New mutants of Drosophila melanogaster

Characteristics are given of 57 Drosophila melanogaster mutants catched in the South and Soeth-West Iran.     

Characterization of lethal Drosophila melanogaster alpha-actinin mutants

We have partially characterized four Drosophila melanogaster alpha-actinin gene mutants, I(1)2Cb1, I(1)2Cb2, I(1)2Cb4, and I(1)2Cb5. We demonstrate that in each case the mutation is caused by a chromosomal rearrangement that precludes normal protein synthesis. In the absence of alpha-actinin, flies complete embryogenesis and develop into flaccid larvae that die within approximately 24 hr. These larvae have noticeable muscle dysfunction at hatching, although they, nevertheless, are capable of escaping from the egg membranes and of subsequent crawling movements. During larval development muscles degenerate, progressively limiting mobility and ultimately causing death. Electron microscopy of mutant muscle fibers reveals that myofibrils are grossly disrupted in one day old larvae and that electron-dense structures reminiscent of those seen in human nemaline myopathies are present throughout larval life. Our work rigorously demonstrates that alpha-actinin deficiencies are the cause of I(1)2Cb muscle defects. We anticipate that the alpha-actinin mutants described herein will facilitate in vivo tests of spectrin superfamily protein domain functions using a combination of directed mutagenesis and germline transformation.     

Genotypic basis of low viability in vestigial mutants of Drosophila melanogaster

The role of a marker mutation and other genes in a decrease in viability was studied in the Drosophila melanogaster vg line. In flies of the C-S line, chromosome 2 was substituted by the homologous chromosome of the vg flies. In addition, the flies of the mutant phenotype with mutant genes partially or completely substituted by the wild-type C-S genes were obtained in saturating crosses C-S x vg. In the reciprocal variant of chromosome 2 substitution, the flies of the C-S phenotype with chromosomes 1, 3, and 4 from the vg line were obtained. Chromosome 2 of the vg line, introduced into C-S fly karyotype, proved to substantially reduce the heat resistance and life span of flies. In the case of reciprocal replacement (C-S line chromosome 2 substituted for the homologous chromosome of vg flies), a significant increase in viability was observed, which, however, never reached the level characteristic of the C-S line. As the vg genotype became saturated with C-S genes, the heat resistance and life span of flies increased substantially. However, even the complete saturation of mutant chromosomes with wild-type genes never resulted in the equal viability of vg and C-S flies. These data suggest that the low viability of the vg mutant is largely accounted for by the gene composition of the second chromosome and, primarily, by the presence of the vg gene. Nevertheless, there is evidence that, along with the pleiotropic effect of the marker mutation, other genes not linked to chromosome 2 are responsible for the studied physiological properties of the vg flies.     

Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen intermediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Catn4, likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Catn1 mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span.     

Neurogenesis in Drosophila melanogaster hemizygotic, heterozygotic, and homozygotic notch mutants

The energy profile of the interaction between the NH2-terminal inactivation domain and the internal mouth of the Shaker H4 K+ channel has been investigated. Macroscopic currents from channels normally inactivating (Shaker H4) and with the inactivation removed (Shaker H4-IR) were recorded at different temperatures using the cut-open oocyte technique. Changes in temperature had a dramatic effect on the inactivation phase. The following parameters were obtained in Shaker H4, lowering the temperature from 20 degrees C to 5 degrees C: (1) the peak amplitude decreased with the temperature coefficient Q10 equal to 1.51; (2) the activation time constant increased with a Q10 equal to 3.14; (3) the decay time constant increased with a Q10 of 7.20, while the recovery from inactivation was less temperature-dependent (Q10=1.57) than the installation of the inactivation phase. At 0 mV, the ratio between the steady state level and the peak amplitude of the current increased with a Q10 of 2.95. These findings indicate that the installation of a fast inactivation process has a strong temperature dependence, while the recovery phase from inactivation is less temperature dependent. These observations support the idea of an NH2-terminal blocking mechanism for inactivation and flexible conformation of the blocking particle.     

A photographic study of development in the living embryo of Drosophila melanogaster

The changes which can be seen occurring during the development of a living embryo of Drosophila melanogaster are described in detail, and represented photographically as a series of developmental stages. This provides an easy, but accurate technique for selecting eggs at precise developmental stages for experiments.     

The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster

We have examined expression of the neurogenic gene, Delta (Dl), and the regulatory relationships between the Delta-Notch signalling pathway and the proneural gene, achaete, during microchaeta development in Drosophila. Delta is expressed in all microchaeta proneural cells and microchaeta sensory organ precursors (SOPs) and is expressed dynamically in SOP progeny. We find that Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that initial Delta expression in the notum is not dependent on proneural gene function. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is regulated, in part, by Delta-Notch signalling activity. We find that Delta signalling is required for correct delineation of early proneural gene expression in developing nota. Within microchaeta proneural stripes, we demonstrate that Delta-Notch signalling prohibits adoption of the SOP fate by repressing expression of proneural genes.     

Changes in gene expression during post-eclosional development in the olfactory system of Drosophila melanogaster

We have found that the expression of some genes in Drosophila melanogaster changes during the life of the adult fly. These changes can be illustrated by the use of enhancer trap lines which mark the expression of particular genes in the adult fly. Although the fly is considered able to perform most necessary adult functions within the first 72 h after eclosion from the pupal case, we find changes in expression over the first 10 days of life in the antennae of several of the genes we have examined. Some genes change by increasing from an initially low level of expression of the marked gene, while other lines, which we have termed 'late-onset' genes, show no expression of the marked gene until 4-5 days following eclosion. In contrast, some genes decrease their expression during the first 10 days of life. The changes in gene expression seen over the first 10 days of the fly's adult life provides molecular evidence of the many maturational changes occurring during the early life of the adult fly.     

Extended reproductive roles of the fruitless gene in Drosophila melanogaster revealed by behavioral analysis of new fru mutants

The fruitless mutants fru3 and fru4 were assessed for sex-specific reproductive-behavioral phenotypes and compared to the previously reported fru mutants. Among the several behavioral anomalies exhibited by males expressing these relatively new mutations, some are unique. fru3 and fru4 males are less stimulated to court females than fru1 and fru2. No courtship pulse song is generated by either fru3 or fru4 males, even though they perform brief wing extensions. fru3 and fru4 males display significantly less chaining behavior than do fru1 males. The hierarchy of courtship responses by fru males directed toward females vs. males, when presented with both sexes simultaneously, is that fru1 males perform vigorous and indiscriminant courtship directed at either sex; fru4 males are similarly indiscriminant, but courtship levels were lower than fru1; fru2 males prefer females; fru3 males show a courtship bias toward males. fru3 and fru4 males essentially lack the Muscle of Lawrence (MOL). On several reproductive criteria, there was no difference between fru-variant females and fru+. The increases in phenotypic severity measured for the new mutants are discussed in the context of the emerging molecular genetics of fru and with regard to the gene's position within the sex-determination pathway.     

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons--the dorsal and ventral lateral neurons--are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that also other cells contribute to the rhythmic control of locomotor activity.     

virilizer regulates Sex-lethal in the germline of Drosophila melanogaster

BACKGROUND AND PURPOSE: Advancing age is associated with declines in motor function; understanding age-related changes in the basal ganglia, therefore, is imperative for comprehension of such functional changes. The purpose of this study was to examine the age, sex, and hemispheric differences in volume of the caudate nucleus, the putamen, and the globus pallidus. METHODS: In a sample of 148 healthy right-handed adults (18-77 years old) with no evidence of age-related motor disorders, we estimated the volume of the head of the caudate nucleus, the putamen, and the globus pallidus from MR images. RESULTS: The analyses revealed bilateral age-related shrinkage of the head of the caudate nucleus and the putamen in both sexes. In men, the age-related shrinkage of the caudate was stronger on the left, whereas, in women, the opposite trend was evident. In both sexes, age-related shrinkage of the right putamen was greater than of its left counterpart. The mild bilateral age-related shrinkage of the globus pallidus was observed only in men. In both sexes, we observed significant rightward asymmetry in the putamen, significant leftward asymmetry in the caudate, and no asymmetry in the globus pallidus. CONCLUSIONS: Bilateral age-related shrinkage of the neostriatum is found in healthy adults. The shrinkage of the globus pallidus is less pronounced and may be restricted to men only.     

Formation of the male pronuclear lamina in Drosophila melanogaster

E. coli homologs of the signal recognition particle (SRP) and its receptor are essential for viability, but their role in protein export is unclear. To elucidate their function, we devised a genome-wide screen to identify genes that encode SRP substrates. Inhibition of the SRP pathway sharply blocked the membrane insertion of several polytopic inner membrane proteins (IMPs) that were predicted to be SRP substrates, but had a smaller effect on the insertion of other IMPs and no significant effect on preprotein translocation. Our results suggest that whereas most E. coli preproteins and some IMPs can utilize SRP-independent targeting pathways effectively, the structural features of a subset of IMPs have required the conservation of an SRP-based targeting machinery.     

The soluble alpha-mannosidases of Drosophila melanogaster

The alpha-mannosidases are implicated in both the catabolism of carbohydrates and the N-linked glycosylation pathway in insects, but little is known of the biochemistry of these glycosidases. In order to study the soluble alpha-mannosidases of Drosophila melanogaster we have used artificial fluorogenic substrates for detection of activity in situ following non-denaturing gel electrophoresis. This approach also permitted examination of the mannosidases present in different tissues and the sensitivity of the enzymes to known mannosidase inhibitors. Fluorogenic substrates were also used to determine the pH optima of partly purified mannosidases. We report that D. melanogaster contains several soluble alpha-mannosidase activities. Acidic mannosidases were detected in the gut, fat body and haemolymph of third-instar larvae. The major activity detected in larval guts was a neutral mannosidase presumed to be involved in digestion.     

Hybrid lethal systems in the Drosophila melanogaster species complex

Lethal phases of the hybrids between Drosophila melanogaster and its sibling species, D. simulans are classified into three types: (1) embryonic lethality in hybrids carrying D. simulans cytoplasm and D. melanogaster X chromosome, (2) larval lethality in hybrids not carrying D. simulans X, and (3) temperature-sensitive pupal lethality in hybrids carrying D. simulans X. The same lethal phases are also observed when either of the two other sibling species, D. mauritiana or D. sechellia, is employed for hybridization with D. melanogaster. Here, we describe genetic analyses of each hybrid lethality, and demonstrate that these three types of lethality are independent phenomena. We then propose two models to interpret the mechanisms of each hybrid lethality. The first model is a modification of the conventional X/autosome imbalance hypothesis assuming a lethal gene and a suppressor gene are involved in the larval lethality, while the second model is for embryonic lethality assuming an interaction between a maternal-effect lethal gene and a suppressor gene.     

Mutation and evolution of microsatellites in Drosophila melanogaster

Levels of nucleotide polymorphism in the Drosophila melanogaster genome are correlated with rates of recombination. This relationship may be due to hitchhiking of advantageous mutations (selective sweeps) or to continual removal of deleterious mutations from the genome (background selection). One test of the relative contributions of selective sweeps and background selection to the observed levels of variation in the genome of D. melanogaster is to compare levels of nucleotide variability (with a mutation rate on the order of 10(-9) per nucleotide per generation) with more rapidly evolving DNA loci such as microsatellites. This test depends critically on details of the mutational process of microsatellites. In this paper, we summarize our studies of microsatellite characteristics and mutation rates in D. melanogaster. We find that D. melanogaster microsatellites are short and have a mutation rate (6.5 x 10(-6) per locus per generation) several orders of magnitude lower than mammals studied to date. We further show that genetic variation at 18 dinucleotide repeat microsatellites in a population of D. melanogaster from Maryland is correlated with regional rates of recombination. These and other microsatellite data suggest that both background selection and selective sweeps may contribute to the correlation between DNA sequence variation and recombination in Drosophila.     

Molecular characterization of hobo-mediated inversions in Drosophila melanogaster

The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-Notch junction fragments indistinguishable in structure from those present in the Uc-1 X chromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobo-Notch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notch junctions present in Uc-1. These results are consistent with a model in which hobo-mediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.     

The transmission of fragmented chromosomes in Drosophila melanogaster

We investigated the fate of dicentric chromosomes in the mitotic divisions of Drosophila melanogaster. We constructed chromosomes that were not required for viability and that carried P elements with inverted repeats of the target sites (FRTs) for the FLP site-specific recombinase. FLP-mediated unequal sister-chromatid exchange between inverted FRTs produced dicentric chromosomes at a high rate. The fate of the dicentric chromosome was evaluated in the mitotic cells of the male germline. We found that dicentric chromosomes break in mitosis, and the broken fragments can be transmitted. Some of these chromosome fragments exhibit dominant semilethality. Nonlethal fragments were broken at many sites along the chromosome, but the semilethal fragments were all broken near the original site of sister-chromatid fusion, and retained P element sequences near their termini. We discuss the implications of the recovery and behavior of broken chromosomes for checkpoints that detect double-strand break damage and the functions of telomeres in Drosophila.     

Genetic dissection of sperm individualization in Drosophila melanogaster

The morphogenesis of spermatids generally takes place within a syncytium, in which all spermatid nuclei descended from a primary spermatocyte remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resolution of the syncytium, or cyst, into individual cells, a process sometimes referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophila spermatids (Tokuyasu, K. T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch 124, 479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms by which this extensive remodeling of the cyst is accomplished. Here, the identification of a major cytoskeletal component of the individualization complex as actin is confirmed with a simple fluorescence assay. Using rhodamine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of highly elongated cysts, then translocating along the length of the cysts. The structure of the individualization complex in a male-sterile clathrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficient phenotype of this mutant. Using the fluorescence assay, a sampling of male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four different phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualization complex around the nuclear bundle, (2) mutations (dud class) in which the individualization complex assembles in/around the nuclear bundle, but fails to translocate down the cyst, (3) mutations (mulet class) that allow the assembly of a morphologically normal individualization complex around the nuclear bundle, but result in a breakdown in the complex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morphologically altered or reduced individualization complex. Individualization also fails in a number of mutants with altered nuclear shape, consistent with the hypothesis that spermatid nuclei provide a physical scaffolding for the assembly of the individualization complex. Genetic analysis suggests that a substantial number of additional loci with phenotypes distinguishable with this assay remain to be identified. The large proportion of male-sterile mutations resulting in a late block to spermatogenesis, in which highly elongated cysts fail to be individualized, suggest a substantial susceptibility of this process to a broad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspondingly complex function requiring exquisite coordination of multiple cytoplasmic functions, and may account for the previously noted high frequency with which Drosophila genes are mutable to male-sterile phenotypes.