Canton S wild-type strain was cloned and characterized. The positions, relative to restriction endonuclease cleavage sites, of several previously characterized chromosomal rearrangement breakpoints that bracket the white locus were mapped. A segment of 14 kilobases was defined containing all of the white locus sequences necessary for the production of a wild-type eye color phenotype. By conventional criteria, no repetitive sequences are present within this 14-kilobase segment; an extremely weak DNA sequence homology between a portion of this segment and a chromosomal region in the vicinity of the zeste locus was identified.

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. 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 2 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. Apparently it was transposed by the action of flanking FB elements, causing the WDZL mutation at its new location.

The lesion in WDZL, a genetically unstable mutant allele of the eye color locus, white, of D. melanogaster was analyzed. The DNA of the white locus region of flies carrying the WDZL allele was cloned and a 13 kilobase insertion not present in the wild-type was found 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.

The white locus has a distal region where structural mutations occur and a proximal region where regulatory mutations occur. To better understand the molecular basis of this genetic organization, white locus transcription was analyzed. A 2.7-kilobase transcript comprising 0.0005% of poly(A)-RNA was detected in RNA prepared from pupae or adults. The structure of this transcript helps clarify some unusual genetic properties of the locus. There is a small 5'' exon separated from the majority of the sequences found in the mature RNA by an intron of .apprxeq. 2.8 kilobases. This 5'' exon is from the proximal region of the locus, whereas the main body of the RNA maps to the distal region. The mutationally silent region between the proximal and distal regions corresponds to the large intron. The family was identified and the exact location of a number of transposable element insertions within the locus was determined. Transposable element insertions within introns can be without phenotypic effect. The effect on the white transcript of the zeste mutation, which represses white locus expression as judged by eye color phenotype, was investigated. The RNA was unchanged in size or abundance in poly(A)-RNA from adult flies. The zeste-white interaction does not occur by simply repressing transcription of the white locus in all tissues.

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 [kilobase] segment of DNA from white successfully rescued the white- eye-color phenotype when inserted in many different chromosomal locations. However, 2 transformants with abnormal eye pigmentation, apparently a consequence of the genomic positions of the transduced white gene, were also recovered. In all 7 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 zeste1 mutation, ranging from greater than normal repression by zeste1 to insensitivity to the presence of zeste1.

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.