Melanogaster Mutants Research Articles

Inheritance Features of Mating Behavior Components in Drosophila melanogaster and Their Significance for Fitness

Components of mating behavior of Drosophila melanogaster mutant and wild-type strains were studied with respect to fitness. The magnitude of the effect of genotype on the male mating activity, female sexual receptivity, fertility and viability was determined. Strong positive correlation was found between the male mating activity and fitness components. It was shown that mating of strains contrasting in sexual behavior features can be accompanied by both heterosis and maternal effect. Inheritance coefficients were determined for sexual behavior components.

Mutations in insulin signaling pathway alter juvenile hormone synthesis in Drosophila melanogaster

Juvenile hormone (JH) is a key endocrine regulator of insect metamorphosis, reproduction, and aging. The synthesis of JH is regulated by neuropeptides and biogenic amines, but the molecular and cellular basis of this control remains largely unknown. Genetic analysis of JH synthesis in Drosophila melanogaster mutant for insulin signaling may provide new and powerful insights. Mutants of the insulin receptor ( InR) are slow to develop, small, infertile, and long-lived. We previously reported that mutants of InR had reduced JH synthesis as young adults, and that normal longevity and vitellogenesis were restored by topical application of a JH analog [Science 292 (2001) 107]. Here, we describe the 10-day adult age course of JH synthesis from isolated corpus allatum (CA) of InR and of chico, the insulin receptor substrate homolog. JH synthesis increased in wildtype flies to a maximum of 30 fmol/gland/h at day 10. In contrast, homozygous InR mutants produced no more than 3 fmol/gland/h JH within the first 5 days, and only 7 fmol/gland/h at day 10. InR mutation disproportionately reduced the synthesis of JH III-bisepoxide, the major JH subtype of the fly. Mutation of chico also reduces body size and extends longevity [Science 292 (2001) 104; Aging Cell 1 (2002a) 75]. Both homozygous and heterozygous chico genotypes reduced JH synthesis, but only to 47 and 67%, respectively, of wildtype and without influencing the ratio of JH subtypes. Because JH synthetic rate does not correlate with the size of CA, it is not likely that insulin signaling mediates JH by impeding endocrine tissue development. Alternatively, we find allatotropin-positive axons to be abundant in the adult brain and in the corpora cardiaca–corpus allatum complex but these neurons are less immunoreactive in the InR mutant genotype, suggesting that insulin signaling may affect JH synthesis through control of JH regulatory neuropeptides.

Expression of genes encoding proteins involved in ecdysteroidogenesis in the female mosquito, Aedes aegypti

A blood meal induces the ovaries of female Aedes aegypti mosquitoes to produce ecdysteroid hormones that regulate many processes required for egg maturation. Various proteins involved in the intracellular transport and biosynthesis of ecdysteroid precursors have been identified by analysis of Drosophila melanogaster mutants and by biochemical and molecular techniques in other insects. To begin examining these processes in mosquito ovaries, complete cDNAs were cloned for putative orthologs of diazepam-binding inhibitor ( DBI), StAR-related lipid transfer domain containing protein ( Start1), aldo/keto reductase ( A/KR), adrenodoxin reductase ( AR), and the cytochrome P450 enzymes, CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase) and CYP314a1 (20-hydroxylase). As shown by RT-PCR, transcripts for all seven genes were present in ovaries and other tissues both before and following a blood meal. Expression of these genes likely supports the low level of ecdysteroids produced in vitro (7–10 pg /tissue/ 6 h) by tissues other than ovaries. Ovaries from females not blood fed and up to 6 h post blood meal (PBM) also produced low amounts of ecdysteroids in vitro, but by 18 and 30 h PBM, ecdysteroid production was greatly increased (75–106 pg /ovary pair/ 6 h) and thereafter (48 and 72 h PBM) returned to low levels. As determined by real-time PCR analysis, gene transcript abundance for AedaeCYP302 and AedaeCYP315a1 was significantly greater (9 and 12 fold, respectively) in ovaries during peak ecdysteroid production relative to that in ovaries from females not blood fed or 2 h PBM. AedaeStart1, AedaeA/KR and AedaeAR also had high transcript levels in ovaries during peak ecdysteroid production, and AedaeDBI transcripts had the greatest increase at 48 h PBM. In contrast, gene transcript abundance of AedaeCYP314a1 decreased PBM. This study shows for the first time that transcription of a few key genes for proteins involved in ecdysteroid biosynthesis is positively correlated with the rise in ecdysteroid production by ovaries of a female insect.

Chemically Induced Drosophila melanogaster Mutants with Changes in Brain Structure

Neurodegenerative human diseases are caused by nerve cell death and anatomical changes in some brain regions. Molecular genetic studies of Drosophila showed that this organism can serve as a valuable test-system for conserved mechanisms underlying human nervous system disorders. Analysis of brain functions is possible when the mutants with disturbed functions are available. In this study, we have developed a unique collection of Drosophila melanogaster mutants with morphological and neurodegenerative changes in brain structure, which were induced by chemical mutagens.

Drosophila long-term memory formation involves regulation of cathepsin activity.

Whereas short-term memory lasts from minutes to hours, long-term memory (LTM) can last for days or even an entire lifetime. LTM generally forms after spaced repeated training sessions and involves the regulation of gene expression, thereby implicating transcription factors in the initial steps of LTM establishment. However, the direct participation of effector genes in memory formation has been rarely documented, and many of the mechanisms involved in LTM formation remain to be understood. Here we describe a Drosophila melanogaster mutant, crammer (cer), which shows a specific LTM defect. The cer gene encodes an inhibitor of a subfamily of cysteine proteinases, named cathepsins, some of which might be involved in human Alzheimer's disease. The Cer peptide was found in the mushroom bodies (MBs), the Drosophila olfactory memory centre and in glial cells around the MBs. The overexpression of cer in glial cells but not in MB neurons induces a decrease in LTM, suggesting that Cer might have a role in glia and that the concentration of the Cer peptide is critical for LTM. In wild-type flies, cer expression transiently decreases after LTM conditioning, indicating that cysteine proteinases are activated early in LTM formation.

Male-specific expression of the fruitless protein is not common to all Drosophila species.

Sex-specific behavioral patterns must be a result of sexual differences in the structure and/or function of the central nervous system (CNS). Male Drosophila melanogaster mutants for the fruitless (fru) locus exhibit enhanced male-to-male courtship. The fru mutant males are accompanied by malformation of the male-specific muscle of Lawrence (MOL), which, in wild-type males, is induced by male motoneurons innervating it. These two phenotypes are the consequences of impaired sex determination of CNS neurons. In D. melanogaster, although the fru mRNAs are transcribed in the CNS of both the male and female, the Fru protein is only translated in the male CNS. This male-specific translation of Fru was also observed in D. simulans, D. yakuba, D. pseudoobscura and D. virilis; however, in D. suzukii, the Fru protein expression was detected even in the female CNS.

Нейродегенеративні мутанти Drosophila melanogaster: отримання, фенотипові особливості, тривалість життя

Нейродегенеративні мутанти Drosophila melanogaster: отримання, фенотипові особливості, тривалість життя

X-linked small GTPase and OXPHOS genes are candidates for the genetic basis of hybrid inviability in Drosophila.

Genetic studies on postmating reproductive isolation in Drosophila have suggested that the genetic basis of hybrid inviability is much less complex than the basis of hybrid sterility, and may be associated with defects affecting the cell cycle. Here I report the identification of a cluster of genes in the middle of the X chromosome of D. melanogaster, which may be responsible for the inviability of hybrids between Drosophila species. Genes from this cluster code for small Ras GTPases proteins, which are hypothesized here to interact with proteins involved in oxidative phosphorylation (OXPHOS), encoded by genes present within the same cluster. At least six genes influencing small Ras GTPases/OXPHOS activity are transcribed from the same strand across 35 kb genomic DNA. This interval is predicted to harbor genes which, when mutated, rescue otherwise inviable hybrids between D. melanogaster and its three most closely related species. Moreover, a total of 16 small GTPase/OXPHOS genes are found within 530 kb genomic DNA encompassing the above cluster. In D. melanogaster mutants which fully rescue lethal hybrids, major lesions have now been identified very near or within untranslated regions of two OXPHOS genes from the above cluster. These observations led to a hypothesis focusing on antagonistic co-evolution between biparentally inherited genes influencing putative GTPase/OXPHOS activity and mitochondrial genes encoding OXPHOS proteins. Alterations in some of these genes are postulated to override hybrid inviability, thus revealing a pathway which implicates mitotic genes as critical players in this barrier to reproduction.

Sensitized Increase of Period Gene Expression in the Mouse Caudate/Putamen Caused by Repeated Injection of Methamphetamine

Methamphetamine (MAP) causes the sensitization phenomena not only in MAP-induced locomotor activity, dopamine release, and Fos expression, but also in MAP-induced circadian rhythm. Cocaine-induced sensitization is reportedly impaired in Drosophila melanogaster mutant for the Period (Per) gene. Thus, sensitization may be related to induction of the Per gene. A rapid induction of mPer1 and/or mPer2 in the suprachiasmatic nucleus after light exposure is believed to be necessary for light-induced behavioral phase shifting. Although the caudate/putamen (CPu) expresses mPer1 and/or mPer2 mRNA, the function of these genes in this nucleus has not yet been elucidated. Therefore, we examined whether MAP affects the expression of mPer1 and/or mPer2 mRNA in the mouse CPu. Injection of MAP augmented the expression of mPer1 but not mPer2 or mPer3 in the CPu, and this MAP-induced increase in mPer1 expression lasted for 2 h. Also, the MAP-induced increase of mPer1 mRNA was strongly antagonized by pretreatment with a dopamine D1 receptor and N-methyl-D-aspartate (NMDA) receptor antagonist, but not by a D2 receptor antagonist. Interestingly, application of either the D1 or the D2 agonist alone did not cause mPer1 expression. The present results demonstrate that activation of both NMDA and D1 receptors is necessary to produce MAP-induced mPer1 expression in the CPu. Repeated injection of MAP caused a sensitization in not only the locomotor activity but also mPer1 expression in the CPu without affecting the level of mPer2, mPer3, or mTim mRNA. Thus, these results suggest that MAP-induced mPer1 gene expression may be related to the mechanism for MAP-induced sensitization in the mouse.

Phenotypic association of spontaneous ovulation and sexual receptivity in virgin females of Drosophila melanogaster mutants.

In Drosophila melanogaster females, mating stimulates ovulation/oviposition and switches off receptivity. To investigate the relationship between ovulation and receptivity, we searched for genetic variants in which ovulation occurs in virgins and characterized their behavioral phenotype. Among a collection of 333 P-element insertion lines, we identified eight lines that showed elevated ovulation in virgins. These females show ovipositor extrusion toward courting males, which is normally observed in mated females. To express the amount of rejection behavior, we defined the extrusion index (EI) as a percentage of time that each female extruded the ovipositor within the total time of being courted. There was a positive correlation between ovulation level and EI, suggesting that the two traits are physiologically associated. Genetic analysis of the variants revealed two regions on the third chromosome responsible for the phenotype.

Very Long-chain Acyl-CoA Synthetases: HUMAN “BUBBLEGUM” REPRESENTS A NEW FAMILY OF PROTEINS CAPABLE OF ACTIVATING VERY LONG-CHAIN FATTY ACIDS

Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids. Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities. The most recently identified family is the very long-chain acyl-CoA synthetases (VLCS). Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins. It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases. Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity. Northern blot analysis showed that hsBG is expressed primarily in brain. Compared with vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates. Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation. Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others. Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound. Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active. hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acyl-CoA synthetases revealed that hsBG along with the D. melanogaster and murine homologs comprise a new family of acyl-CoA synthetases. Thus, two protein families are now known that contain enzymes capable of activating VLCFA. Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis.

Complex alterations of the ribosomal gene spacers in mutant sc 8 of Drosophila melanogaster

Extreme changes in the proportions of the rDNA intergenic spacers (IGSs) have previously been demonstrated by us in the X-chromosomal rDNA array of the Drosophila melanogaster mutant sc8, where nearly all IGSs were found to be reduced in size. We have cloned different structural variants of the Xsc8 ribosomal units and mapped their spacers by restriction endonuclease analysis. Most of the IGSs exhibited complex rearrangements within the subrepeated region at their 5' terminus. The sequence data revealed the presence of an additional 100 bp subrepeat. In addition, extended deletions/substitutions down to the 18S gene were also found. Examination of the Ysc8 IGS polymorphism indicates that some of the X-chromosomal IGS variants are probably the result of X-Y interchanges. The rarity of alternative recombination products implies that the overabundant deleted spacers in sc8 are generated by nonreciprocal recombination. Our results demonstrate nonrandom distribution of deletional breakpoints within the "1900" region in sc8 and show that the deletions do not truncate the internal IGS subregions at either breakpoint but eliminate them as discrete blocks.

Autosomal mutations in Drosophila which reduce operant learning ability.

A collection of Drosophila melanogaster mutants was created by insertion of a P-element into autosomes at a rate of one copy per genome. The abilities of 64 homozygous P-insertion mutants to produce a form of associative behavior were determined. Testing was based on an original paradigm of operant learning: interactions between Drosophila individuals when placed in a group situation in which the flies learned to inhibit their own activity to avoid punishment in the form of conflict with other individuals. Four lines were found in which, like the known learning mutants dunce and rutabaga, individuals did not show changes in their initial responses to each other. These lines were also studied using other paradigms of associative learning.

An eye-specific G beta subunit essential for termination of the phototransduction cascade.

Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes: (Drosophila mutant/profilin/gametogenesis/bristle).

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee. We named the allele chickadeebin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadeewc57 and WF57 (2). In addition, the egg chamber of chickadeebin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.

Selective neuroanatomical plasticity and division of labour in the honeybee.

The mushroom bodies in the protocerebrum are believed to be the structures of the insect brain most closely associated with higher-order sensory integration and learning. Drosophila melanogaster mutants with olfactory learning deficits have anatomically abnormal mushroom bodies or altered patterns of gene expression in mushroom body neurons. In addition, anatomical reorganization of the mushroom bodies occurs in adult flies, and possibly in adult honeybees; disturbance of electrical activity in this region disrupts memory formation in honeybees. Little is known, however, about the relationship of naturally occurring anatomical changes in the mushroom bodies to naturally occurring behavioural plasticity. We now report that age-based division of labour in adult worker honeybees (Apis mellifera) is associated with substantial changes in certain brain regions, notably the mushroom bodies. Moreover, these striking changes in brain structure are dependent, not on the age of the bee, but on its foraging experience, thus demonstrating a robust anatomical plasticity associated with complex behaviour in an adult insect.

Relationship between catalase activity, life span and some parameters associated with antioxidant defenses in Drosophila melanogaster

This study was conducted on Drosophila melanogaster mutants with different levels of catalase activity in order to assess the role of antioxidant defenses in the aging process. We present here the analysis of two mutant strains: the cat n1 / cat n4 heterozygote which exhibits no detectable catalase activity and the cat n2 homozygote which exhibits approximately 14% that of the parent reference strain. Since insects lack glutathione peroxidase activity, catalase activity provides the sole enzymatic mechanism for the removal of H 2O 2. Average and maximum life spans of flies were unaffected by the absence or low levels of catalase activity. The mutants however exhibited adaptive responses in their metabolic rate or glutathione content. The metabolic rate of flies was significantly lowered in the null mutants. Glutathione concentration tended to increase in flies with the hypomorphic catalase allele (exhibiting 14% of the normal catalase activity). Gamma-glutamylcysteine synthetase activity was significantly higher in the null flies. Activities of superoxide dismutase and glutathione reductase were unaffected. Results of this study indicate that 14% of the normal level of catalase activity allows flies to achieve both a normal life span and a normal metabolic potential. Small decreases in certain antioxidant defenses, frequently observed during aging, may be functionally not very consequential.

Genetic and biochemical characterization of little isoxanthopterin (lix), a gene controlling dihydropterin oxidase activity in Drosophila melanogaster

Dihydropterin oxidase catalyses the oxidation of 7,8-dihydropteridines into their fully oxidized products, and is involved in the biosynthesis of isoxanthopterin. Fifteen Drosophila melanogaster mutants, selected for their low pterin and isoxanthopterin content, were assayed for dihydropterin oxidase activity. The activity was around 100% in most mutants tested, slightly reduced in red, g and dke, and undetectable in lix. In flies carrying various doses of the lix+ allele, a correlation was found between enzyme activity and the number of lix+ copies in the genome. The results suggest that lix is the structural gene for the dihydropterin oxidase enzyme. Isoxanthopterin was quantitated in strains carrying deficiencies for the region in which lix has been mapped by recombination. This allowed us to assign the lix locus to the 7D10-7F1-2 segment of the X chromosome.

Lack of response to family selection for directional asymmetry inDrosophila melanogaster: left and right are not distinguished in development

Family selection for directional asymmetry in the expression of the Drosophila melanogaster mutant scute had no result. Fluctuating asymmetry did not show a selection differential correlated with directional asymmetry. The unfolding of bilateral symmetry in embryogenesis can be used to explain the lack of genetic variation for directional asymmetry. Directional asymmetry provides a well-understood example of a developmental constraint in evolution. It is proposed that as no evidence is available for an independent left-right gradient in the embryo, quantitative traits can only be expressed variably along an existing gradient of positional information or a morphogen.

Encapsulation of parasitoid eggs in phenoloxidase-deficient mutants of Drosophila melanogaster

Eggs of the parasitoid Leptopilina boulardi, strain L104, are routinely encapsulated by haemocytes in Drosophila melanogaster larvae, and the capsules subsequently melanize. In D. melanogaster mutant strains deficient for phenoloxidase activity, L104 eggs are encapsulated by host haemocytes but the cellular capsules do not melanize and harden. These observations suggest that phenoloxidases are not essential for recognition of nonself and encapsulation of foreign objects in D. melanogaster, but they are required for blackening and hardening of haemocytic capsules.