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

Abstract

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.[Key Words: P element; lacZ; mutagenesis; cell market; Drosophila; pattern]Received May 30, 1989; revised version accepted July 11, 1989. One approach to studying development is to obtain genetic variants that are defective in some crucial step. This type of genetic analysis has been very successful in identifying virtually all of the zygotic loci required for the early stages of segmentation during embryogenesis in Drosophila melanogaster Niisslein-Volhard et al. 1984;Wieschaus et al. 1984). Besides chemical mutagenesis, transposon tagging has been used as a mutagen and allows rapid cloning of genes of interest (Bingham et al. 1981;Kidwell 1986).Recently, a scheme wherein single P elements are mobilized to new chromosomal locations has been implemented successfully (Cooley et al. 1988). The essential nature of this approach is to use two separate P elements to provide the two functions necessary for transposition. The first is a genetically marked P element that is defective in production of transposase but contains the ends required for its own transposition. The second is a P element with functional transposase activity but a much reduced likelihood for its own transposition (Robertson et al. 1988). Transposition of the marked P element then is initiated by crossing flies that carry only the marked P element to those that harbor only transposase. Insertions generated by this scheme are recovered in flies lacking tranposase activity and are therefore genetically stable.P-element vectors also have been used recently to search for cis-acting sequences which confer tissue-specific expression of a p-galactosidase [lacZ] fusion gene driven by the weak promoter of the P-element transpoPresent addresses:

GAL4 drivers expression in the whole adult fly

“…Six different X-linked pGawB insertions were tested for transposition efficiency and ranged from 9.3% (MS1096) to 84.3% (Mz376.1). In contrast with previous reports (Brand and Perrimon, 1993;Gustafson and Boulianne, 1996), it is possible to achieve high frequency of GAL4 P-element comparable to pLacZ transposons (Bellen et al, 1989;Bier et al, 1989), indicating that the lower frequencies observed previously probably resulted from the genomic location of the starting pGawB insertion rather than a intrinsic feature. The chromosomal location and the effects of the insertion on the viability and the reproductive ability were determined for 468 lines (Table 1).…”

Mutations in the β-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain

“…In this experiment we used X-gal staining to detect b-galactosidase activity. This staining is mostly nuclear due to the nuclear localization signal of the transposase-b-galactosidase fused protein (Bier et al, 1989). In the heterozygous normal brain, the inner region of the brain hemispheres was stained (Figure 7a).…”

“…A third brat P-element strain, l(2)K11538, is probably a duplicate of strain l(2)K11523 (Spradling et al, 1999) since both were obtained from the same parents and may be derived from a premeiotic cluster. We veri®ed that each strain carries only one P-element used in the mutagenesis experiment (the P-lacW transposon, Bier et al, 1989), by restricting genomic DNA from these strains with enzymes that cut once within the transposon, and once in the adjacent genomic DNA. Under these conditions, restriction of transposons inserted in di erent sites yielded distinct restriction fragments, as predicted by the detailed map of the brat region (Figure 2a; data not shown).…”

“…In parallel, we stained embryos from strain brat K06028 , carrying the P-lacW transposon, with anti b-galactosidase antibody (Figure 6j ± t). This transposon sits in the untranslated exon 4 of brat (Figure 2a), and it carries a bacterial b-galactosidase gene fused to the P-element transposase, under the control of the transposase promoter (Bier et al, 1989). We suspected that this transposon can be utilized as a brat enhancer trap (Bellen et al, 1989;Bier et al, 1989), namely that the b-galactosidase expression mimics brat expression due to the activity of nearby putative brat enhancer elements.…”

GETDB, a database compiling expression patterns and molecular locations of a collection of gal4 enhancer traps

“…Transcriptional enhancers are often difficult to identify by a simple survey of genomic sequences since their nucleotide sequence are not well conserved and may be located several tens of kilobases from the site of transcription initiation. Enhancer trapping detects chromosomal enhancer elements in situ with minimal perturbations of the genome structure (Bellen et al, 1989;Bier et al, 1989;O'Kane and Gehring, 1987). A database of enhancer trap elements compiling their expression patterns and precise genomic locations should be extremely useful for studies of gene expression and genome analyses.…”