Copia RNA levels are elevated in dunce mutants and modulated by cAMP

Clones carrying sequences expressed at altered abundance levels in dunce mutants were isolated by differentially screening a genomic library with cDNA probes representing the RNA population from dunce+ flies and the RNA population from dunce mutant flies. These mutants have an elevated cAMP content, so some isolates potentially contain cAMP responsive genes. Two classes of clones were isolated. One class contains genes expressed at a higher steady state abundance level in dunce mutants compared to dunce+ flies and the other contains genes expressed at a lower steady state level in the mutants. The recovery of clones from the differential screen demonstrates that in addition to altering normal behavior, fertility, and cAMP metabolism, dunce mutation confers an alteration in the level of expression of certain genes. The class of clones carrying sequences which are overexpressed in the mutants have been characterized. These clones carry a common repetitive sequence which codes for a 5.5 kb poly(A)+ RNA - the RNA species found to be overexpressed in the mutants. Restriction analysis and hybridization experiments show these repetitive sequences to be members of the copia family of transposable elements. Administration of pharmacological agents to normal flies to increase cAMP levels leads to an increased steady state level of copia RNA. Thus, copia RNA metabolism appears to be influenced by cAMP levels.     

Isolation of the Drosophila melanogaster dunce chromosomal region and recombinational mapping of dunce sequences with restriction site polymorphisms as genetic markers.

Using the method of chromosomal walking, we have isolated a contiguous region of the Drosophila melanogaster X chromosome which corresponds to salivary gland chromosome bands 3C12 to 3D4. This five-band region contains approximately 100 kilobases of DNA, including those sequences comprising dunce, a gene which functions in memory and cyclic nucleotide metabolism. Genome blots of DNA from flies carrying several different chromosomal aberrations with breakpoints in the region have been probed with the isolated clones to map the breakpoints on the cloned DNA and to delimit dunce sequences. This has localized dunce to a 50-kilobase region. In addition, we have searched this 50-kilobase region for restriction site polymorphisms between X chromosomes from different Drosophila strains by genome blotting experiments, and we have followed the segregation of detected polymorphisms and dunce alleles after meiotic recombination. The data map one dunce mutation between two polymorphisms located 10 to 12 kilobases apart.     

Two Drosophila learning mutants, dunce and rutabaga, provide evidence of a maternal role for cAMP on embryogenesis

The dunce gene of Drosophila melanogaster encodes a cAMP-specific phosphodiesterase (form II). Mutant dunce flies have elevated levels of cAMP and exhibit a number of defects including learning deficiencies and female sterility. Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells.     

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.     

Sexual Hyperactivity and Reduced Longevity of dunce Females of Drosophila melanogaster

The dunce gene of Drosophila melanogaster codes for a cyclic adenosine-3',5'-monophosphate-specific phosphodiesterase. Mutations of dunce alter or abolish the activity of this enzyme, produce elevated cAMP levels, cause recessive female sterility, and produce learning deficiencies in both sexes. Aberrant male sexual behavior has also been associated with the memory defects of dunce mutants. Here we show that the longevity of dunce mutant females, homozygous for null-enzyme alleles, is reduced by 50% in the presence of males compared to control dunce females kept without males. Mutant dunce females, mate every 22-24 hr. We propose a cause-effect relationship between mating and reduced longevity. Pheromones or peptides transferred during mating may activate adenylate cyclase and create an increase in cAMP levels that cannot be damped in dunce females. This increase may affect basic physiological functions and lead to reduced longevity.     

Effect of dunce and amnesiac genes on signal learning in Drosophila melanogaster: experiments with odors]

The behavior of normal Drosophila and of X-linked olfactory conditioning mutants, dunce and amnesiac, was analyzed using an olfactory search task. Normal (C-S) flies quickly learn and remember which of two odors signals the presence of food and they are capable of retaining this information for at last eight hours. Both dunce and amnesiac mutants are able to learn, whereas mutant dunce do not reach the learning level of wild type C-S flies. Also dunce flies require more than one learning trial for sizeable learning effect. Reversal learning experiments showed that normal C-S flies and amnesiac are able to switch to a new food signal in response to a new experience, while the dunce mutation inhibits the acquisition of new information in reversal learning experiments.     

Gprk2 controls cAMP levels in Drosophila development

G protein-coupled receptors mediate their biological responses through the generation of second messengers, such as cAMP. The down-regulation of their activity (desensitization) is carried out, in part, by the family of G protein-coupled receptor kinases, which phosphorylate activated receptors. The Gprk2 gene in Drosophila melanogaster is a putative member of this family. The GPRK2 protein is expressed most abundantly in the ovaries and in the mushroom bodies, the brain region that is implicated in learning and memory in insects. Many of the genes that are involved in learning in Drosophila are members of a cAMP-signaling pathway and are also expressed in the mushroom bodies. These observations suggest that the Gprk2 gene may be involved in a cAMP-mediated pathway. To investigate this possibility, we tested for a genetic interaction between Gprk2 and dunce (which encodes cAMP-specific phosphodiesterase). A mutant allele of Gprk2, called gprk2(6936), has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2(6936). Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2(6936) background suppressed all of these defects to a significant degree. Suppression was also observed when a single copy of gprk2(6936) was introduced into a dunce background. Like mutants of rutabaga (which encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2(6936) has reduced levels of cAMP. Ovaries from gprk2(6936) females contain about one third of the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels.     

Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system

The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.     

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster

The protein phosphorylation patterns of wild type and dunce mutant strains of Drosophila melanogaster, as detected by sodium dodecylsulfate-gel electrophoresis and autoradiography, have been compared. After labelling in vivo with 32Pi or in vitro in homogenates with [gamma-32P]ATP, radioactive bands at and above apparent polypeptide mol. wt approximately 110,000 were more pronounced in dunce fly heads than in wild type heads. When labelling in vitro, in dunceM11 there appeared a radioactive band at apparent mol. wt approximately equal to 53,000 that was faintly visible in the wild strain. The same band could be intensified in both strains by adding cyclic AMP to the homogenate or by performing homogenization in the presence of theophylline. The data suggest that the mol. wt approximately equal to 53,000 protein is a substrate for cyclic AMP-dependent protein kinase.     

Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila

The rutabaga and dunce genes, encode two enzymes of the cyclic adenosine monophosphate transduction pathway in Drosophila, adenylyl cyclase and cyclic adenosine monophosphate phosphodiesterase, respectively. Two main second messenger systems, depending on inositol 1,4,5-triphosphate and cyclic adenosine monophosphate, have been associated with olfaction in vertebrates as well as invertebrates. A relationship between the cyclic adenosine monophosphate signaling pathway and olfactory reception in Drosophila is suggested by the presence of cyclic nucleotide gated channels and cyclic-nucleotide modulated K+ channels in the antennae, the main olfactory organs. In this report, molecular, electrophysiological and behavioral data support the role of cyclic adenosine monophosphate in olfactory function for this species. Expression of both genes in the antennae has been shown by messenger ribonucleic acid analysis. Changes in the electroantennogram kinetics have been observed specifically on the slope of the initial rising phase, as predicted for processes that affect cyclic adenosine monophosphate concentration. Olfactory behavior changes due to both mutations were coherent with a functional meaning of the reported electrophysiological phenotype in olfactory perception. Sensitivity level increases or decreases for the mutants compared to the control line depending on the odorant. These results are compatible with some olfactory coding at the reception level by differential activation of a dual transduction system involving the inositol 1,4,5-triphosphate and cyclic adenosine monophosphate cascades.     

The cyclic AMP system andDrosophila learning

Molecular and Cellular Biochemistry - The cyclic AMP (cAMP) system plays a critical role in olfactory learning in the fruit fly,Drosophila melanogaster, as evidenced by the following: [1] The dunce...     

SSB, an antigen that selectively labels morphologically distinct synaptic boutons at the Drosophila larval neuromuscular junction.

In this report we describe the expression of Small Synaptic Bouton (SSB), an antigen that is selectively expressed in a specific subset of neuromuscular junction terminals in the body wall of Drosophila larva. The expression of SSB was studied with a polyclonal antibody raised against the cAMP phosphodiesterase of the Drosophila learning mutant dunce (Nighorn et al., 1991, Neuron 6:455-467); however, immunoreactivity was not abolished by the dunce (dnc) alleles dncM14 and dncM11 or deficiencies of the dnc gene, indicating that the antigen labelled could not be the dnc gene product, but another antigen that we termed SSB. Immunoreactivity was localized in the body wall muscles to a specific subset of neuromuscular junction terminals that have been implicated in activity-dependent plasticity. This demonstrates that these morphologically distinct terminals can be immunocytochemically distinguished and that they probably represent innervation by a distinct neuronal population. Confocal and electron microscopic examination demonstrated that staining was restricted to the synaptic boutons themselves, not to neurites or motor axons. Ultrastructural analysis showed label close to synaptic vesicles in the presynaptic terminal and in the surrounding subsynaptic reticulum. Central nervous system (CNS) staining was restricted to a segmentally repeated pattern of cell bodies in the ventral ganglion and to a few small groups of cells in the brain lobes.     

Cooperation between Drosophila flies in searching behavior

In Drosophila melanogaster food search behaviour, groups of flies swarm around and aggregate on patches of food. We wondered whether flies explore their environment in a cooperative way as interactions between individual flies within a population might influence the flies' ability to locate food sources. We have shown that the food search behavior in the fruit fly Drosophila is a two-step process. Firstly, 'primer' flies search the environment and randomly land on different food patches. Secondly, the remaining group of flies move to the most favorable food source and aggregate there. We call this a 'search–aggregation' cycle. Our data demonstrate that flies do not individually assess all available food resources. Rather, social interactions between flies appear to affect their choice of a specific food patch. A genetic analysis of this 'search–aggregation' behavior shows that flies carrying mutations in specific genes (for example, the dunce ( dnc ) gene which codes for a phosphodiesterase) were defective in this search–aggregation behavior when compared to normal flies. Future investigations of the neuronal signaling involved in this behavior will help us to understand the complexities of this aspect of Drosophila social behaviour.     

Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.     

Synaptic ultrastructure in nerve terminals of Drosophila larvae overexpressing the learning gene dunce

We investigated synaptic ultrastructure of individual nerve ending varicosities at the Drosophila larval neuromuscular junction in transgenic larvae overexpressing the learning gene dunce (dnc) in the nervous system. It was previously shown that cAMP is reduced to one-third normal in these larvae and that they have fewer nerve terminal varicosities and smaller junction potentials, although transmitter release from individual nerve ending varicosities is not significantly altered. We tested the hypothesis that synaptic ultrastructure is modified to compensate for possible reduced efficacy of synaptic transmission resulting from lower than normal cAMP. Synaptic size and number of presynaptic dense bodies (active zone structures) per synapse are modestly enhanced in transgenic larvae overexpressing the dnc gene product and in rutabaga (rut(1)) mutant larvae, which have reduced adenylyl cyclase activity and reduced neural cAMP. The incidence of complex synapses (possessing 2 or more presynaptic dense bodies) was not consistently different in experimental larvae compared to controls. The observations suggest that chronic reduction of cAMP levels in the nervous system of Drosophila larvae, although leading to a modest compensatory change in synaptic structure, does not markedly alter several synaptic ultrastructural parameters which are thought to influence the strength of transmitter release; thus, homeostatic mechanisms do not act to maintain normal-sized junction potentials by altering synaptic structure.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.