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Biomedical subjects

A A James

Publications and source records attributed to A A James.

At least 37 records · Page 2Linked to original sources
PubMed2000

Plasmodium falciparum: generation of a cDNA library enriched in sporozoite-specific transcripts by directional tag subtractive hybridization.

We have adapted the "directional tag subtractive hybridization" technique as a means of investigating stage-specific gene expression in Plasmodium falciparum. This technique utilizes unidirectional cDNA libraries cloned into separate lambda vectors and involves hydroxyapatite chromatographic separation of target antisense cDNA and driver sense strand cRNA followed by PCR amplification of cDNA sequences specific to the target stage. This technique enabled efficient subtraction of asexual blood stage sequences from a P. falciparum sporozoite cDNA library and led to identification of novel sporozoite sequences. This technique can be applied to study gene expression in parasite stages that are difficult to obtain routinely.

Animals↗
PubMed2000

Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti.

Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.

Aedes↗
PubMed2000

Structure of hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti.

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8-bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut-and-paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT-like element in the mosquito genome.

Aedes↗
PubMed2000

Expression patterns of the larval and adult hexamerin genes of Musca domestica.

Hexamerins are proteins found in high abundance in the haemolymph of larval and adult insects. The expression patterns of the genes encoding the house fly, Musca domestica, hexamerins were determined by Northern analyses using cDNAs as probes. A cDNA, A1, hybridized to a fat body-specific messenger RNA (mRNA) which is detectable in larvae until pupation. Antibodies raised to the larval-specific hexamerin, Hex-L, bind recombinant protein encoded by a 5' rapid amplification of cDNA ends (RACE) product of A1, A2, indicating that the A cDNAs likely represent the genes encoding Hex-L. The F1, F2 and F3 cDNAs, corresponding to genes encoding an adult, female-enriched hexamerin, Hex-F, hybridized with an mRNA isolated from protein-fed females which has a temporal expression profile similar to that observed for the accumulation of Hex-F. Furthermore, expression of the mRNAs hybridizing to the F cDNAs is correlated with the abundance of Hex-F protein during the gonotrophic cycles. The mRNA transcription profiles indicate that the Hex-L and Hex-F genes are regulated in a sex-, tissue- and developmental phase-dependent manner. This stage-specific expression of hexamerins contrasts with the expression patterns of hexamerins seen in other insects. The conceptual translation products of larval hexamerin cDNAs showed identity with larval serum protein 1 (LSP1)-type hexamerins while the deduced products of the female hexamerin cDNAs showed the highest identity with LSP2-type hexamerins. Genomic analyses showed that the larval hexamerin and female hexamerin genes from M. domestica belong to two distinct multigenic families.

Amino Acid Sequence↗
PubMed2000

Genetics of mosquito vector competence.

Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.

Animals↗
PubMed2000

Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti.

Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.

Aedes↗
PubMed1999

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Aedes↗
PubMed1999

Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae.

The signal sequence trap method was used to isolate cDNAs corresponding to proteins containing secretory leader peptides and whose genes are expressed specifically in the salivary glands of the malaria vector Anopheles gambiae. Fifteen unique cDNA fragments, ranging in size from 150 to 550 bp, were isolated and sequenced in a first round of immunoscreening in COS-7 cells. All but one of the cDNAs contained putative signal sequences at their 5' ends, suggesting that they were likely to encode secreted or transmembrane proteins. Expression analysis by reverse transcription-PCR showed that at least six cDNA fragments were expressed specifically in the salivary glands. Fragments showing a high degree of similarity to D7 and apyrase, two salivary gland-specific genes previously found in Aedes aegypti, were identified. Of interest, three different D7-related cDNAs that are likely to represent a new gene family were found in An. gambiae. Moreover, three salivary gland-specific cDNA fragments that do not show similarity to known proteins in the databases were identified, and the corresponding full length cDNAs were cloned and sequenced. RNA in situ hybridization to whole female salivary glands showed patterns of expression that overlap only in part those observed in the culicine mosquito A. aegypti.

Amino Acid Sequence↗
PubMed1999

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

Aedes↗
PubMed1999

Oxidation of 3-hydroxykynurenine to produce xanthommatin for eye pigmentation: a major branch pathway of tryptophan catabolism during pupal development in the yellow fever mosquito, Aedes aegypti.

This study concerns the metabolic pathways of 3-hydroxykynurenine in Aedes aegypti mosquitoes during development with emphasis on its oxidation pathway to produce xanthommatin during eye pigmentation. Oxidation of tryptophan to 3-hydroxykynurenine is the major pathway of tryptophan catabolism in Aedes aegypti, but 3-hydroxykynurenine oxidizes easily under physiological conditions, which stimulate the production of reactive oxygen species. Our data show that in Aedes aegypti, the chemically reactive 3-hydroxykynurenine is converted to the chemically stable xanthurenic acid by a transaminase-catalyzed reaction during larval development, while 3-hydroxykynurenine is transported to the compound eyes for eye pigmentation during pupal development. Our data suggest that (1) the transamination pathway of 3-hydroxykynurenine is down-regulated during the pupal development, (2) 3-hydroxykynurenine produced in other body tissues is actively transported to the compound eyes during the pupal stage, (3) the compound eye is the place where ommochromes are produced, and (4) formation of ommochromes results from nonenzymatic oxidation of 3-hydroxykynurenine in the compound eyes.

Aedes↗
PubMed1999

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

Aedes↗
PubMed1999

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system.

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.

Aedes↗
PubMed1999

Salivary gland-specific gene expression in the malaria vector Anopheles gambiae.

Molecular studies on the tissue-specific gene expression in the salivary glands of Anopheles gambiae may provide useful tools for the development of new strategies for the control of the most efficient malaria vector in the sub-Saharan Africa. We summarize here the results of a recent investigation focused on the isolation of secreted factors and putative receptors from the salivary glands of An. gambiae. Using the Signal Sequence Trap technique we have identified the first cDNAs specifically expressed in the An. gambiae salivary glands. Among these, four are exclusively expressed in female glands and encode factors presumably involved in blood-feeding, whereas two other cDNAs seem to be expressed both in male and in female glands and are likely implicated in sugar-feeding. Homologues of genes previously identified in the yellow fever mosquito Aedes aegypti, like the apyrase and D7, as well as novel salivary gland-specific cDNAs, were identified. The isolation and characterization of promoter sequences from the corresponding genes may prove useful for the expression of anti parasitic agents in the salivary glands of transgenic mosquitoes.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the gene encoding a novel factor Xa-directed anticoagulant from the yellow fever mosquito, Aedes aegypti.

Mosquito salivary glands secrete a number of proteins that inhibit mammalian hemostasis and facilitate blood feeding. We have isolated the protein product and corresponding cDNA of a gene designated Anticoagulant-factor Xa (AFXa), that encodes the factor Xa (FXa)-directed anticoagulant of the yellow fever mosquito, Aedes aegypti. The protein was purified partially by cation exchange chromatography and shown by enzyme activity profiles and SDS-polyacrylamide gel electrophoresis analysis to have an Mr = 54, 000. The protein was purified further by preparative SDS-polyacrylamide gel electrophoresis and subjected to internal protein sequencing, and the sequence of five peptides was determined. Degenerate oligonucleotides were designed based on three of the peptide sequences, and these were used to screen an adult female salivary gland cDNA library from A. aegypti. A 1.8-kilobase pair cDNA was isolated and shown to encode a 415-amino acid conceptual translation product with a predicted molecular mass of 47.8 kDa that contains the five sequenced peptides. Hydrophobicity analysis predicts a 19-amino acid signal peptide typical for secreted proteins. Northern analysis demonstrated that AFXa is expressed only in female salivary glands. Baculovirus-expressed AFXa protein has the appropriate size and expected FXa-directed anticoagulant activity. Analysis of the primary amino acid sequence shows that the AFXa gene product has similarities to the serpin superfamily of serine protease inhibitors and may represent a novel, highly diverged member of this family.

Aedes↗
PubMed1998

Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly.

The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn+-carrying transposon. The cn+ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.

Aedes↗
PubMed1998

Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti.

The mariner transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito, Aedes aegypti. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified mariner element marked with a wild-type allele of the Drosophila melanogaster cinnabar gene was microinjected into embryos of a kynurenine hydroxylase-deficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a mariner transformation system greatly enhances our ability to study and manipulate this important vector species.

Aedes↗