Neuropathology in Drosophila membrane excitability mutants

Mutations affecting ion channels and neuronal membrane excitability have been identified in Drosophila as well as in other organisms and characterized for their acute effects on behavior and neuronal function. However, the long-term effect of these perturbations on the maintenance of neuronal viability has not been studied in detail. Here we perform an initial survey of mutations affecting Na+ channels and K+ channels in Drosophila to investigate their effects on life span and neuronal viability as a function of age. We find that mutations that decrease membrane excitability as well as those that increase excitability can trigger neurodegeneration to varying degrees. Results of double-mutant interactions with dominant Na+/K+ ATPase mutations, which themselves cause severe neurodegeneration, suggest that excitotoxicity owing to hyperexcitability is insufficient to explain the resultant phenotype. Although the exact mechanisms remain unclear, our results suggest that there is an important link between maintenance of proper neuronal signaling and maintenance of long-term neuronal viability. Disruption of these signaling mechanisms in any of a variety of ways increases the incidence of neurodegeneration.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.     

Postreplication repair defects in mutants of Drosophila melanogaster

Summary Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with ³H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brainganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].Abbreviations MMS methyl methanesulfonate - HN2 nitrogen mustard - AAF 2-acetylaminofluorene - AAAF N-acetoxy-2-acetylaminofluorene - DMSO dimethyl sulfoxide     

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.     

Searching for sleep mutants of Drosophila melanogaster

The functions of sleep are still unknown, but are probably related to cellular and molecular aspects of neural function. To better understand the benefits that sleep may bring at the cellular level, recent studies have employed Drosophila melanogaster as a model system and shown that fruit flies share the fundamental features of mammalian sleep. As in mammals, sleep in Drosophila is characterized by increased arousal threshold and by changes in brain electrical activity. Fly sleep is homeostatically regulated independent of the circadian clock, is modulated by stimulants and hypnotics, and is affected by age. Also, fly sleep is associated with changes in brain gene expression similar to those observed in mammals. While Drosophila neurobiology is sufficiently complex to permit meaningful generalizations to mammals and humans, Drosophila genetics is simple enough to allow a rapid mutagenesis screening. An ongoing mutagenesis study has screened approximately 5000 mutant Drosophila lines and found that sleep amount, sleep pattern, and the homeostatic regulation of sleep are highly conserved phenotypes in flies. So far, this study has identified 10 short sleeper lines and 4 lines that show no sleep rebound after sleep deprivation. Ultimately, the characterization of these lines should help identifying crucial cellular pathways involved in the regulatory mechanisms of sleep and its functional consequences.     

Coagulation and survival in Drosophila melanogaster fondue mutants

Drosophila larval coagulation factors have been identified in vitro. Better understanding of insect hemolymph coagulation calls for experiments in vivo. We have characterized a fondue (fon) mutation and null alleles isolated by imprecise excision of a transposable element. Loss of fon was pupal lethal, but adults could be recovered by expressing the UAS::fonGFP construct of Lindgren et al. (2008). Despite their lethality, fon mutations did not affect larval survival after wounding either when tested alone or in combination with a mutation in the hemolectin clotting factor gene. This reinforces the idea of redundant hemostatic mechanisms in Drosophila larvae, and independent pleiotropic functions of the fondue protein in coagulation and a vital process in metamorphosis.     

Mutagen-sensitive mutants in Drosophila melanogaster: effects on premutational damage.

Drosophila melanogaster males from a Basc stock were mutagenized with either X-rays, ethyl methanesulfonate (EMS), or nitrogen mustard (HN2). Groups of identically treated males were crossed to different types of female. Sex-linked recessive lethals were scored as a genetic end point. The females used were homozygous for X-chromosomal mutations (mus(1)101D1, mus(1)104D1, mei-9 or mei-41D5) which lead to defective DNA repair and which increase the mutagen sensitivity of larvae. Females from a white stock with normal DNA repair capacities served as controls. The premutational lesions induced in mature sperm are only processed after insemination by the maternal enzyme systems present in the oocytes. Differences in the efficiency of the processing of lesions can lead to maternal effects on the frequency of mutations recovered from mutagenized sperm. It was found that, with the exception of mus(1)104D1, all mutants analysed significantly modify the mutation fixation of one or more types of premutational lesions. The most drastic effect is found with the mus(1)101D1 stock in which HN2-induced DNA cross-links do not lead to sex-linked recessive lethals. It is assumed that mus(1)101D1 is defective in an early step of DNA cross-link repair. Our first set of data clearly demonstrates that the study of maternal effects in Drosophila is an efficient tool to analyse the in vivo function of repair mutations on chemically induced mutagenesis.     

Evolutionary consequences of altered atmospheric oxygen in Drosophila melanogaster

Twelve replicate populations of Drosophila melanogaster, all derived from a common ancestor, were independently evolved for 34+ generations in one of three treatment environments of varying PO(2): hypoxia (5.0-10.1 kPa), normoxia (21.3 kPa), and hyperoxia (40.5 kPa). Several traits related to whole animal performance and metabolism were assayed at various stages via "common garden" and reciprocal transplant assays to directly compare evolved and acclimatory differences among treatments. Results clearly demonstrate the evolution of a greater tolerance to acute hypoxia in the hypoxia-evolved populations, consistent with adaptation to this environment. Greater hypoxia tolerance was associated with an increase in citrate synthase activity in fly homogenate when compared to normoxic (control) populations, suggesting an increase in mitochondrial volume density in these populations. In contrast, no direct evidence of increased performance of the hyperoxia-evolved populations was detected, although a significant decrease in the tolerance of these populations to acute hypoxia suggests a cost to adaptation to hyperoxia. Hyperoxia-evolved populations had lower productivity overall (i.e., across treatment environments) and there was no evidence that hypoxia or hyperoxia-evolved populations had greatest productivity or longevity in their respective treatment environments, suggesting that these assays failed to capture the components of fitness relevant to adaptation.     

Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster.

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.     

Developmental defects of female-sterile mutants of Drosophila melanogaster

Gans et al. (1975) isolated female-sterile mutants, viable and normal in the homozygous state, producing apparently normal eggs which were unable to develop or developed into defective embryos or adults, even when fertilized with wild-type sperm. Developmental abnormalities of these mutants were surveyed by observing living and fixed material. The following types of mutants were distinguished according to the predominant developmental defect: (i) Eggs not developing at all, mostly remaining unfertilized. (ii) Eggs stopping development after a few cleavages, some with polyploid nuclei. (iii) Eggs stopping development at various embryonic stages, with haploid nuclei. (iv) Eggs with abnormal blastoderm, not developing further or giving abnormal embryos. (v) Eggs with abnormal gastrula, in one case with excessive invaginations, in another case with germ band failing to elongate. (vi) Eggs with embryos dying at different stages, without easily visible effects. Several of the mutants were temperature sensitive. In all the mutants there were eggs that died without developing. Most of the developmental defects appear to be due to general metabolic disturbances of the egg, not directly related to morphogenesis. There were no mutants affecting determination of a particular adult organ. The closest to a morphological type of mutation were those with abnormal blastoderm having successive bands of nuclei of different sizes. Those mutants were thermosensitive; at permissive termperatures they developed into agametic adults or adults with various defects of abdomens, wings or eyes. The nature of maternal genetic control of early morphogensis was discussed.     

Pyrimidine biosynthesis in the dumpy mutants of Drosophila melanogaster

Summary The status of de novo pyrimidine synthesis in the dp mutant of Drosophila melanogaster was examined by measuring the activity of the rate-limiting orotate phosphribosyl transferase (OPRT) enzyme. Activity is significantly elevated in late third instar larvae of 5 different dp mutant strains. A more detailed analysis of a dp ^ovc allele has shown that this elevation arises at about mid-larval life and persists until pupation.A low nucleotide diet causes a depression in OPRT activity in dp ^ovc larvae which can be reversed by dietary supplementation of uracil. However, neither the low nucleotide diet nor uracil supplementation results in a change in the expressivity of the dp mutant phenotypes.Changes in expressivity are produced by 6-azauracil and by elevated temperature although, in those cases, the effect on OPRT activity is minimal.The significance of the observations is discussed in relation to the role of pyrimidine biosynthesis in dp expressivity and chitin synthesis.