Wingless mutation inDrosophila melanogaster

A temperature sensitive lethal allele of thewingless locus ofDrosophila melanogaster together with previously studied lethal and viable alleles in this locus, has been used to study some properties of this locus. These studies show the existence of two lethal phases for thewingless lesion; one during embryogenesis and another during pupation. By growing embryos with temperature sensitivewingless lesion at the permissive temperature and letting the larvae develop at non-permissive temperature, a large-scale cell death and subsequent regeneration were seen to occur in the mutant wing discs. This cell death followed by regeneration alters the normal developmental potential of the wing disc. Disc transplantation experiments show that these discs are incapable of differentiating into wing blade structures.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

Evidence for myosin heterogeneity inDrosophila melanogaster

Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca²⁺ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles.The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.     

Development of thoracic curvature inDrosophila melanogaster

Summary The influence of muscle development on thorax morphogenesis has been investigated inDrosophila melanogaster. The development of an indirect flight muscle, the dorsal longitudinal muscle (DLM), has been thought to be responsible for the formation of the distinct thoracic curvature. Using aDrosophila mutant (sr/Df(3)sr) in which the DLM is completely missing, we have shown that a normally curved thorax still is produced. Such results indicate that an external structure (epidermis) is capable of developing wholly independent of an absent internal structure (muscle).     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

A high-resolution morphogenetic map of the second-leg basitarsus inDrosophila melanogaster

Summary The lineages of cells on the second-leg basitarsus ofDrosophila melanogaster were analyzed by examining gynandromorphs andMinute mosaics. Bracts lie proximal to bristles on the adult basitarsus, yet bract precursor cells were found to originate lateral to bristle precursor cells. In 6 of the 8 longitudinal rows of bristles on this segment, the bract cells arise ventral to the bristle cells; in the others they arise dorsally. The lateral cell origins are interpreted as reflecting a pattern of lateral cell movements associated with evagination of the leg disc. An unusual discrepancy was observed in the relative frequencies of male vs. female bracts and bristles in gynandromorphs. The discrepancy suggests that there is a cell-autonomous sexual difference in either the time at which cells begin moving during evagination or the speed with which they move.On the basis of the results, it is reasoned that the bristle pattern of the basitarsus does not originate in its final form. Prior to evagination, the bristle cells of each row are apparently closer together than in the final pattern, and the rows are farther apart. Evidence is presented which suggests that the bristle cells of each row may originally be arranged in a jagged line which is later straightened by cell movements.The two locations where the anterior/posterior compartment boundary of the second leg passes through the basitarsus were found to vary relative to the bristle pattern. If this boundary is assumed to be a fixed line of positional values, then the extent of the observed variability — which is estimated to be ± 1 or 2 cell diameters — provides a measure of the precision of patterning around the circumference.     

Genomic imprinting of chromatin inDrosophila melanogaster

During gametogenesis, chromosomes may become imprinted with information which facilitates proper expression of the DNA in offspring. We have used a position effect variegation mutant as a reporter system to investigate the possibility of imprinting inDrosophila melanogaster. Genetic crosses were performed in which the variegating gene and a strong modifier of variegation were present either within the same parental genome or in opposite parental genomes in all possible combinations. Our results indicate that the presence of the variegating chromosome and a modifier chromosome in the same parental genome can alter the amount of variegation formed in progeny. The genomic imprinting we observed is not determined by the parental origin of the variegating chromosome but is instead determined by the genetic background the variegating chromosome is subjected to during gametogenesis.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

Probing the evolution of senescence inDrosophila melanogaster withP-element tagging

Natural populations host a wealth of genetic variation in longevity and age-specific schedules of reproduction. This variation provides critical information for inferring the evolutionary origin of senescence. Patterns of mutational effects on age-specific fecundity and survival provide additional insight to distinguish alternative models of senescence. In this study,P-elements bearing thewhite minigene were inserted at random into a common genetic background, generating lines ofD. melanogaster with single, stable transposon inserts. A series of 48 single-P-element lines revealed statistically significant heterogeneity in both longevity and fecundity. Longevity and early fecundity were only weakly positively correlated (r=0.286,P=0.0398). Both the pooled sample and 30 of the individual lines exhibited a leveling of age-specific mortality at advanced ages, in opposition to the classical demographic models. To the extent that these mutational effects are representative of naturally-occurring mutations in heterogeneous populations, this result presents a problem for the evolutionary theory of senescence. Natural selection is inefficient at removing deleterious mutations that are expressed only at late ages, and selection may not differentiate between mutations whose effects on longevity are post-reproductive. A leveling of the mortality rate would also be seen if mutations whose expression is delayed until very late simply do not occur. A simulation of mutation-selection balance among the 48P-element tagged lines shows that the mean longevity declines monotonically with increasing mutation rate, consistent with the mutation-accumulation model.     

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.     

A comparative study of embryonic gene expression inDrosophila andEphestia

Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and -glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.     

Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost,Oryzias latipes

Summary In many organisms, the germinal dense bodies (GDBs) are known to be organelles unique to the cells of germ-line. In the present study, GDBs in primordial germ cells (PGCs) of the teleost, Oryzias latipes, were examined by electron microscopy. An obvious change was noticed in the morphology of GDBs. In PGCs situated in the endoderm, GDBs consisted of a loosely woven strand-like structure, whereas, GDBs in PGCs in the gonadal anlage, which were amorphous bodies of various sizes and shapes, were composed of electron-dense fine fibrils. The changes in the morphology of GDBs proceeded gradually according to the progress of the stages in migration of the PGCs. GDBs of intermediate morphology were found. The change in the morphology of the GDBs began at the stage of movement of the PGCs from endoderm to mesoderm. It is suggested that the differentiation of PGCs proceeds during their migratory stages under the influence of surrounding somatic cells.     

A qualitative analysis of some life-history correlates of longevity inDrosophila melanogaster

Summary Three likely traits were examined for their possible connection with increased life span in strains ofDrosophila melanogaster selected for longevity. First, pairing with males caused a substantial reduction in survival of females from the short-lived control strain but, long-lived females were relatively unaffected. A significant component of the improvement in selected females is, therefore, increased tolerance to the presence of mates. Females only slightly affected male survival in either long- or short-lived populations. Selected strains survive substantially better than controls independently of any effect of mate presence.The (dry) weight of whole flies is equivalent in long- and short-lived populations. Variation in body size does not appear to contribute significantly to extended longevity here.A third character, the duration of tethered flight, was found to be from three to five times greater in long-lived populations than controls. This suggests the existence of a common physiological basis of longevity to which multiple components contribute in adaptive improvement.     

Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster

Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.     

The peripheral nervous system of mutants of early neurogenesis inDrosophila melanogaster

Summary Mutations previously known to affect early neurogenesis inDrosophila melanogaster have been found also to affect the development of the peripheral nervous system. Anti-HRP antibody staining has shown that larval epidermal sensilla of homozygous mutant embryos occur in increased numbers, which depend on the allele considered. This increase is apparently due to the development into sensory organs of cells which in the wild-type would have developed as non-sensory epidermis. Thus, neurogenic genes act whenever developing cells have to decide between neurogenic and epidermogenic fates, both in central and peripheral nervous systems. Different regions of the ectodermal germ layer are distinguished with respect to their neurogenic abilities.     

Immunocytochemistry of a novel GABA receptor subunitRdl inDrosophila melanogaster

Following our recent cloning of a novel γ-aminobutyric acid (GABA) receptor subunit geneResistance to dieldrin orRdl from the cyclodiene resistance locus inDrosophila melanogaster, we were interested in defining its pattern of expression during development. Here we report the raising of an anti-Rdl polyclonal antibody that recognizes a single protein of the expected 65 kDa size in immunoblots ofDrosophila head homogenates.In situ hybridization usingRdl cDNA probes and the anti-Rdl antibody shows thatRdl message and protein are highly expressed in the developing central nervous system (CNS) of 15–17 h embryos. Interestingly, despite the use of GABA in both the peripheral and CNS of insects,Rdl GABA receptor subunits appear to be confined to the CNS. Detailed immunocytochemistry ofDrosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes. Results are compared with the distribution of staining observed in the insect CNS with antibodies against GABA itself and synaptotagmin, a synaptic vesicle protein.     

The genetic basis of adaptation to selection for longevity inDrosophila melanogaster

Summary Analysis of electrophoretic loci shows that at least four differences exist in isozymes of long- and short-lived populations ofD. melanogaster, descended by selection from a common ancestral stock. Adults of longlived populations differ in gene dosage of phosphoglucomutase (PGM), NAD malate dehydrogenase (MHD), NADP malic enzyme (ME) and by additional mobility variants of glucose-6-phosphate dehydrogenase (G6PD). Larvae, however, differ only by variants of G6PD. The differences in these enzymes, considered together with the greater flight endurance that long-lived populations have shown elsewhere, suggest that increased glycogen synthesis plays a significant role in the improved life span of selected populations. Adaptation to selection for increased life span may, therefore, derive from an improved ability to use dietary sucrose in the media provided. The distribution of electrophoretic loci agrees with the results of a study indicating the position of genetic elements contributing to life span.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Clonal analysis ofsex-lethal,a gene needed for female sexual development inDrosophila melanogaster

Summary The mutationSxl ^f , located on the X-chromosome, is a sex-limited recessive lethal that specifically kills 2X; 2A flies while it does not affect X; 2A flies (Cline 1978). We have analyzed the role ofSxl ^f on sex determination by a clonal analysis of a new spontaneous allele,Sxl ^fLS . Female embryos and larvae heterozygous forSxl ^fLS were irradiated at different times of development to generate homozygousSxl ^fLS clones which were recognized by linked marker mutations. We have studied the phenotype of such clones on sexually dimorphic regions of the fly (foreleg basitarsus, 5th, 6th and 7th tergites, analia and external genitalia). Despite their female (2X; 2A) chromosomal constitution, clones homozygous forSxl ^fLS differentiated male structures. These results confirm and extend the preliminary report of Cline (1979). They show that the wildtype product ofSxl ^f is required for female development.     

Suppression of abdominal legs inDrosophila melanogaster

Summary Drosophila melanogaster normally have six thoracic legs and no abdominal legs. However, one or two legs often appear in the first abdominal segment ofbithoraxoid mutants. The extent to which these extra legs develop is determined both by thecis-regulatory action ofbithoraxoid lesions onUltrabithorax and by the number of copies of the adjacent homeotic geneabdominal-A. Thebithoraxoid region does notcis-regulateabdominal-A.