Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.     

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.     

Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction

Genomic sequences provide powerful new tools in genetic analysis, making it possible to combine classical genetics with genomics to characterize the genes in a particular chromosome region. These approaches have been applied successfully to the euchromatin, but analysis of the heterochromatin has lagged somewhat behind. We describe a combined genetic and bioinformatics approach to the base of the right arm of the Drosophila melanogaster second chromosome, at the boundary between pericentric heterochromatin and euchromatin. We used resources provided by the genome project to derive a physical map of the region, examine gene density, and estimate the number of potential genes. We also carried out a large-scale genetic screen for lethal mutations in the region. We identified new alleles of the known essential genes and also identified mutations in 21 novel loci. Fourteen complementation groups map proximal to the assembled sequence. We used PCR to map the endpoints of several deficiencies and used the same set of deficiencies to order the essential genes, correlating the genetic and physical map. This allowed us to assign two of the complementation groups to particular "computed/curated genes" (CGs), one of which is Nipped-A, which our evidence suggests encodes Drosophila Tra1/TRRAP.     

Mapping candidate genes for Drosophila melanogaster resistance to the parasitoid wasp Leptopilina boulardi

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.     

Studies of the Genetic Organization of the Vestigial Microregion of Drosophila Melanogaster

The region of the second chromosome of Drosophila melanogaster defined by Df(2R)vgB was screened for recessive lethal and visible mutations. Fifty-eight new recessive alleles fall into 17 complementation groups. Many new vg alleles were also isolated in a screen for new vg deficiencies. The breakpoints of the new vg deficiencies were nonrandomly distributed. The distal breakpoints of twelve of 20 deficiencies overlapping Df(2R)vgB are genetically identical to that of Df(2R)vgD, coinciding with the position of a complex, pleiotropic locus, l(2)49Ea-Psc-Su(z)2.     

Genetic Analysis of the Antennapedia Gene Complex (Ant-C) and Adjacent Chromosomal Regions of DROSOPHILA MELANOGASTER. II. Polytene Chromosome Segments 84A-84B1,2

The existence of a gene complex in the proximal right arm of chromosome 3 of Drosophila melanogaster involved in the development of the head and thorax was originally suggested by the phenotypes of several dominant homoeotic mutations and their revertants. A screen for mutations utilizing Df(3R) Antp^Ns+R17 (proximally broken in salivary region 84B1,2) yielded, among 102 recovered mutations, 17 localized by deficiency mapping to the putative homoeotic cluster. These fell into four complementation groups, two of which were characterized by homoeotic phenotypes. To explore the limits of the Antennapedia gene complex (ANT-C) more proximally, a second screen has been undertaken utilizing Df(3R)Scr, a deficiency of 84A1–B1,2.—Of 2832 chromosomes screened, 21 bearing alterations localized to polytene interval 84A–84B1,2 have been recovered. Sixteen are recessive lethals, and five showing reduced viability display a visible phenotype in surviving individuals. Complementation and phenotypic analyses revealed four complementation groups proximal to those identified in the previous screen, including two new alleles of the recessive homoeotic mutation, proboscipedia (pb). Ten of the new mutations correspond to complementation groups defined previously in the Df(3R)Antp^Ns+R17 screen four to the EbR11 group, two to the Scr group and four to the Antp group.—On the basis of the phenotypes of the 39 mutations localized to this region, plus their interactions with extant homoeotic mutations, we postulate that there are at least five functional sites comprising the ANT-C. Three have been demonstrated to be homoeotic in nature. The specific homoeotic transformations thus far observed suggest that these loci are critical for normal development of adult labial, maxillary and thoracic structures.     

Deficient protein kinase C activity in turnip, a Drosophila learning mutant

The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.     

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.     

The Genetics of the Dras3-Roughened-Ecdysoneless Chromosomal Region (62b3-4 to 62d3-4) in Drosophila Melanogaster: Analysis of Recessive Lethal Mutations

The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.     

Cytogenetic Analysis of Chromosome Region 73ad of Drosophila Melanogaster

The 73AD salivary chromosome region of Drosophila melanogaster was subjected to mutational analysis in order to (1) generate a collection of chromosome breakpoints that would allow a correlation between the genetic, cytological and molecular maps of the region and (2) define the number and gross organization of complementation groups within this interval. Eighteen complementation groups were defined and mapped to the 73A2-73B7 region, which is comprised of 17 polytene bands. These complementation groups include the previously known scarlet (st), transformer (tra) and Dominant temperature-sensitive lethal-5 (DTS-5) genes, as well as 13 new recessive lethal complementation groups and one male and female sterile locus. One of the newly identified lethal complementation groups corresponds to the molecularly identified abl locus, and another gene is defined by mutant alleles that exhibit an interaction with the abl mutants. We also recovered several mutations in the 73C1-D1.2 interval, representing two lethal complementation groups, one new visible mutant, plucked (plk), and a previously known visible, dark body (db). There is no evidence of a complex of sex determination genes in the region near tra.     

A Genetic Analysis of the Rose-Gespleten Region (68c8-69b5) of Drosophila Melanogaster

We describe a genetic analysis of the region 68C8-69B5 defined by Df(3L)vin-7. We have induced 35 new lethal mutations in this region, which together with 20 existing lethal mutations, visible mutations, genes identified by protein products and one gene deduced from complementation data fall into 37 complementation groups in this 35-band interval. Using existing and newly induced deficiencies we have assigned these to 11 intervals defined by deficiency breakpoints. Those mutations which fell in the same breakpoint interval as the Lsp-2 gene, which codes for the abundant larval serum protein 2, were the subject of detailed study. None was rescued by the active Lsp-2 gene transformed on to chromosome II and we conclude that, as yet, we have no lethal mutations of Lsp-2.     

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. II. Lethal Mutations in the Region

Forty-seven lethal mutations and alleles of nine visible loci (including alcohol dehydrogenase) have been mapped by both deficiency mapping and, in most cases, by recombination mapping to a small region (34D-35C) of chromosome arm 2L of Drosophila melanogaster. The lethals fall into approximately 21 complementation groups, and we estimate that the total number of lethal plus visible complementation groups within the 34-band deficiency, Df(2L)64j, is approximately 34, a remarkable numerical coincidence. The possible genetic significance of this coincidence is discussed. Lethals mapping close to the structural gene for alcohol dehydrogenase, both distally and proximally, have been identified and will be used for the construction of selective crosses for the study of exchange within this locus. Despite many abnormal cytological features (e.g., ectopic pairing, weak points) region 35 of chromosome arm 2L does not display any unusual genetic features; indeed, in terms of the amount of recombination per band and the average map distance between adjacent loci, this region is similar to that between zeste and white on the X chromosome.     

Deletions encompassing the manganese superoxide dismutase gene in the Drosophila melanogaster genome.

Two deletions, Df(2R)Sod2-11 and Df(2R)Sod2-332, are recovered that encompass the manganese superoxide dismutase (MnSOD) gene or a null mutant referred to as SOD2n283 in Drosophila. Molecular analysis has revealed that the Df(2R)Sod2-332 deletion completely uncovered both MnSOD and its adjacent gene, Arp53D, whereas Df(2R)Sod2-11 was missing the promoter region of MnSOD gene. As a consequence of reduced MnSOD expression, these deletion heterozygotes are now sensitive to oxidative stress. Complementation analysis with some recently recovered deletions in the 53C/D region has established that other essential loci exist in this interval, and second, that Arp53D function is not essential for the survival of the organism. These deletions will be instrumental in the recovery of missense substitutions in the MnSOD peptide and their influence on oxidative stress resistance.     

Defects in embryogenesis in mutants associated with the antennapedia gene complex of Drosophila melanogaster

Embryogenesis in individuals with mutations or deficiencies of the genes in the polytene interval 84A-84B1,2 of Drosophila melanogaster was examined using scanning electron microscopy (SEM). The developmental function of this region of chromosome 3 is of particular interest since it contains the Antennapedia Gene Complex (ANT-C), a gene cluster that includes the homoeotic proboscipedia (pb), Sex combs reduced (Scr), and Antennapedia (Antp) loci. The results of SEM studies, clonal analyses, and temperature-shift experiments show that the fushi tarazu (ftz) and zerknullt (zen) genes, which map between pb and Scr, are involved in processes initiated during embryogenesis. The activity of ftz+ appears to be required within the first 4 hr of development for the establishment of the proper number of segments in the embryonic germ band. Individuals with ftz mutations or deficiencies produce only half the normal number of segments. Each of the segments is twice the normal width and is apparently comprised of cells that would normally form two separate metameres. The zen allele is required from about 2-4 hr of embryogenesis. Mutations of this gene result in disturbances of morphogenetic movements during gastrulation. The mutant phenotype is characterized by the absence of the optic lobe, defects in involution of the head segments, and in some cases, failure of germ band elongation. A requirement during embryogenesis for the activities of other genes residing in the 84A-84B1,2 polytene interval is suggested by the phenotypes of individuals heterozygous or homozygous for chromosomal deficiencies. Using the deficiencies Df(3R)AntpNs+R17, Df(3R)Scr, and Df(3R)ScxW+RX2, we examined the effects of deleting the distal portions or all of the 84A-84B1,2 interval. The defects in deletion heterozygotes suggest that the wild-type activity of some gene(s) other than zen, within or just adjacent to the 84B1,2 doublet, is required to complete normal head involution. The deletion of all the loci in the 84A5-84B1,2 interval results in grossly abnormal morphology and morphogenesis of the gnathocephalic appendages of the embryo. From these studies we conclude that mutations and deficiencies of genes associated with the ANT-C have profound effects on embryogenesis. The mutant phenotypes suggest, in addition to ensuring proper segment identity, the wild-type alleles of the 84A-84B1,2 genes are necessary for normal segmentation and elongation of the germ band and normal head involution.