We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

The Drosophila gene eyeless (ey) encodes a transcription factor with both a paired domain and a homeodomain. It is homologous to the mouse Small eye (Pax-6) gene and to the Aniridia gene in humans. These genes share extensive sequence identity, the position of three intron splice sites is conserved, and these genes are expressed similarly in the developing nervous system and in the eye during morphogenesis. Loss-of-function mutations in both the insect and in the mammalian genes have been shown to lead to a reduction or absence of eye structures, which suggests that ey functions in eye morphogenesis. By targeted expression of the ey complementary DNA in various imaginal disc primordia of Drosophila, ectopic eye structures were induced on the wings, the legs, and on the antennae. The ectopic eyes appeared morphologically normal and consisted of groups of fully differentiated ommatidia with a complete set of photoreceptor cells. These results support the proposition that ey is the master control gene for eye morphogenesis. Because homologous genes are present in vertebrates, ascidians, insects, cephalopods, and nemerteans, ey may function as a master control gene throughout the metazoa.

/pdf/induction-of-ectopic-eyes-by-targeted-expression-of-the-4ia2fio7dp.pdf

Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila

A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.

/pdf/promoter-traps-in-embryonic-stem-cells-a-genetic-screen-to-19fifgltiy.pdf

Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

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.

/pdf/the-bdgp-gene-disruption-project-single-transposon-4j3rm9e1ix.pdf

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene. The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions. It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants. These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases. Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas. Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.

/pdf/the-drosophila-tumor-suppressor-gene-warts-encodes-a-homolog-35z4nnod6u.pdf

The Drosophila Tumor Suppressor Gene Warts Encodes a Homolog of Human Myotonic Dystrophy Kinase and Is Required for the Control of Cell Shape and Proliferation

A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.

/pdf/searching-for-pattern-and-mutation-in-the-drosophila-genome-5d8a9pa9o0.pdf

R Carretto

Papers

Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.