A direct screen identifies new flight muscle mutants on the Drosophila second chromosome.

An ethyl methanesulfonate mutagenesis of Drosophila melanogaster was undertaken, and >3000 mutagenized second chromosomes were generated. More than 800 homozygous viable lines were established, and adults were screened directly under polarized light for muscle defects. A total of 16 mutant strains in which the indirect flight muscles were reduced in volume or disorganized or were otherwise abnormal were identified. These fell into seven recessive and one semidominant complementation groups. Five of these eight complementation groups, including the semidominant mutation, have been mapped using chromosomal deficiencies and meiotic recombination. Two complementation groups mapped close to the Myosin heavy chain gene, but they are shown to be in different loci. Developmental analysis of three mutations showed that two of these are involved in the early stages of adult myogenesis while the other showed late defects. This is the first report of results from a systematic and direct screen for recessive flight muscle defects. This mutant screen identifies genes affecting the flight muscles, which are distinct from those identified when screening for flightlessness.     

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     

Two Recessive Suppressors of SACCHAROMYCES CEREVISIAE CHO1 That Are Unlinked but Fall in the Same Complementation Group

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.     

Characterization of third chromosome dominant alpha-methyl dopa resistant mutants (Tcr) and their interactions with l(2)amd alpha-methyl dopa hypersensitive alleles in Drosophila melanogaster

In Drosophila melanogaster two alleles at the Third chromosome resistance locus (Tcr; 3-39-6) were isolated in a screen of EMS mutagenized third chromosomes for dominant resistance to dietary alpha-methyl dopa, alpha-MD, a structural analogue of DOPA. Both alleles of Tcr are recessive lethals exhibiting partial complementation. Almost half (48.3%) of the Tcr40/Tcr45 heterozygotes die as embryos but some survive past adult eclosion. Both the embryonic lethal phenotype and the adult phenotype suggest that Tcr is involved in cuticle synthesis. Tcr mutants suppress the lethality of partially complementing alleles at the alpha-MD hypersensitive locus, l(2)amd. The viability of Tcr40/Tcr45, however, is not increased by the presence of a l(2)amd allele. The possibility that the Tcr and l(2)amd mutations reveal a catecholamine metabolic pathway involved in cuticle structure is discussed.     

Co-Localization to Chromosome Bands 99e1-3 of the Drosophila Melanogaster Myosin Light Chain-2 Gene and a Haplo-Insufficient Locus That Affects Flight Behavior

Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.     

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 genetic analysis of lethal alleles at the ewg locus and their effects on muscle development in Drosophila melanogaster

The recessive X-linked mutation erect wing (ewg), in Drosophila melanogaster, was characterized as a flightless behavioral mutant which specifically lacked the dorsal longitudinal flight muscles [1]. This mutation was mapped distal to the X chromosomal locus yellow, and further to the cytological segment 1 A 1 to 1 B2-3 [2]. Several lethal complementation groups have been mapped to this interval [3]. Our complementation tests show that ewg is allelic to one lethal complementation group in the region 1 A 1 to 1 B2-3. A further analysis of ewg and several lethal alleles isolated at this locus was undertaken in the present investigation. Most of the lethal alleles at this locus lead to a late embryonic or early larval lethal phase, indicating that the ewg⁺ gene product is necessary for the development of more than just the dorsal longitudinal flight muscles. Intragenic complementation was observed for some of the ewg lethal alleles. Genetic mosaics with ewg lethal alleles showed that mutant cell clones in cuticular structures are viable. Mosaic analysis is consistent with a mesodermal defect associated with the locus.     

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.     

Mutants of Paramecium Defective in Chemokinesis to Folate

Ten mutant lines of Paramecium tetraurelia defective in attraction to folate were isolated and examined. All mutants were normal in response to other attractants and repellents tested. One mutant was able to accumulate in folate given sufficient time. All mutations were recessive and behaved as single site Mendelian lesions. Complementation tests indicate that the mutants fall into three complementation groups. Mutants of Group 2 fall into two phenotypic classes and probably represent two alleles of the mutated fol² gene. Possible sites of the mutants'' blocks in chemoresponse are discussed.     

Genetic Analysis of Olfactory Behavior in Drosophila: A New Screen Yields the Ota Mutants

A simple means of measuring Drosophila olfactory response is described, and the behavior which it measures is characterized. The assay was used to screen for X-linked mutants defective in olfactory function. Six ota mutants were isolated and characterized (ota = olfactory trap abnormal). Four of the mutants were found to be abnormal in another chemosensory behavior as well. Two of the mutant phenotypes extend to include another sensory system: they are defective in visual system physiology. All were normal, however, in a test of giant fiber system physiology. Two of the mutations are dominant, and the recessive mutations define two complementation groups. Mutations representing each complementation group, as well as one of the dominant mutations, were mapped. For the mutants with defective visual system physiology, the visual defects were shown to cosegregate with olfactory phenotypes.     

Chaser (Csr), a New Gene Affecting Larval Foraging Behavior in Drosophila Melanogaster

Chaser (Csr) was uncovered in a gamma mutagenesis screen to identify genes that modify the larval foraging behavior of sitters to rovers. Rover larvae have significantly longer path lenghts than sitters while foraging on a yeast and water paste. This difference is influenced by one major gene, foraging (for), which has two naturally occurring alleles, for(R) (rover) and for(s) (sitter). In a mutagenesis screen for modifiers of for, we identified three lines with viable mutations on chromosome 3 that alter foraging behavior. Each of these mutations increased larval path lengths in for(s)/for(s) larvae in a dominant fashion, and were not separable by recombination. These mutations are therefore probably allelic and define a new gene that we have called Csr. Csr was genetically localized using the lethal-tagging technique. This technique resulted in seven lines with a significant decrease in larval path-length and recessive lethal mutations on chromosome 3. We refer to these as reverted Csr (Csr(rv)) lines. Deficiencies that uncovered cytologically visible chromosome rearrangements in three of the seven reverted lines were used in a complementation analysis. In this way we mapped the lethal mutations in the Csr(rv) lines to cytological region 95F7-96A1 on the right arm of chromosome 3.     

Mutational analysis of glutamine synthetase response to the ammonium analogue ethylene diamine in the cyanobacterium Nostoc muscorum

Tunicamycin is a nucleoside antibiotic complex produced by Streptomyces lysosuperficus which inhibits glycosylation. Several mutants have been isolated in this laboratory that are resistant to tunicamycin, of which the majority are recessive and a few are dominant. The mutations are possibly due to some loss of transport function or alteration in the membrane. These recessive mutations have been mapped to chromosome 1 by the 2 mu mapping method. Studies are underway to map the dominant mutations as well and to group these mutations into its complementation groups and to characterize them biochemically. Both mating types of these mutant strains have been generated in our laboratory.     

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.     

Latheo, a New Gene Involved in Associative Learning and Memory in Drosophila Melanogaster, Identified from P Element Mutagenesis

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.     

Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha.

We have isolated a collection of peroxisome degradation-deficient (Pdd-) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.     

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.     

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.     

Salt-tolerant mutants in glycophytic salinity response (GSR) genes in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The periwinkle Catharanthus roseus shares glycophytic properties of crop plants. To contribute towards an understanding of the glycophytic response to salinity, large populations of M(2) seeds having an origin in nitroso-methyl urea and ethyl methane sulphonate treatments were screened for germination with 250 mM of NaCl. Out of the nine mutant lines so recovered, which tolerated salt stress due to loss of the normal glycophytic salinity response ( GSR), the characteristics of six gsr mutants are reported here. All six, gsr-1 to gsr-6, differed from the wild-type in both seedling and adult-plant morphological characters beside being salt tolerant. The mutations in them were inherited as monogenic recessive alleles at the corresponding wild-type loci. The trans-complementation tests revealed that the gsr-1 to gsr-6 mutants specified six complementation groups. The mutant seedlings generally accumulated more proline and glycine betaine, constitutively, than the wild-type. The mutant plants transpired lower amounts of water and accumulated higher amounts of proline under drought stress. It was inferred that the products of the six GSR genes defined here are involved in the regulation of salt stress, as well as cell division, developmental and/or morphogenetic pathway(s), in C. roseus.     

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.