Physical map of the white locus of Drosophila melanogaster.

Abstract

The white locus of Drosophila 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, we have cloned and characterized the DNA of a 48-kilobase chromosomal region ofthe Canton S wild-type strain. We have mapped the positions, relative to restriction endonuclease cleavage sites, of several previously characterized chromosomal rearrangement breakpoints that bracket the white locus. These results define a segment of 14 kilobases that contains 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; however, we have identified an extremely weak DNA sequence homology between a portion of this segment and a chromosomal region in the vicinity of the zeste locus.The wl mutation at the white (w) locus was the first mutation reported in Drosophila melanogaster (1) We have previously isolated, by recombinant DNA techniques, a single-copy DNA segment from the white locus of a strain carrying the white-apricot (wa) allele (7). In this report, we describe the molecular cloning of overlapping segments of the Canton S wild-type white locus region. This has allowed us to construct a physical map ofrestriction enzyme cleavage sites in this 48-kilobase (kb) segment, to orient this physical map with respect to the genetic map, and to define a segment of 14 kb that contains all ofthe white locus sequences necessary to allow the production of a w+ (wild-type eye color) phenotype.MATERLALS AND METHODS Drosophila Strains and DNA Preparation. The Drosophila strains used have been described (7). DNA was prepared from adult flies that were anesthetized with C02, collected on ice, rapidly frozen, and stored at -700C. Flies were disrupted and a crude nuclear pellet was prepared as described (8) except that all steps subsequent to grinding were done on ice and Nitex 44 filters were used in place of milk filters. The nuclei were suspended in a small volume of homogenization buffer to which was added 10 vol of 30 mM Tris'HCl, pH 8.0/100 mM EDTA containing proteinase K at 0.5 mg/ml followed by N-lauroylsarcosine to 1%. After incubation overnight at 370C, the DNA was banded in CsCl gradients (9).Recombinant Phage and DNA Preparation. Phage from a Charon 4/Canton S D. melanogaster library (10) were screened by plaque hybridization (11) at a density of 2 x 104 plaques per 24 X 24 cm agar plate as described (7). For DNA preparation, phage were grown in 200 ml of liquid culture (7), debris was removed by low-speed centrifugation, and the phage were precipitated with polyethylene glycol (12), suspended in 2 ml of 10 mM Tris HCl, pH 7.5/10 mM MgSO4 containing 20 ,ug of DNase (Sigma), extracted with an equal volume of CHC13, and purified by two cycles ofCsCl step-gradient centrifugation (13). DNA was isolated from purified phage by phenol ...

Insulation of Enhancer-Promoter Communication by a Gypsy Transposon Insert in the Drosophila cut Gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 Proteins

“…The mod(mdg4) bpd1 and mod(mdg4) bpdEX57 mutations were balanced over In(3LR)TM6,Tb ubi-GFP (provided by Kathryn Anderson, Sloan-Kettering Institute, New York, N.Y.), and homozygous mutant embryos and larvae lacking green fluorescent protein were collected using a fluorescence dissection microscope. Genomic DNA was prepared as described elsewhere (37). Genomic DNA samples were analyzed for the presence or absence of specific sequences by PCR using several different primers.…”

Isolation of cDNAs encoding the Drosophila GAGA transcription factor.

“…Drosophila Oregon R genomic DNA was prepared as described by Levis et al (34). Ten micrograms of genomic DNA was digested with restriction endonuclease EcoRI, BglII, or HindIII and electrophoresed through a 0.8% agarose gel (10 by 6 cm) for 400 V-h.…”

Species-specific signals for the splicing of a short Drosophila intron in vitro.

“…Plasmids pMG1 and pMG2 ( Fig. 1) were constructed by inserting the PvuII (white nucleotide 11078)-SalI (position 11867) DNA fragments from plasmids pml2.5 (35) and pClLTR (46), respectively, between the SmaI and SalI sites of the vector pIBI24 (International Biotechnologies, Inc.), downstream of the promoter for T7 RNA polymerase. The resulting clones contained portions of the second and third exons of the wild-type white gene with either the wild-type second intron (pMG1) or the same intron carrying an sLTR insertion (pMG2).…”