Charles W. Roth

Genome sequence of Aedes aegypti, a major arbovirus vector

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at ∼1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of ∼4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of ∼2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.

Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae.

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Molecular analysis of multiple cytochrome P450 genes from the malaria vector, Anopheles gambiae

Cytochrome P450s are a superfamily of haemoproteins, important in the metabolism of endogenous compounds and xenobiotics. As a first step to elucidating the role of this family in insecticide resistance in the malaria mosquito, Anopheles gambiae, we have cloned and mapped multiple P450 genes. Sixteen cDNAs encoding full‐length P450s were cloned and physically mapped to the mosquitos polytene chromosomes. Fourteen of these encode putative CYP6 proteins and two encode P450s belonging to the CYP9 class. Eighteen new A. gambiae Cyp4 P450 genes were identified using degenerate PCR primers, cDNAs were detected for ten and in situ locations for thirteen members of this gene family.

Characterization and detection of plant trypanosomatids by sequence analysis of the small subunit ribosomal RNA gene

The complete sequences of the genomic small subunit ribosomal RNA gene from two Phytomonas isolates: one associated with palm pathologies (P. cocos FGuiana) and one found in lactiferous plants with no apparent pathology (P. Euphorbe Senegal), were analyzed. Partial sequences from a number of other Phytomonas isolates were also determined. The sequences obtained were used to determine the phylogenetic relationships between Phytomonas and other trypanosomatids as well as within the genus Phytomonas. The analysis showed that the intraphloemic isolates associated with pathologies in palm trees formed a homogeneous group that diverged from the more heterogeneous group of non-pathogenic isolates found in latex plant. Sequence comparisons of the full and partial SSU rRNA gene, identified sequences which are specific to the genus Phytomonas and an EcoRI restriction nuclease site which specifically identifies the Phytomonas isolates associated with diseases in palm trees.

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion.

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160 000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune‐related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

The Drosophila melanogaster multidrug-resistance protein 1 (MRP1) homolog has a novel gene structure containing two variable internal exons.

Drosophila melanogaster has a gene very similar to human MRP1 that encodes a full ABC-transporter containing three membrane-spanning domains and two nucleotide-binding domains. This 19 exon insect gene, dMRP (FBgn0032456), spans slightly more than 22 kb. The cDNA SD07655 representing this gene was sequenced and found to contain sequences from 12 exons including single copies of two exons having multiple genomic copies. The gene contains two variant copies of exon 4 and seven of exon 8. While a cDNA contains only one version of each variable exon, all forms of these variable exons were detected in adult fly mRNA. These results predict that Drosophila could make 14 different MRP isoforms from a single gene by substituting different variable exons. This is the first report of any organism using differential splicing of alternative, internal exons, to produce such a large array of MRP isoforms having the same size, but with limited and defined internal variations. Defining the functional differences in the dMRP isoforms should provide clues to the structure/function relationships of the amino acids in these MRP domains, both for the insect enzyme and for those of other species.

Pilot Anopheles gambiae full-length cDNA study: sequencing and initial characterization of 35,575 clones

We describe the preliminary analysis of over 35,000 clones from a full-length enriched cDNA library from the malaria mosquito vector Anopheles gambiae. The clones define nearly 3,700 genes, of which around 2,600 significantly improve current gene definitions. An additional 17% of the genes were not previously annotated, suggesting that an equal percentage may be missing from the current Anopheles genome annotation.

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry.

BACKGROUND Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.