Drosophila melanogaster deficient in protein kinase A manifests behavior-specific arrhythmia but normal clock function.

Drosophila melanogaster bearing mutations in the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), displays arrhythmic locomotor activity strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, we examined the protein and mRNA products from the clock gene period (per) in PKA-deficient flies. The per protein (PER) and mRNA products undergo daily cycles in the heads and bodies of DCO mutants that are indistinguishable from those observed in control wild-type flies. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. Our results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors.     

Heat shock during the development of brain structures of Drosophila: the memory development in the l(1)ts403 mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophila and humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Dropping like flies: environmentally induced impairment and protection of locomotor performance in adult Drosophila melanogaster

In Drosophila, heat shock (HS) during the pupal stage chronically hinders adult locomotor performance by disrupting wing development and cellular and/or tissue-level mechanisms that support walking and flight. Furthermore, heat pretreatment (PT) protects locomotor function against these disruptions. HS flies with abnormal wings were less able to alter trajectory in free fall relative to control, PT-only, and PT+HS wild-type flies. This deficit was less severe but still present in HS-only flies with wild-type wings. Transgenic increases in the copies of genes encoding the major inducible heat-shock protein of Drosophila melanogaster, Hsp70, also protected walking ability from disruption due to pupal HS. Walking velocity did not differ between excision (five natural hsp70 copies) and extra-copy (five natural and six transgenic hsp70 copies) flies in the control, PT, and PT+HS groups, nor did velocity vary among these thermal treatment groups. HS dramatically reduced walking velocity, however, but this effect occurred primarily in the excision flies. These results suggest that Hsp70 and other mechanisms protect against heat-induced locomotor impairment.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

Prevention of apoptosis by mitochondrial phosphatase PGAM5 in the mushroom body is crucial for heat shock resistance in Drosophila melanogaster

The heat shock (HS) response is essential for survival of all organisms. Although the machinery of the HS response has been extensively investigated at the cellular level, it is poorly understood at the level of the organism. Here, we show the crucial role of the mushroom body (MB) in the HS response in Drosophila. Null mutants of the mitochondrial phosphatase Drosophila PGAM5 (dPGAM5) exhibited increased vulnerability to HS, which was reversed by MB-specific expression of the caspase inhibitor p35, and similar vulnerability was induced in wild-type flies by knockdown of MB dPGAM5. Elimination of the MB did not affect the HS response of wild-type flies, but did increase the resistance of dPGAM5-deficient flies to HS. Thus, the MB may possess an apoptosis-dependent toxic function, the suppression of which by dPGAM5 appears to be crucial for HS resistance.     

Phototransduction genes are up-regulated in a global gene expression study of Drosophila melanogaster selected for heat resistance

The genetic architecture underlying heat resistance remains partly unclear despite the well-documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64 hours after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance-selected flies. We found 108 genes up-regulated and 10 down-regulated using the Affymetrix gene expression platform. Among the up-regulated genes, a substantial number are involved in the phototransduction process. Another group of genes up-regulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to nonstress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress-sensing mechanisms. In addition, the results suggest yet-uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.     

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.     

Evolution of Senescence: Longevity and the Expression of Heat Shock Proteins

Senescence is the progressive deterioration of organismal functionleading to accelerating rates of mortality. Cumulative extrinsicand intrinsic stresses are thought to contribute to senescence.Molecular chaperones, such as heat shock proteins, are hypothesizedto modulate senescence through their ability to mitigate proteindamage. Recent discoveries made with the nematode Caenorhabditiselegans and the fruit fly Drosophila melanogaster lend strongsupport to this theory. Longevity extending mutants of the nematodealso increase intrinsic and inducible thermotolerance, and theyoverexpress heat shock proteins upon thermal shock. Intriguingly,these genes regulate dauer (diapause) formation, and are associatedwith an insulin-like dependent signal transduction pathway.Direct evidence for a casual role of hsp70 in aging is providedby analysis of transgenic fruit flies. When hsp70 is inducedby mild heat shock, flies that overexpress the protein havegreatly reduced mortality rates during subsequent weeks of agingat normal temperatures. Current work with fruit flies focuseson the relationship between insulin-like receptors, ovariandiapause, heat shock and aging.     

Deficient protein kinase C activity in turnip, a Drosophila learning mutant

The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.     

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.     

Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.     

苯甲醛对果蝇视觉联想记忆的阻断

采用飞行模拟系统,以视觉模式为线索、热惩罚为负强化因子,对于在不同发育时期经受苯甲醛处理过的果蝇的视觉飞行定向条件化进行了检验。苯甲醛气味分别作用于果蝇幼虫和成虫阶段,将阻断果蝇成虫建立视觉联想记忆的能力;雌性果蝇在处女期对苯甲醛气味的接触,会阻断其子代建立视觉联想记忆,这种视觉联想记忆的能力可以通过对其子代连续３代的正常饲养而逐渐得到恢复。     

Heat shock induces changes in the expression and binding of ubiquitin in senescent Drosophila melanogaster

We examined the effect of aging on the expression of ubiquitin RNA and the binding of the ubiquitin polypeptide to proteins following heat shock in Drosophila melanogaster. Heat-shocked adult flies transcribe two major RNA species-one of 4.4 kb and one of about 6 kb that hybridize to the polyubiquitin-encoding probe. Several less abundant RNAs were also observed but the 4.4-kb band was present as the major RNA species in both stressed and nonstressed flies of both ages. The 6-kb fragment was more abundant in heat shocked aged flies than in younger flies. The quantitative expression of the polyubiquitin gene increased in proportion to the duration of the heat stress. Moreover, the induction of the polyubiquitin RNA was markedly elevated during aging following heat shock. Hybridization of Northern blots with the monoubiquitin gene probe revealed a band of 0.9 kb that was not significantly affected by heat stress. We also investigated the relationship between the changes in polyubiquitin gene expression and the formation of ubiquitin-protein complexes in aging heat-shocked flies. Heat shock to old flies results in a significant increase in the level of proteins immunoprecipitated by anti-ubiquitin antibodies. In the case of proteins synthesized 2 h before heat shock, most of the ubiquitinated proteins were of high molecular weight. For those proteins synthesized during a 30-min heat shock and the 2 h following heat shock, two major immunoprecipitated bands were observed: an 80-kD and a 70-kD polypeptide. The ubiquitination of a 60 kD protein was also observed in nonstressed flies, but its for mation was drastically reduced following heat shock. For proteins synthesized during and after heat shock from both age groups, the major ubiquitinated polypeptide is 70 kD. In all age groups, more ubiquitin complexes were formed with proteins synthesized before heat shock, than with proteins synthesized either during or after heat shock. This suggests that cellular proteins synthesized at physiological temperatures are more sensitive to heat induced damage than those synthesized during stress. These data support the hypothesis that in aging flies, heat shock induces an unusually high concentration of abnormal proteins which are targeted for degradation by the ubiquitin-dependent proteolytic system. © 1993Wiley-Liss, Inc.     

Dissociation of visual associative and motor learning in Drosophila at the flight simulator

Ever since operant conditioning was studied experimentally, the relationship between associative learning and possible motor learning has become controversial. Although motor learning and its underlying neural substrates have been extensively studied in mammals, it is still poorly understood in invertebrates. The visual discriminative avoidance paradigm of Drosophila at the flight simulator has been widely used to study the flies' visual associative learning and related functions, but it has not been used to study the motor learning process. In this study, newly-designed data analysis was employed to examine the flies' solitary behavioural variable that was recorded at the flight simulator-yaw torque. Analysis was conducted to explore torque distributions of both wild-type and mutant flies in conditioning, with the following results: (1) Wild-type Canton-S flies had motor learning performance in conditioning, which was proved by modifications of the animal's behavioural mode in conditioning. (2) Repetition of training improved the motor learning performance of wild-type Canton-S flies. (3) Although mutant dunce(1) flies were defective in visual associative learning, they showed essentially normal motor learning performance in terms of yaw torque distribution in conditioning. Finally, we tentatively proposed that both visual associative learning and motor learning were involved in the visual operant conditioning of Drosophila at the flight simulator, that the two learning forms could be dissociated and they might have different neural bases.     

Interaction of hormones in controlling reproductive function of Drosophila females under stressful conditions is genetically determined

The effect of heat stress (38 degrees C) on the content of octopamine (OA) and 20-hydroxyecdysone (20HE) was studied under normal and stressful conditions in adult flies of Drosophila virilis lines contrasting in the level of the juvenile hormone (JH). The wild-type flies (line 101) exhibited a pronounced sex dimorphism for the content of both OA and 20HE, which was substantially lower in this line than in flies of the mutant line 147. The level of both hormones increased in flies of line 101 exposed to heat stress, whereas it remained unchanged in flies of line 147 under the same conditions. The effect of heat stress on the level of JH metabolism and fertility was also studied in D. melanogaster wild-type lines and lines carrying mutations in genes responsible for OA and DA syntheses. In octopamineless females of the T beta hnM18 line and in females of the Ste line characterized by a doubled content of DA, JH degradation differed from normal: it was increased in both young and mature T beta hnM18 females, while decreased in young and increased in mature Ste flies. Fertility was substantially lower in the Ste than in the wild-type line. Flies of all of the D. melanogaster lines produced a stress response; however, in mutant lines, both fertility and stress reactivity of the systems controlling JH metabolism differed significantly from that of the wild-type lines. The role of JH, 20HE, OA, and DA interaction in regulation of Drosophila reproduction under stressful conditions is discussed.     

Heat shock gene activation by mutant actin is independent of myofibril degeneration in Drosophila muscle

Artificially mutagenized Drosophila Act88F actin genes with triple and double mutations were expressed in the indirect flight muscles of transgenic flies. The triple mutant actin, GD245T (Gly-36----Glu, Glu-83----Asp, and Gly-245----Asp), induced heat shock protein (hsp) synthesis without affecting flight ability. On the other hand, the double mutation, GD245D (Gly-36----Glu and Glu-83----Asp), disrupted myofibrils but induced little hsp synthesis. These results demonstrate that myofibril degeneration is not the primary cause of the anomalous heat shock gene activation by mutant actins.