The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome

The Drosophila dopa decarboxylase gene, Ddc, functions normally when reintroduced into flies. DNA containing a cloned Ddc gene inserted into a P element transposon was injected into early embryos. Transformants were identified by suppression of the cuticular phenotype of a Ddc mutant allele. The reintegrated genes are expressed in the proper tissue and at the proper stages during development even though their positions within the genome are different from that of the wild-type Ddc gene. Absolute levels of DDC enzyme activity are within 35% of that found in wild-type Canton S flies, the source of the transforming DNA. The transformants' Ddc RNA is indistinguishable from that of wild type. One reintegrated Ddc gene, inserted on the X chromosome, is affected by the dosage compensation mechanism that leads to sex-specific differences in the expression of many X-chromosome genes.     

A Genetic Analysis of the 63e-64a Genomic Region of Drosophila Melanogaster: Identification of Mutations in a Replication Factor C Subunit

We have performed and F(2) genetic screen to identify lethal mutations within the 63E-64A genomic region. We have isolated 122 mutations in 20 different complementation groups. Of these groups, 16 are represented by multiple alleles. We have also established that the Rop and Ras2 genes are located within the 63E-64A genomic domain at 64A10,11. We have sequenced 10.2 kb of DNA surrounding this gene pair and find that in addition to Rop and Ras2 there is another gene located within this DNA sequence. The gene product, which we have named Rfc40, shows 68% identity to the 40-kDa subunit of replication factor C. We find that the members of one complementation group (13 alleles) derived from our screen correspond to mutations in the Rop gene, whereas the members of another (five alleles) correspond to mutations in the Rfc40 gene. In addition we have isolated 11 new mutant alleles of the disembodied gene.     

A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. II. Molecular Localization of the Dox-A2 Coding Region

Mutations at the Dox-A2 (2-53.9) locus alter the A2 component of diphenol oxidase, an enzyme having an important role in cuticle formation. This locus is in the dopa decarboxylase, Df(2L)TW130 region, which contains a cluster of at least 14 genes involved in catecholamine metabolism and the formation, sclerotization and melanization of cuticle in Drosophila. The region is subdivided by deficiencies, and localization of breakpoints in cloned DNA reveals a dense subcluster of six genes in the 23 kb proximal to Ddc. Five lethal loci distal to Ddc comprise a second such subcluster. The proximal breakpoints of deficiencies Df(2L)hk18 and Df(2L)OD15 define a 14.3- to 16.8-kb region containing Dox-A2 and l(2)37Bb, and those of Df(2L)OD15 and Df(2L)TW203 define a 9.3- to 12.1-kb region containing l(2)37Ba, l(2)37Bc and l(2)37Be. Southern blots show two of the Dox-A2 mutations are small deletions (0.1 and 1.1 kb). The Dox-A2 locus mRNA is 1.7 kb. cDNA clones indicate that the 3' end is centromere proximal and that the coding region contains at least one small intron. The Dox-A2 locus is within 3.4 to 4.4 kb of the Df(2L)OD15 breakpoint, placing four of the vital loci within a maximum of 15.5 kb. The location of Dox-A2 in a cluster of genes affecting cuticle formation is discussed.     

Molecular Genetics of the ROSY-ACE Region of DROSOPHILA MELANOGASTER

Three hundred and fifteen kilobases of DNA from the rosy-Ace region on chromosome 3R of D. melanogaster have previously been cloned and extensively characterized. We describe the isolation of nine new deficiency mutants that break within the 315-kb interval. The position of these breakpoints on the DNA map was determined by in situ and Southern hybridization. Further, we more precisely mapped the breakpoints of several deletions previously analyzed. The results permit us to delimit sequences essential to the known complementation groups in the region within approximately 20 kb in most cases. However, one gene, B16-1, is shown to contain essential sequences that span about 50 kb. Also, we demonstrate by overlapping deficiencies that a 45-kb DNA segment from the region, which includes one known complementation group, allows limited survival when deleted.     

Molecular mapping of genetic and chromomeric units in Drosophila melanogaster

We have used a set of overlapping cloned segments defining a 315 kb (X 10(3) base-pairs) region of Drosophila melanogaster chromosomal DNA to map the sequences associated with the polytene band-interbands (chromomeric units) and with the lethal complementation groups contained within this region. The molecular map positions of the 13 +/- 1 chromomeric units from the 87D5-6 to 87E5, 6 region of the third chromosome were determined by in situ hybridization of selected segments to the polytene chromosomes. The length of the largest chromomeric unit within the 315 kb region is approximately 160 kb, while that for the smallest is less than 7 kb and may be as short as 3 kb. By mapping the breakpoints of deletions within the 315 kb region, we have located its 12 lethal complementation groups, which include the genes coding for acetylcholinesterase (Ace) and xanthine dehydrogenase (rosy). Comparison of the two molecular maps indicates a one-to-one topographical correlation between the genetic and chromomeric units.     

A P Element Chimera Containing Captured Genomic Sequences Was Recovered at the Vestigial Locus in Drosophila following Targeted Transposition

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.     

Genetic and Molecular Mapping of Chromosome Region 85a in Drosophila Melanogaster

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.     

Genomic Structure ofPEX13,a Candidate Peroxisome Biogenesis Disorder Gene

The peroxisome biogenesis disorders (PBDs) are a set of lethal genetic diseases characterized by peroxisomal metabolic deficiencies, multisystem abnormalities, mental retardation, and premature death. These disorders are genetically heterogeneous and are caused by mutations in genes, termedPEXgenes, required for import of proteins into the peroxisomal matrix. We have previously reported the identification of humanPEX13,the gene encoding the docking factor for the PTS1 receptor, or PEX5 protein. As such, mutations inPEX13would be expected to abrogate peroxisomal protein import and result in PBD phenotypes. We report here the structure of the humanPEX13gene.PEX13spans approximately 11 kb on chromosome 2 and contains four exons, one more than previously thought. The corrected PEX13 cDNA is predicted to encode a protein product with a molecular mass of 44,312 Da. We examined the ability ofPEX13expression to rescue the peroxisomal protein import defects of fibroblast cells representing all known PBD complementation groups. No complementation was observed, suggesting that this gene is not mutated in any set of existing patients. However, given that complementation group assignments have been determined for only a subset of PBD patients, it is possible thatPEX13-deficient patients may exist at a low frequency within our existing PBD patient population or within ethnic groups underrepresented in our patient pool.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.