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F C Kafatos

Publications and source records attributed to F C Kafatos.

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

Stable germline transformation of the malaria mosquito Anopheles stephensi.

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi, through a transposase-mediated process.

Animals↗
PubMed2000

A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae.

Sp22D, a modular serine protease encompassing chitin binding, low density lipoprotein receptor, and scavenger receptor cysteine-rich domains, was identified by molecular cloning in the malaria vector, Anopheles gambiae. It is expressed in multiple body parts and during much of development, most intensely in hemocytes. The protein appears to be posttranslationally modified. Its integral, putatively glycosylated form is secreted in the hemolymph, whereas a smaller form potentially generated by proteolytic processing is associated with the tissues. Bacterial challenge or wounding result in low-level RNA induction, but the protein does not bind to bacteria, nor is its processing affected by infection. However, Sp22D binds to chitin with high affinity and undergoes transient changes in processing during pupal to adult metamorphosis; it may respond to exposure to naked chitin during tissue remodeling or damage.

Animals↗
PubMed2000

Anopheles gambiae pilot gene discovery project: identification of mosquito innate immunity genes from expressed sequence tags generated from immune-competent cell lines.

Together with AIDS and tuberculosis, malaria is at the top of the list of devastating infectious diseases. However, molecular genetic studies of its major vector, Anopheles gambiae, are still quite limited. We have conducted a pilot gene discovery project to accelerate progress in the molecular analysis of vector biology, with emphasis on the mosquito's antimalarial immune defense. A total of 5,925 expressed sequence tags were determined from normalized cDNA libraries derived from immune-responsive hemocyte-like cell lines. The 3,242 expressed sequence tag-containing cDNA clones were grouped into 2,380 clone clusters, potentially representing unique genes. Of these, 1,118 showed similarities to known genes from other organisms, but only 27 were identical to previously known mosquito genes. We identified 38 candidate genes, based on sequence similarity, that may be implicated in immune reactions including antimalarial defense; 19 of these were shown experimentally to be inducible by bacterial challenge, lending support to their proposed involvement in mosquito immunity.

Animals↗
PubMed2000

From sequence to chromosome: the tip of the X chromosome of D. melanogaster.

One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.

Animals↗
PubMed2000

Toward Anopheles transformation: Minos element activity in anopheline cells and embryos.

The ability of the Minos transposable element to function as a transformation vector in anopheline mosquitoes was assessed. Two recently established Anopheles gambiae cell lines were stably transformed by using marked Minos transposons in the presence of a helper plasmid expressing transposase. The markers were either the green fluorescent protein or the hygromycin B phosphotransferase gene driven by the Drosophila Hsp70 promoter. Cloning and sequencing of the integration sites demonstrated that insertions in the cell genome occurred through the action of Minos transposase. Furthermore, an interplasmid transposition assay established that Minos transposase is active in the cytoplasmic environment of Anopheles stephensi embryos: interplasmid transposition events isolated from injected preblastoderm embryos were identified as Minos transposase-mediated integrations, and no events were recorded in the absence of an active transposase. These results demonstrate that Minos vectors are suitable candidates for germ-line transformation of anopheline mosquitoes.

Animals↗
PubMed2000

BAC trimming: minimizing clone overlaps.

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.

Arabidopsis↗
PubMed2000

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae.

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.

Amino Acid Sequence↗
PubMed1999

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae.

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5' flanking sequence (-855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5' flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Animals↗
PubMed1999

CTRP is essential for mosquito infection by malaria ookinetes.

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.

Animals↗
PubMed1999

Identification of regulatory regions driving the expression of the Drosophila spalt complex at different developmental stages.

The zinc finger transcription factors Spalt and Spalt-related have been implicated in multiple developmental processes. In the wing they are regulated by the secreted protein Decapentaplegic and participate in the positioning of the wing veins. The function of Spalt has been also analyzed during tracheal development and embryonic segmentation. Here, we present the isolation and characterization of novel spalt/spalt-related alleles, which analysis indicates that these genes cannot substitute for each other in the developmental processes studied. The mutants present embryonic or pupal lethality, with phenotypes consistent with the loss of spalt function. We also present a detailed functional analysis of the DNA regions implicated in the regulation of these genes. This regulation is complex, integrating the information from both negative and positive regulators, and it is modular, with discrete fragments of DNA directing expression to discrete regions in embryonic and larval tissues.

Alleles↗
PubMed1999

Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA.

BACKGROUND: NF-kappa B/Rel transcription factors play important roles in immunity and development in mammals and insects. Their activity is regulated by their cellular localization, homo- and heterodimerization and association with other factors on their target gene promoters. Gambif1 from Anopheles gambiae is a member of the Rel family and a close homologue of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. RESULTS: We present the crystal structure of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. CONCLUSIONS: The Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel-DNA complex structures leads to a general model for DNA recognition by Rel proteins.

Amino Acid Sequence↗
PubMed1999

Phylogenetic perspectives in innate immunity.

The concept of innate immunity refers to the first-line host defense that serves to limit infection in the early hours after exposure to microorganisms. Recent data have highlighted similarities between pathogen recognition, signaling pathways, and effector mechanisms of innate immunity in Drosophila and mammals, pointing to a common ancestry of these defenses. In addition to its role in the early phase of defense, innate immunity in mammals appears to play a key role in stimulating the subsequent, clonal response of adaptive immunity.

Animals↗
PubMed1999

A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae.

An invertebrate intestinal mucin gene, AgMuc1, was isolated from the malaria vector mosquito Anopheles gambiae. The predicted 122-residue protein consists of a central core of seven repeating TTTTVAP motifs flanked by hydrophobic N- and C-terminal domains. This structure is similar to that of mucins that coat the protozoan parasite Trypanosoma cruzi. Northern blot analysis indicated that the gene is expressed exclusively in the midgut of adult mosquitoes. A length polymorphism and in situ hybridization were used to genetically and cytogenetically map AgMuc1 to division 7A of the right arm of the second chromosome. The subcellular localization of the encoded protein in tissue culture cells was examined by using a baculovirus vector to express AgMuc1 protein tagged with the green fluorescent protein (GFP). The results indicated that this protein is found at the cell surface and that both hydrophobic domains are required for cell surface targeting. We propose that AgMuc1 is an abundant mucin-like protein that lines the surface of the midgut microvilli, potentially protecting the intestinal epithelium from the proteinase-rich environment of the gut lumen. An intriguing possibility is that, as an abundant surface protein, AgMuc1 may also interact with the malaria parasite during its invasion of the mosquito midgut.

Amino Acid Sequence↗
PubMed1999

A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes.

Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae.

Amino Acid Sequence↗
PubMed1999

Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection.

A new insect member of the STAT family of transcription factors (Ag-STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag-STAT is most similar to Drosophila D-STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag-STAT protein in fat body cells and induction of DNA-binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag-STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects.

Amino Acid Sequence↗
PubMed1999

Muscle structure and innervation are affected by loss of Dorsal in the fruit fly, Drosophila melanogaster.

In Drosophila, the Rel-protein Dorsal and its inhibitor, Cactus, act in signal transduction pathways that control the establishment of dorsoventral polarity during embryogenesis and the immune response during postembryonic life. Here we present data indicating that Dorsal is also involved in the control of development and maintenance of innervation in somatic muscles. Dorsal and Cactus are colocalized in all somatic muscles during postembryonic development. In larvae and adults, these proteins are distributed at low levels in the cytoplasm and nuclei and at much higher levels in the postsynaptic component of glutamatergic neuromuscular junctions. Absence of Dorsal, in homozygous dorsal mutant larvae results in muscle misinsertions, duplications, nuclear hypotrophy, disorganization of actin bundles, and altered subcellular distribution of Cactus. Some muscles show very abnormal neuromuscular junctions, and some motor axon terminals are transformed into growth cone-like structures embedded in synaptotagmin-enriched vesicles. The detailed phenotype suggests a role of Dorsal signalling in the maintenance and plasticity of the NMJ.

Animals↗
PubMed1999

Microsatellite markers and genotyping procedures for Anopheles gambiae.

The procedures outlined in this article by Rui Wang, Fotis Kafatos and Liangbiao Zheng are well suited to studies of field mosquito populations, and also to the genetic mapping of qualitative and quantitative traits of Anopheles gambiae, a major malaria vector in Africa. An accurate, reproducible and high-throughput microsatellite genotyping procedure has been established.

Animals↗