Julian M. Crampton

Genetic transformation of the mosquito Aedes aegypti by micro-injection of DNA

ABSTRACT. We report the successful introduction of heterologous DNA sequences into embryos of the mosquito Aedes aegypti (L.) by microinjection. The injected DNA carrried P transposable element sequences, derived from and known to facilitate transformation in Drosophila melanogaster. Two plasmids, one of which carried a dominant selectable marker, were introduced into the posterior of embryos prior to pole cell formation and subsequently taken up into the germ line of transformed individuals. Stable transfer of the selectable marker (G418 resistance) was demonstrated over two generations. The precise nature of these putative P mediated integration events is currently being investigated. However, the results presented here establish the technique of DNA transformation for the genetic manipulation of Aedes aegypti.

Evolution of Disintegrin Cysteine-Rich and Mammalian Matrix-Degrading Metalloproteinases: Gene Duplication and Divergence of a Common Ancestor Rather Than Convergent Evolution

The evolution of the Metalloproteinase Disintegrin Cysteine-rich (MDC) gene family and that of the mammalian Matrix-degrading Metalloproteinases (MMPs) are compared. The alignment of snake venom and mammalian MDC and MMP precursor sequences generated a phylogenetic tree that grouped these proteins mainly according to their function. Based on this observation, a common ancestry is suggested for mammalian and snake venom MDCs; it is also possible that gene duplication of the already-assembled domain structure, followed by divergence of the copies, may have significantly contributed to the evolution of the functionally diverse MDC proteins. The data also suggest that the structural resemblance of the zinc-binding motif of venom MDCs and MMPs may best be explained by common ancestry and conservation of the proteolytic motifs during the divergence of the proteins rather than through convergent evolution.

Transgenic mosquitoes: A future vector control strategy?

Transgenic mosquitoes may provide a new way of dealing with the old problem of diseases transmitted by insects. Although many technical, and perhaps ethical, problems associated with the wild-release of transgenic insects have yet to be overcome, Julian Crampton and colleagues explore the potential of this technology in the continuing battle to control insect-borne disease.

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

The Aedes aegypti genome: complexity and organization

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2s. A striking feature of the cDNA is the high sequence conservation of the 5′ and 3′ untranslated regions in cDNAs coding for PLA2s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2s.

Genetic manipulation of insect vectors as a strategy for the control of vector-borne disease

A variety of very effective methods have been employed for suppressing insect vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess alternative strategies for vector control. What is required is a completely novel approach to either suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for genome manipulation in insects and the progress which has been made to create transgenic vector insects are reviewed. The potential applications of this methodology are then explored in the context of its future use for the control of vector-borne diseases.

Gene expression in Echis carinatus (carpet viper) venom glands following milking

RNA was extracted from the venom glands of Echis carinatus at different times after milking, and the temporal pattern and nature of mRNA transcribed during venom regeneration was examined by in vitro translation. Venom products were immunoprecipitated with E. carinatus venom polyclonal antiserum. Maximum transcriptional activity occurred 3 days after milking. Electrophoretic analysis of the translation products showed minimal differences in the banding patterns at each time interval. Analysis of the translation products from Kenyan, Nigerian and Saudi Arabian carpet vipers, however, revealed differences which suggest that the observed heterogeneity in E. carinatus venom occurs at the level of the genome.

The changing response of Plasmodium falciparum to antimalarial drugs in East Africa

For the past 20 years, chloroquine chemotherapy has been the single most effective malaria control measure in East Africa. The advent of chloroquine-resistant Plasmodium falciparum has reduced the clinical effectiveness of chloroquine and this trend is likely to continue. Combinations of antifol drugs are at present effective inhibitors of most P. falciparum infections in the region, in spite of widespread resistance to pyrimethamine. The development of (i) sensitive methods for monitoring changes in sensitivity to antifol combinations, (ii) more effective and less costly alternatives to commercially available combinations, and (iii) investigation of host and parasite factors leading to drug treatment failure in P. falciparum infections has been the primary goal of the Wellcome Trust Research Laboratories in Kenya (directed by Dr W.M. Watkins) within the malaria programme of the Kenya Medical Research Institute, and collaborating laboratories at the School of Tropical Medicine and the University of Liverpool.