Search

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3- parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3- ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3- sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3. more...

International audience; A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families. more...

AnoBase (http://www.anobase.org) is an integrated, relational database of basic biological and genetic data on anopheline species, with a particular emphasis on Anopheles gambiae. It has been designed as an information source and research support tool for the broad vector biology community. Although AnoBase is not a primary genomic database that develops and provides tools to access the genome of the malaria mosquito, it nevertheless contains several sections that offer data of genomic interest such as in situ hybridization images, an integrated gene tool and direct online access to AnoXcel, the proteomic database of An. gambiae. Moreover, AnoBase also contains information on non-gambiae mosquito species and a novel section on studies related to insecticide resistance. more...

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium. more...

Using an in vitro development system for Plasmodium berghei sporogonic stages and microarray technology we examined parasite gene expression during ookinete invasion of Aedes cells and the ensuing oocyst development. A number of genes were found to be differentially expressed. The most prominent class of up-regulated elements corresponded to products involved in protein synthesis and metabolism. Furthermore, several previously studied genes with a known in vivo developmental profile matched published data. A large number of genes with a hitherto unknown function during the life cycle stages studied also show a differential pattern of expression, indicating the involvement of their products in control and execution of active developmental processes. more...

Of the RNA labelled after incubation of hepatoma cells with radioactive precursors for 20 and 150 min. 35% and 70%, respectively, can be isolated from nuclei by two consecutive extractions with 0.14 M NaCl at pH 8. The isolated RNA is complexed with nuclear proteins forming structures with sedimentation coefficients of less than 30 S to greater than 100 S. Similar complexes from rat liver isolated under the same experimental conditions show coefficients of 30-40 S. The RNA-associated proteins are similar, on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis, to the respective proteins of other cell types. The presence on these RNP complexes of six discrete small nuclear RNAs (snRNA) has been established. Experiments with a reversible inhibitor of RNA synthesis, D-galactosamine, demonstrated, differences in the turnover of hnRNA and snRNA. The half-lives of the six snRNA species has been determined, varying from 32 h for snRNA species a, b and d, to 22 h for snRNA species e and f and to 13 h for snRNA species c. Treatment of the nuclear extracts with 0.7 M and 1 M NaCl results in dissociation of hnRNA from the 'core' and other polypeptides, whereas snRNA remains complexed with polypeptides of Mr 54 000-59 000. Incubation of the nuclear extracts at 0 C with low doses of pancreatic R Nase (up to 1.5 micrograms/ml), which renders approximately 80% of the hnRNA acid-soluble and cleaves most of the snRNA, results in conversion of the high-molecular-weight hnRNPs to 30-S structures, without disrupting the 30-S RNP. Treatment of the nuclear extracts with higher doses of RNase (3 micrograms/ml) leads to disruption of the 30-S RNP and release of the hnRNA-associated proteins, underlining the importance of hnRNA-protein interaction for the retainment of the hnRNP structures. more...

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes. more...

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed. more...

The AGER gene encoding the epidermal growth factor receptor (EGFR) of the malaria mosquito Anopheles gambiae was cloned and sequenced. It represents a canonical member of this family of tyrosine kinase proteins exhibiting many similarities to orthologues from other species, both on the level of genomic organization and protein structure. The mRNA can be detected throughout development. Western analysis with an antibody raised against the extracellular domain of the mosquito protein suggests developmental variation in protein size and location that may be involved in the function of EGFR in the mosquito. more...

We have isolated an mRNA encoding a beta integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to betaPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete. more...

The sequence of a 2.3 kb long DNA segment derived from the 5′-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako. more...

The complete sequence of the mitochondrial genome of Ceratitis capitata has been determined. The circular genome is 15 980 bp long and contains a standard gene complement, i.e. the large and small ribosomal RNA subunits, twenty-two transfer RNA (tRNA) genes and thirteen genes encoding mitochondrial proteins. When comparing the sequence to fragments previously sequenced from other isolates it becomes apparent that interstrain polymorphisms are not rare. These differences are potentially useful for the development of diagnostic tools for population analysis applications, such as determining the source of recent introductions. Moreover, they could help obtain a solution to the long-lasting controversy on the possible eradication of the Medfly from certain locations. more...

VectorBase, an integrated, relational database that manages genomic and other genetic/biological data pertaining to arthropod vectors of disease, has recently embarked on the construction of ontologies and controlled vocabularies (CVs). It aims, thus, at providing all necessary tools for the complete annotation of vector genomes and, in particular, the annotation of functional genomic data. This task was initiated with the development of anatomical ontologies of mosquitoes and ticks, both of which were made compliant to CARO, the common anatomy reference ontology. The ontologies are complemented by the development of novel web-based browsers that can show figures for anatomical terms, something that is especially helpful for fully illustrating the controlled vocabularies of anatomy. more...

Three genes encoding serine proteases (Sp6A, Sp6T and Sp8T) were isolated from the malaria mosquito An. gambiae. The proteins that are conceptually translated from these genes contain all amino acids that have been described for this class of proteolytic enzymes, namely the His, Asp and Ser residues at the active site, and the six cysteine residues that form the three disulphide bridges in invertebrate serine proteases. The genes are expressed at low levels and the transcripts were detected only by PCR. Analysis of the nucleotide sequences of the three genes and their pattern of expression indicate that none of the genes code for digestive enzymes, but rather that the proteins have features of the tethered type of serine proteases. more...

Extracellular stages of apicomplexan parasites utilize their own actin myosin motor machinery for gliding locomotion, penetration of cell barriers, and host cell invasion. Thus far, filamentous actin could not be visualized by standard microscopic techniques in vivo. Here, we describe the generation of a novel peptide antibody against the divergent amino-terminal portion of the major Plasmodium isoform, actin I. We show that our antiserum, termed Ab-actinI-I, is conformation-specific. In motile ookinetes it recognizes actin in rod-like structures, which are sensitive to inhibitors interfering with actin polymerization. The average size of the rods is 600 nm, which is considerably longer than what has been detected in in vitro studies of actin filaments. more...

Actin-related proteins (Arps) constitute a family of eukaryotic cytoskeletal proteins involved in such diverse events as cell motility, cytokinesis, vesicle transport, and chromatin remodelling. Previously, in a study of Plasmodium berghei gene expression in ookinetes and oocysts, we detected stage-specific increased expression of a gene encoding an Arp. Here we further characterize this gene and the encoded protein. We present a phylogenetic and three-dimensional modelling analysis as well as cell biological and genetic data that support classification of this gene as being an orthologue of the actin-related protein 1 (Arp1). This gene was found to be expressed in asexual stages as well as in the mosquito stages of the parasite, both on the transcript and protein level. Our attempts to delete the gene in the parasite for functional studies were unsuccessful, suggesting that it may be essential. The protein was localized apically of the nucleus in ookinetes, and in combination with the known function of Arp1 proteins, this suggests a role in vesicular transport. Expression of the gene in Saccharomyces cerevisiae resulted in toxic effects and interference with the yeast cytoskeleton. more...

We have isolated theDrosophila melanogaster locus coding forl-glutamate dehydrogenase (EC 1.4.1.3) by virtue of its similarity to the corresponding human gene. There is only one copy of this gene in the fruit fly genome, located on the right arm of chromosome 3 (95D1-4). The transcript includes at least one large intron and matures to a ∼2.4-kb-long polyadenylated RNA whose expression is under developmental control. more...

International audience; A large scale microarray (20k MMC1) from the African malaria vector Anopheles gambiae was used to monitor gene expression in insecticide resistant and susceptible strains of the Asian mosquito Anopheles stephensi. Heterologous hybridization at slightly reduced stringency yielded ∼7000 significant signals. Thirty-six putative genes were differentially transcribed between the pyrethroid-resistant (DUB-R) and the susceptible (BEECH) strains. The expression profiles of selected transcripts were verified by real-time PCR. A gene putatively involved in the thickening of the adult cuticle showed the most striking up-regulation in DUB-R. A more specialized microarray containing 231 An. gambiae genes putatively involved in insecticide detoxification was used to further analyse classical insecticide resistance genes. Three glutathione S-transferase (GST) transcripts, one esterase and a cytochrome P450 were up-regulated in the resistant strain, while two peroxidases were down-regulated. more...

Ookinetes of the genus Plasmodium are motile, invasive cells that develop in the mosquito midgut following ingestion of a parasite-infected blood meal. We show here that ookinetes display gliding motility on glass slides in the presence of insect cells. Moreover, in addition to stationary “flexing” and “twirling” of the cells, two distinct types of movements occur: productive forward translocational motility in straight segment that progresses with an average speed of approximately 6 μm/min and rotational motility, which does not lead to forward translocation. Locomotion is reduced by treatment with butanedione monoxime, an inhibitor of myosin ATPase, and by three different actin inhibitors. We also studied the expression during ookinete development of genes encoding actin and two small class XIV myosins, PbMyoA, and PbMyoB. Western immunoblots revealed that PbMyoA is only present in fully mature ookinetes, whilst the other two proteins are additionally expressed in gametocytes and zygotes. Immunofluorescence experiments reveal that MyoA and actin co-localize in the apical tip of the parasite whereas MyoB displays a punctate pattern of expression around the entire cell periphery. Following treatment with jasplakinolide, the apparent level of detectable actin appears to substantially increase and becomes concentrated in a discrete area in the basal pole of the ookinete. more...

Contains fulltext : 35755.pdf (Publisher’s version ) (Closed access) Mosquitoes use effective immune responses, including phagocytosis, to fight microbial infection. Here we show that in an Anopheles gambiae immune responsive cell line, RGD recognizing receptors play an important role in the phagocytic response, suggesting overlap between molecular components implicated in adhesion and phagocytosis. Integrins are a major class of adhesive receptors that recognize ligands containing an RGD motif. We have cloned a gene encoding a new beta integrin, BINT2, and demonstrated its involvement in Escherichia coli engulfment. Based on molecular modeling, we propose a structural reason for the role of BINT2, but not BINT1, on phagocytosis of Gram-negative bacteria. Using bioinformatic tools, we have identified and compared the complete A. gambiae integrin repertoire as a prelude to a future systematic functional study. more...

Annexins belong to a class of proteins that are known to bind to, and hold together structures such as membranes. Interestingly, Anopheles gambiae (and Drosophila melanogaster) annexins bind Plasmodium ookinetes in vitro. In the malaria mosquito three genes in two cytogenetic loci on chromosome arm 2R encode annexin homologues; their expression, monitored by quantitative real-time PCR during mosquito development, as well as in various tissues, revealed little fluctuation in patterns of expression during all life stages. A different mode of transcription was observed for the three genes in the midgut in relation to the uptake of a blood meal. Immunohistochemical staining of midguts and ovaries with polyclonal anti-annexin sera reveals that the Anopheles polypeptides are present in the epithelial cells of both tissues and associated with the plasma membrane. more...

SUMMARY During the invasion of the mosquito midgut epithelium, Plasmodiumookinetes come to rest on the basal lamina, where they transform into the sporozoite-producing oocysts. Laminin, one of the basal lamina's major components, has previously been shown to bind several surface proteins of Plasmodium ookinetes. Here, using the recently developed RNAi technique in mosquitoes, we used a specific dsRNA construct targeted against the LANB2 gene (laminin γ1) of Anopheles gambiae to reduce its mRNA levels, leading to a substantial reduction in the number of successfully developed oocysts in the mosquito midgut. Moreover, this molecular relationship is corroborated by the intimate association of developing P. berghei parasites and laminin in the gut, as observed using confocal microscopy. Our data support the notion of laminin playing a functional role in the development of the malaria parasite within the mosquito midgut. more...

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium-Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland-specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue-specific expression in mosquito female salivary glands. more...

The proteome of the mosquito Anopheles gambiae was organized on a hyperlinked spreadsheet format containing one protein per row and several pieces of information in each column. The information for each protein ranges from the presence or absence of signal peptide indicative of secretion, presence of transmembrane domains, similarities to several databases, chromosomal location, and relatedness to other An. gambiae proteins, etc. Hosted by AnoBase (http://www.anobase.org/), the whole spreadsheet or segments of it can be downloaded or searched from http://www.anobase.org/AnoBase/Genes/Ano-Xcel by the scientist dealing with the annotation of proteome subsets such as those deriving from transcriptomes, nucleotide microarrays or high throughput mass spectrometry data. more...

This review examines what is presently known of the molecular interactions between Plasmodium and Anopheles that take place in the latter's midgut upon ingestion of the parasites with an infectious blood meal. In order to become 'established' in the gut and to transform into a sporozoite-producing oocyst, the malaria parasite needs to undergo different developmental steps that are often characterized by the use of selected resources provided by the mosquito vector. Moreover, some of these resources may be used by the parasite in order to overcome the insect host's defence mechanisms. The molecular partners of this interplay are now in the process of being defined and analyzed for both Plasmodium and mosquito and, thus, understood; these will be presented here in some detail. more...

Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst. In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin gamma 1 polypeptide and follow its expression during mosquito development. To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system. Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro. Laminin gamma 1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants. This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin gamma 1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector. more...

The saliva of blood-feeding arthropods contains an apyrase that facilitates hematophagy by inhibiting the ADP-induced aggregation of the host platelets. We report here the isolation of a salivary gland-specific cDNA encoding a secreted protein that likely represents the Anopheles gambiae apyrase. We describe also two additional members of the apyrase/5′-nucleotidase family. The cDNA corresponding to the AgApyL1 gene encodes a secreted protein that is closely related in sequence to the apyrase of the yellow fever mosquito, Aedes aegypti, and whose expression appears enriched in, but not restricted to, female salivary glands. The AgApyL2 gene was found searching anA. gambiae data base, and its expression is restricted to larval stages. We isolated the gene encoding the presumed A. gambiae apyrase (AgApy) and we tested its putative promoter for the tissue-specific expression of the LacZgene from Escherichia coli in transgenic Drosophila melanogaster. All the transgenic lines analyzed showed a weak but unambiguous staining of the adult glands, indicating that some of the salivary gland-specific transcriptional regulatory elements are conserved between the malaria mosquito and the fruit fly. The availability of salivary gland-specific promoters may be useful both for studies on vector-parasite interactions and, potentially, for the targeted tissue-specific expression of anti-parasite genes in the mosquito. more...

I begin by stating that I have not regretted coming back to Greece after 17 years of working abroad and that I have no plans to seek employment in another country. I still enjoy both living in and doing science in Greece. Having said that, let me say that most non-Greeks to whom I describe my life as a researcher tend to see in me a clear-cut case of schizophrenia.How can one plan anything in a country where, over the past 15 years, there have been 10 or so different Secretaries-General for Research and Technology and a slightly larger number of Ministers of Education? How can one do research when the annual funds allocated to the universities for that purpose arrive in August, or even later (if ever)? How can one hope to obtain the necessary funds if, in order for a research grant proposal to be accepted, one has almost always to include a partner from the private sector, even on the most basic science projects? And this, in a country in which the private industry hardly invests any money in development. The solution to these and other similar problems was provided years ago by Nikos Kazantzakis, Greece's most famous modern writer: act like Zorba the Greek — be ebullient.Unpredictability is the most prominent characteristic of modern Greek life, and science is no exception. As a matter of fact, unpredictability is itself so predictable that most of us have learned to live with it. For example, there are always transport strikes during the days before Christmas or Easter, so you plan your travel accordingly. There are always power failures in Crete in the winter months, so you use sophisticated back-up systems to ensure that your sequencer will be running uninterrupted. You know that a letter from Greece to Brussels will take anything from one day to two weeks, so you use an overnight courier when you need to meet a grant deadline. You not only learn to cope with these ‘simple’ problems, but you also wonder why foreigners can't.Are these the only problems encountered? Obviously not, but I don't think it would help to write a catalogue of examples of how things should not be. Several of these problems are not unique to Greek scientists; indeed, some of them have already been mentioned in this column by my colleagues of different nationalities when they described the situation in their own home countries.There is the ever-present problem of the brain drain towards the United States and other more developed countries, affecting all levels of science from graduate students to established researchers. There are the usual complaints about some particular rules of European Union research funding — coping with these often requires a very good sense of humour. And the biggest problem is the exhaustion arising from the constant need to overcome these difficulties. This fatigue leads many excellent scientists to feel so defeated that they're likely to give up the struggle — for ever.Am I really schizophrenic when I say that I enjoy my job, then? I don't think so. Things do improve in Greece. There might still be four ministries that are responsible for research but, although for several years the complete lack of communication between them was a fact of life, the Secretariat General for Research and Technology (SGRT) will soon be heading a coordination committee. Furthermore, the SGRT is perhaps the least bureaucratic Government agency and the Secretary General was, before his appointment, an active researcher himself. The fact that he's been in office for more than two years, an aeon by Greek standards, offers the hope of a degree of consistency in the Government's policies.Moreover, structural funds from the European Union, co-administered by national agencies, provide the opportunity to acquire equipment that could not otherwise be purchased. In addition, there is also a small but measurable influx of young postdocs who take advantage of some of the European Union's mobility programmes. These postdocs come to work in some of the already existing excellent Greek laboratories, which help act as a nucleation core as well as reversing, to some extent, the brain drain. All of these facts, combined with several everyday improvements in Greek life, will help, sooner or later, to bring research in my country much closer to what are considered to be ‘international standards’.As I said, I still enjoy it here. And if something unpredictable happens that will prevent me from working for a few hours, well, the beach is 10 minutes drive from the lab and you can reach the snow in less than an hour. There are not many places that offer these advantages and Zorba the Greek could only thrive here! more...

Control of malaria by a methodology that would permit the effective blockage of the Anopheles gambiae midgut wall penetration by Plasmodium parasites requires a detailed understanding of both the physiology of the mosquito's digestion, and of the interactions between the parasite and its host. We have transformed Drosophila melanogaster with several constructs that allow the study of the promoter region of two of the major late trypsin genes of A. gambiae. Using several deletions, we have identified, for both genes, small genomic segments that are sufficient to confer tissue specificity to the promoter in a species that is far away in evolution from the mosquito. This will allow further studies that will enable both the understanding of the blood meal digestion, and may potentially be useful for the design of anti-plasmodial constructs at a later stage. more...

Different strains of transgenic Drosophila melanogaster carrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against the white gene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, a D. melanogaster strain, deficient in eye pigmentation caused by a deletion of the white gene, was used for P element-mediated germline transformation: the transposon used contained the hsp70-driven, white-directed ribozyme gene and, on the same DNA, the mini-white gene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon. more...

We have transformed Drosophila melanogaster with a DNA construct encoding a catalytic antisense RNA directed against the primary white gene RNA transcript. Total RNA isolated from transformed flies was shown to catalyze the specific cleavage of in vitro transcribed substrate RNA, indicating the expression of a functional ribozyme. Moreover, transgenic lines carrying homozygous copies of this construct depict a further clear-cut reduction in eye pigmentation when present in a genetic background that has a priori reduced levels of white gene expression. more...

Two spontaneous mutations of rudimentary, the gene encoding the first steps of de novo pyrimidine biosynthesis in Drosophila, are suppressed by mutant alleles of the suppressor of Hairy-wing locus. This interaction differs from typical su(Hw) suppression in that neither rudimentary allele is associated with an insertion of the gypsy retrotransposon. One allele, rsP1, appears to be a point mutation. Adult rsP1 homozygous females accumulate substantially less 7.3 kb rudimentary transcript than do wild-type females. The other allele, rsP2, is an insertion of an mdg3 retrotransposon in the sixth exon of rudimentary and in the opposite transcriptional orientation. This insertion divides the rudimentary locus into two separate, yet functional, transcription units by truncating transcription from the rudimentary promoter and promoting transcription of downstream rudimentary sequences. Phenotypic suppression of both rsP1 and rsP2 by mutant alleles of the suppressor of Hairy-wing locus correlates with enhanced levels of the rsP1 and rsP2 transcripts. more...

The 5S gene cluster of Drosophia melanogaster is a tandem array of a repeat unit made up of a 135 bp gene plus a 238 bp spacer. The length of the 5S repeat (373 bp) equals the average length of a Drosophila dinucleosome. Digestion of Drosophila nuclei with micrococcal nuclease generates discrete 5S RNA gene subfragments when the purified DNA is further cleaved with a single-cut restriction enzyme. We have mapped four micrococcal nuclease-sensitive sites within the 5S repeat: A1, centered at bp −110 (+1 being the G:C bp at the start of the gene); A1′, at bp −80; A2, at bp +80, within the intragenic control region; and B, at −190 bp. These findings suggest that nucleosomes can be positioned on the 5S gene repeat in one of two possible phases, A or B. In the A phase a potential regulatory sequence near the center of the gene is exposed in one of the two linkers of the repeat. In the B phase, in contrast, one of the linkers includes the 5′ end of the gene. more...

In Drosophila melanogaster the rudimentary locus encodes the first three enzymes in de novo pyrimidine biosynthesis: CPSase, ATCase and DHOase. rudimentary is located towards the proximal end of the X chromosome at the cytogenetic locus 15A1. A variety of genetic and molecular studies have suggested that all three enzymatic activities are encoded in a single transcription unit whose mRNA is translated into a multifunctional polypeptide with a molecular weight of approximately 220,000 D. In this paper we describe the isolation of a 90 kb D. melanogaster DNA segment which contains the rudimentary gene. This was achieved by screening a D. melanogaster genomic phage library with a cDNA probe homologous to the corresponding genetic unit of hamster (pyr 1–3). One of the phage recombinants homologous to this hamster probe, BS313 was found to be derived from the 15A1 region of the X chromosome. Starting from BS313 we “walked” in both directions along the X chromosome to isolate the entire rudimentary locus plus flanking DNA segments. To positively identify the rudimentary gene we in situ hybridized the various phage recombinants obtained in this walk to an inversion chromosome that has one breakpoint at 15A1 which inactivates the gene. The phage immediately adjacent to BS313 was found to span the inversion breakpoint and we were able to determine the orientation of the phage walk on the basis of this in situ hybridization result. Finally we indentify a portion of the rudimentary mRNA coding region on the basis of homology first to the hamster cDNA probe and to total Drosophila RNA and second, to a 7.3 kb long poly A+ transcript which is found both in adult flies and in embryos. more...

We have examined the chromatin organization of the Drosophila melanogaster ribosomal RNA genes using both micrococcal nuclease and DNase I. Several findings are of interest. First, the transcribed DNA segments of the rRNA repeat unit appear to be packaged into an unstable or "multiphasic" nucleosome structure. Second, the 5' end of the transcription unit is preferentially exposed to nuclease attack. Third, the non-transcribed spacer immediately upstream from the transcription start site has a novel chromatin organization with micrococcal nuclease and DNase I cleavage sites spaced at intervals of about 240 base-pairs. This unusual fragment distribution appears to reflect the underlying sequence organization of the spacer DNA segment, which consists of a series of tandemly repeated 239 base-pair sequence blocks. We have also examined the chromatin structure of the rRNA repeat unit after extraction of nuclei with different concentrations of salt. Our results suggest that the higher order structures may be of importance in determining the novel chromatin organization of the rRNA repeat unit. more...

P strains of Drosophila melanogaster are characterized by the presence of both full-length and deletion derivatives of the transposable element P in their genome, and by their ability to induce the syndrome of hybrid dysgenesis among the progeny of certain intra-strain crosses, when introduced through the male parents. In contrast, strains belonging to the M' class, and which were also found to bear P element-homologous sequences, lack this ability and this has been attributed to the presence in the genome of most of these strains of a distinct class of deletion derivatives termed KP, which can suppress the action of functional P factors. Here we demonstrate that KP elements are present, next to full-length ones, in the genome of at least three strains which induce P-M-like dysgenic symptoms, including GD sterility. KP elements form the majority of the P-homologous sequences in the strains MR-h12, 23.5/CyL4 and the latter's derivative 23.5*/Cy. While the first one is a genuine P strain and the second one depicts a strong P cytotype, the third is a genuine M' strain. The hybrid dysgenesis induced by the two 23.5 MRF strains seems to be due, not primarily to the P elements, but to the action of hobo elements. more...

1. Introduction In previous papers [l-3] , we have presented evidence that the nuclear RNP-particles carrying the HnRNA (informoferes) [4] are composed, in addition to proteins and HnRNA, also of RNA species with a slower turnover. These conclusions were reached on the basis of double labelling of the RNA with pre- cursors [l-3] and of RNAase digestion of the RNA of the informoferes [ 1,2] as well as on the effects of inhibitors of RNA synthesis on the RNA moiety of the RNP-particles 131. In the present report we demon- strate by acrylamide gel electrophoresis the existence in informoferes of small molecular weight RNAs, which represent the metabolically more stable RNA species of the RNP-particles. 2. Materials and methods 2.1. more...

In this communication, we report the isolation of seven new recombinants derived from the 5S gene locus of Drosophila melanogaster. These recombinants can be divided into three different classes. There are four clones derived from within the 5S gene cluster, two which contain non-5S sequences representing the 5'flanking segment of the gene array and have a structure similar to 12D8 described by Artavanis-Tsakonas et al. (1977), and finally one (22A8) which is shown to contain a DNA segment that is located adjacent to the 3' end of the 5S gene cluster. Analysis of these recombinants supports the model in which all 5S genes of our D. melanogaster Oregon-R wild type strain are arranged in one uninterrupted cluster and are transcribed in the same direction. Interestingly, the 2.5 kb of non-5S RNA coding sequences on the recombinant derived from the 3' edge of the cluster contains at least four genes coding for tRNAs and one of these is located less than 300 bp downstream from the last 5S transcription unit. These tRNA genes are shown to be functional on the basis of the ability of 22A8 DNA to direct the synthesis of tRNA in an in vitro transcription system. more...

In the studies reported here, we have examined the molecular organization of the rudimentary gene of Drosophila melanogaster. rudimentary encodes a 220,000 Mr polypeptide catalyzing the first three steps in pyrimidine biosynthesis: carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase. We have determined the direction of transcription of the gene relative to the genetic map of the locus. The 5' end maps to the distal end of the locus (relative to the centromere) and transcription proceeds through the domains encoding dihydroorotase, carbamyl phosphate synthetase and finally aspartate transcarbamylase. The rudimentary transcription unit spans a DNA segment of 13.2 X 10(3) base-pairs and encodes a mature messenger RNA of 7.3 X 10(3) base-pairs. Three intervening sequences have been identified, one of which is over 4 X 10(3) base-pairs in length. Finally, we have compared the DNA sequence organization of the Drosophila rudimentary gene with the corresponding loci of yeast and hamster. more...

The molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlates well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females. more...

RAPD analysis technique is used as a rapid and reliable tool for genome analysis in the malaria vector Anopheles gambiae. Using more than eighty different commercially available primers we identified more than sixty different DNA segments that were differentially amplified in different strains of An. gambiae s.s. and An. arabiensis. An estimate of the cytogenetic position of these markers is provided by their hybridization to divisional dot-blot filters. Potentially useful RAPD markers can be cytogenetically mapped with more precision by in situ hybridization and, as they segregate as dominant markers in a Mendelian fashion, they can also be genetically mapped relative to other genes or rearrangements. Finally, we identified markers for their potential use in the identification of different mosquito strains. more...