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.     

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 screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase.     

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.     

A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase. Received: 25 April 1998 / Accepted: 27 May 1998     

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 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.     

Developmental Genetics of Loci at the Base of the X Chromosome of Drosophila Melanogaster

We have conducted a genetic and developmental analysis of the 26 contiguous genetic complementation groups within the 19D3-20F2 interval of the base of the X chromosome, a region of 34 polytene bands delimited by the maroon-like and suppressor of forked loci. Within this region there are four loci which cause visible phenotypes but which have little or no effect on zygotic viability (maroon-like, little fly, small optic lobes and sluggish). There are 22 loci which, when mutated, are zygotic lethals and three of these, legless/runt, folded gastrulation and 13E3, have severe effects on embryonic development. In addition, three visible phenotypes have been defined only by overlapping deficiencies (melanized-like, tumorous head, and varied outspread). We have analyzed the lethal phases and maternal requirement of 58 mutations at 22 of the zygotic lethal loci by means of germline clone analysis using the dominant female sterile technique. Additionally, all lethal complementation groups, as well as a specific subset of deficiencies, have been studied histologically for defects in the development of the central and peripheral embryonic nervous systems.     

Mutations Affecting Functions of the Drosophila Gene Glued

Glued mutations in Drosophila comprise an essential complementation group with complex developmental effects. The original Glued mutation (Gl) has dominant nonlethal effects in heterozygous flies, principally on the morphogenesis of the visual system. Gl also has a recessive lethal effect early in development. Mutations that reverse the dominant visual effects of Gl (GlR mutations) were induced by gamma-radiation or by insertions of the transposable P element. The GlR(G) mutations induced by gamma-radiation do not reverse the lethal effect of Gl; these appear to be null mutations, some of which (and possibly all) delete segments of the Glued region. The GlR(P) mutations induced by insertion of the P element also reverse concomitantly a recessive lethal effect of Gl, suggesting that both the recessive and dominant effects are controlled by the same gene. The reversal of a lethal effect of Gl by the P element is remarkable, since it indicates that an essential gene function can be restored by insertion of unrelated DNA. Another class of lethal Glued mutations was induced in the normal Gl+ strain by ethyl methanesulfonate (EMS). The EMS mutations belong to the same essential complementation group as Gl, but do not have the strong dominant effects of Gl on the visual system. The GlR(P) mutations provide a molecular marker for the Glued gene, which was used to map the gene to the 70C2 band of chromosome 3L by in situ hybridization of a P element probe to polytene chromosomes from the GlR(P) strains and also to isolate clones of Glued genomic DNA for molecular studies of the normal gene and the various Glued mutations.     

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.     

Genetic Characterization of the Region between 86f1,2 and 87b15 on Chromosome 3 of DROSOPHILA MELANOGASTER

The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.     

Genetic Analysis of the Cha-1-Unc-17 Gene Complex in Caenorhabditis

In C. elegans, the gene cha-1 is the structural gene for choline acetyltransferase, the enzyme which synthesizes acetylcholine. cha-1 is a complex gene which includes the previously described unc-17 locus; it has been hypothesized that a single protein is encoded which consists of several discrete structural domains. Mutations of the cha-1-unc-17 locus can be assigned to one of four classes on the basis of phenotype and complementation properties. A fine-structure map of this region has now been obtained by recombinational mapping. It is a large locus, spanning at least 0.035 map unit. On the map, the mutations lie in four contiguous, nonoverlapping regions, corresponding exactly to the different classes as defined by complementation and phenotype. Several new cha-1 mutations are described and mapped in the present study, including temperature-sensitive and lethal alleles.     

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.     

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.     

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 determinants of glutamine synthetase inDrosophila melanogaster: A gene for glutamine synthetase I resides in the 21B3-6 region

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.