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.     

Genetic analyses of essential genes in cytological region 61D1-2 to 61F1-2 of Drosophila melanogaster

We performed a systematic mutagenesis screen for lethals in the genomic region 61D1-2 to 61F1-2 on chromosomal arm 3L of Drosophila melanogaster. Our genetic analyses revealed that this region contains eight essential complementation groups including trio, Glut1 and extra macrochaetae (emc). For the trio locus, 22 mutant alleles were identified, and all of the alleles analyzed resulted in defects in the central nervous system of embryos, indicating that trio functions in the control of axon extension or guidance. Western analysis showed that at least three proteins are derived from trio and also suggested that a polypeptide of over 200 kDa plays a crucial role in embryonic or larval development. In addition, a newly identified emc allele was associated with several defects in embryonic morphogenesis, including abnormalities in head involution, gut formation and dorsal closure, thus revealing multiple roles for emc in embryonic development. We also performed preliminary phenotypic analyses on stocks bearing mutations belonging to the other lethal complementation groups. These genes function in essential biological events, but the mutations do not result in gross morphological changes during embryonic stages. The present study extends our knowledge of the Drosophila gene set, by identifying most of the essential genes in the chromosomal region 61D1-2 to 61F1-2.     

The Genetics of the DORSAL-BICAUDAL-D Region of DROSOPHILA MELANOGASTER

The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.     

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     

A Cytogenetic Analysis of Chromosomal Region 31 of Drosophila Melanogaster

Cytogenetic region 31 of the second chromosome of Drosophila melanogaster was screened for recessive lethal mutations. One hundred and thirty nine new recessive lethal alleles were isolated that fail to complement Df(2L)J2 (31A-32A). These new alleles, combined with preexisting mutations in the region, define 52 complementation groups, 35 of which have not previously been described. Among the new mutations were alleles of the cdc2 and mfs(2)31 genes. Six new deficiencies were also isolated and characterized identifying 16 deficiency subintervals within region 31. The new deficiencies were used to further localize three loci believed to encode non-histone chromosomal proteins. Suvar(2)1/Su(var)214, a dominant suppressor of position-effect variegation (PEV), maps to 31A-B, while the recessive suppressors of PEV mfs(2)31 and wdl were localized to regions 31E and 31F-32A, respectively. In addition, the cytological position of several mutations that interact with heterochromatin were more precisely defined.     

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.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

Genetic Characterization of the 87c Region of the Third Chromosome of DROSOPHILA MELANOGASTER

Ethyl methanesulphonate (EMS) was used to induce 39 lethal and 13 karmoisin mutations within Df(3R)kar^3J, a nine-band deficiency extending from 87C1 to 87C9 (inclusive). Five complementation groups (four lethal and one visible) were identified and cytologically mapped between 87C4–5 and 87C9, one complementation group per band, with the exception of complementation group A, which is localized to 87C4–5. These positions were determined using a set of overlapping deficiencies, each having at least one breakpoint in the 87C1–9 region. Mutations within a single complementation group have similar lethal phases or subvital phenotypes, consistent with the notion that each complementation group represents a single functional locus. No mutations localized to 87C1–C3. The inability to induce mutations in the 87C1 heat-shock puff locus is consistent with the current interpretation of a duplication of coding sequences at the 87A7 and 87C1 heat-shock puffs.     

Genetic Analysis of Chromosomal Region 67a-D of Drosophila Melanogaster

In an effort to (1) characterize the 67 interval of chromosome 3 of Drosophila melanogaster genetically and (2) isolate mutations of the 67B1 small heat shock protein (hsp) gene cluster specifically, we undertook a mutational analysis of the 67A-D subinterval. Using a deficiency of the 67A2 to 67D11-13 region, Df(3L)AC1, we screened 8700 diepoxybutane-treated chromosomes and 7800 ethyl methanesulfonate-treated chromosomes for visible and lethal mutations throughout this interval and recovered 74 independent recessive lethal mutations, but no visible mutations. One of the lethal mutations, d29A6, was identified as an overlapping deficiency extending from 66F3 to 67B1. An additional 6000 diepoxybutane-treated chromosomes were screened for lethality over d29A6, yielding another four lethal mutations within the 67A2-B1 subinterval. These 78 lethal mutations, along with two others isolated in other laboratories, define 23 essential loci--6 within the 67A2-B1 subinterval and 17 within the 67A2 to D11-13 subinterval. Many of these loci appear to be required for imaginal development only, exhibiting late larval to pharate adult lethal phases. Examination of the 67A2-B1 lethal complementation groups for (1) earlier onset of lethality following a heat shock, (2) missing or altered small hsps on two-dimensional protein gels, and (3) restoration of viability by transformed wild-type copies of the small hsp genes indicates that none of these mutations affect the small hsps. On the basis of this analysis and the known homology of the genes, we conclude that the small hsps are functionally equivalent.     

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.     

Cytogenetics of Notch Mutations Arising in the Unstable X Chromosome Uc of Drosophila melanogaster

A derivative of the unstable X chromosome, Uc, isolated in 1978 is still unstable and exhibits most of the genetic properties characteristic of the original Uc. This derivative, Df(1)cm-In, contains an inversion of the genes between bands 6F1-2 and 3D3-5 and a lethal deficiency between 6D5-7 and 6F1-2. This chromosome generated Notch mutations at a rate of 3.47 +/- 0.32% during seven consecutive generations. Cytological analysis of 50 Notch mutations of independent origin in the Df(1)cm-In chromosome showed that all of the 50 had an apparently identical deletion involving the region between 3D3-5 and 3C7-8 of the X chromosome. The results of in situ hybridization indicated that the extent of deletion in all of the 20 Notch deficiencies sampled from the 50 mentioned above involves about 10 kb of the sequences from the 3' end of the Notch locus. In addition to hypermutability and the accumulation of site-specific chromosome breaks, the Df(1)cm-In chromosome reinverts its inversion to the normal sequence and exhibits use of the existing chromosome breakpoints to generate new rearrangements.     

The Unc-22(iv) Region of Caenorhabditis Elegans: Genetic Analysis of Lethal Mutations

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.     

Genetic maps of the proximal half of chromosome arm 2L of Drosophila melanogaster.

Drosophila mutants have played an important role in elucidating the physiologic function of genes. Large-scale projects have succeeded in producing mutations in a large proportion of Drosophila genes. Many mutant fly lines have also been produced through the efforts of individual laboratories over the past century. In an effort to make some of these mutants more useful to the research community, we systematically mapped a large number of mutations affecting genes in the proximal half of chromosome arm 2L to more precisely defined regions, defined by deficiency intervals, and, when possible, by individual complementation groups. To further analyze regions 36 and 39-40, we produced 11 new deficiencies with gamma irradiation, and we constructed 6 new deficiencies in region 30-33, using the DrosDel system. trans-heterozygous combinations of deficiencies revealed 5 additional functions, essential for viability or fertility.     

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.     

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.     

Genetic characterization of the region of the Drosophila genome known to include the histone structural gene sequences

This report describes a genetic study of salivary map region 39DE of the Drosophila genome, which is known to include the histone gene sequences (Pardue 1975; Liftonet al. 1977). Small deficiences extending proximally into 39DE were constructed by the segmental aneuploid method of Lindsleyet al. (1972). The translocational deficiencies obtained in this manner were gamma-irradiated to remove the Y translocational arms. One of these newly reconstituted deficiencies was then used to screen 10,000 gamma-irradiated second chromosomes for lethal mutations. The 32 lethals recovered from the screen were tested against several deficiencies and markers, crossed inter se and categorized according to their genetic properties. From these data, a preliminary complementation map was constructed of salivary region 39A-39F. The salivary map positions of certain of the complementation groups suggest that the mutants in these groups may affect histone gene functions.     

Genetic and Phenotypic Analysis of Thirteen Essential Genes in Cytological Interval 22f1-2; 23b1-2 Reveals Novel Genes Required for Neural Development in Drosophila

In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.