Ann Fallon
University of Minnesota - Twin Cities, Entomology, Faculty Member
The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma... more
The obligate intracellular microbe, Wolbachia pipientis (Rickettsiales; Anaplasmataceae), is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate hosts in the clade Ecdysozoa. Originally described in Culex pipiens mosquitoes, Wolbachia is currently represented by 17 supergroups and is believed to occur in half of all insect species. In mosquitoes, Wolbachia acts as a gene drive agent, with the potential to modify vector populations; in filarial worms, Wolbachia functions as a symbiont, and is a target for drug therapy. A small number of Wolbachia strains from supergroups A, B, and F have been maintained in insect cell lines, which are thought to provide a more permissive environment than the natural host. When transferred back to an insect host, Wolbachia produced in cultured cells are infectio...
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Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell... more
Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell lines. C/wStr1 cells from the mosquito Aedes albopictus maintain a robust infection with Wolbachia strain wStr, originally isolated from the planthopper, Laodelphax striatellus. To explore possible functions of penicillin-binding proteins expressed from the wStr genome, C/wStr1 cells were exposed to ampicillin. Absolute levels of Wolbachia increased 3.5-fold in ampicillin-treated cells and fivefold in naive cells newly infected with wStr. Because cell numbers were depressed by ampicillin treatment, Wolbachia yield on a per-cell basis increased by 15-fold. The absence of a similar effect on wAlbB in Aa23 host cells suggests that the Wolbachia strain, the presence/absence of genes encoding penicillin-binding proteins, or the interaction between wAlbB and its host cells may modulate the effects of ampicillin.
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Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric... more
Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric profiles to define optimal conditions for harvesting an infectious inoculum. Wolbachia recovered from the cell culture supernatant after gentle pipetting of infected cells represented about 1% of the total bacterial population and were more infectious than Wolbachia that remained associated with intact cells and/or membranes after low-speed centrifugation. Optimal establishment of a robust infection in naïve cells required 6 d, at a ratio of 80 to 160 bacteria per cell. Among Aedes albopictus mosquito cell lines, an aneuploid line with a 4n + 1 karyotype was more susceptible to infection than diploid lines. These findings contribute to the in vitro manipulation of Wolbachia, illustrate some of the many factors that influence infectivity, and identify areas for future investigation.
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Treatment with tetracycline or rifampicin to eliminate the obligate intracellular bacterium, Wolbachia pipientis, provides an important tool for validating Wolbachia’s diverse effects on arthropod reproduction, including cytoplasmic... more
Treatment with tetracycline or rifampicin to eliminate the obligate intracellular bacterium, Wolbachia pipientis, provides an important tool for validating Wolbachia’s diverse effects on arthropod reproduction, including cytoplasmic incompatibility, feminization, male killing and parthenogenesis. For the soil collembolan, Folsomia candida (Entomobryomorpha: Isotomidae) efforts to establish the role of Wolbachia in parthenogenesis using tetracycline have been ambiguous, possibly reflecting variation in overall experimental design, mode of antibiotic application and duration of treatment. By maintaining F. candida populations on agar plates containing dissolved antibiotics we show that the EC50 (effective concentration for 50% reduction in biomass) for tetracycline is 50-fold higher than for rifampicin. Experiments with the fluorescent dye rhodamine further show that tetracycline deters feeding. Using individual Collembola, we show that reproductively mature F. candida survive more than 60 days exposure to tetracycline at concentrations 4-fold higher than the EC50 and that its effect on egg production is reversible, consistent with persistence of Wolbachia during tetracycline treatment. As has been shown with other microbes, our results suggest that in F. candida, Wolbachia survives antibiotic exposure by entering a metabolically dormant persister state, from which it can recover under favorable conditions. This possibility is of particular interest because persister cells are induced by toxin-antitoxin (TA) gene pairs, which have recently been associated with Wolbachia-induced cytoplasmic incompatibility in Culex pipiens mosquitoes.
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Research Interests: Zoology, Immune response, Biology, Innate immunity, Medicine, and 15 moreAedes aegypti, Cell line, Defensins, Animals, Transferrin, Aedes albopictus, Immune system, Culicidae, Insect Immunity, Insect Biochemistry and Molecular Biology, Immune function, Amino Acid Sequence, Biochemistry and cell biology, Molecular Sequence Data, and innate immune response
Research Interests: Zoology, Biology, Protein synthesis, Cell line, Animals, and 15 moreTandem Mass Spectrometry, Aedes albopictus, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Trypanosoma brucei, Time Factors, Western blot, Insect Biochemistry and Molecular Biology, Adaptive Response, Amino Acid Sequence, Cell Survival, Aedes, Insect Biochemistry, Biochemistry and cell biology, Molecular Sequence Data, and Cell count
Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. From Aedes albopictus (mosquito) cells, cycloheximide-resistant mutants... more
Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. From Aedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5 degrees C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show increased sensitivity to G-418, another antibiotic that interacts with the eukaryotic ribosome. However, there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%.
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Research Interests: Molecular Biology, Mass Spectrometry, Biology, Drosophila melanogaster, Gene expression, and 15 moreDrosophila, Cell line, Animals, Peptides, Aedes albopictus, High Performance Liquid Chromatography, Molecular cloning, Antimicrobial Cationic Peptides, Culicidae, Amino Acid Sequence, Base Sequence, Reverse Transcriptase, Full Length Movies, Biochemistry and cell biology, and Molecular Sequence Data
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Page 1. OPTIMIZATION OF GENE TRANSFER IN CULTURED INSECT CELLS Ann Marie Falton Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, Minnesota 55108 SUMMARY: The use of ...
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Mosquito cells from the C7-10 Aedes albopictus line were transfected with a recombinant plasmid containing the Escherichia coli galactokinase gene under control of the promoter from the Drosophila melanogaster metallothionein gene, Mtn.... more
Mosquito cells from the C7-10 Aedes albopictus line were transfected with a recombinant plasmid containing the Escherichia coli galactokinase gene under control of the promoter from the Drosophila melanogaster metallothionein gene, Mtn. Consistent with what has been observed with heterologous metallothionein promoters in several vertebrate systems, treatment of transiently transfected mosquito cells with CuSO4 or CdCl2 induced a 2- to 5-fold increase in galactokinase gene expression. Levels of enzyme activity were not increased in tests using stably transformed lines despite wide ranges in the number of transfected gene copies detected in Southern blots. The importance of comparative studies with gene constructs that may eventually be used to produce genetically modified mosquitoes is underscored by the apparent variability in activity of heterologous promoters from D. melanogaster in different mosquito cell lines.
Research Interests: Biology, Drosophila melanogaster, Comparative Study, Medicine, Gene expression, and 15 moreCell Division, Cell line, Escherichia coli, Animals, Metallothionein, Aedes albopictus, Cadmium, Enzyme, Genetically Modified, Enzyme activity, Heat Shock Protein, Copper sulfate, Aedes, Biochemistry and cell biology, and Dihydrofolate Reductase
Research Interests: Mass Spectrometry, Biology, Drosophila melanogaster, Medicine, Cell Culture, and 15 moreCell line, Animals, Peptides, Aedes albopictus, High Performance Liquid Chromatography, Antibacterial activity, Antimicrobial Cationic Peptides, Diptera, Amino Acid Sequence, Human Disease, Aedes, Biochemistry and cell biology, Molecular Sequence Data, Amino acid composition, and Medical biochemistry and metabolomics
We have analyzed cell cycle parameters for the Aedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In... more
We have analyzed cell cycle parameters for the Aedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10-12 h. The duration of mitosis (M) was < or = 1 h, and the DNA synthesis phase (S) required 6 h. Unlike Drosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2 h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.
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In Drosophila melanogaster, ribosomal protein RpS3 has extra-ribosomal activities including apurinic/apyrimidinic lyase activity and N-glycosylase activity that participate in DNA repair. It has been suggested that these activities couple... more
In Drosophila melanogaster, ribosomal protein RpS3 has extra-ribosomal activities including apurinic/apyrimidinic lyase activity and N-glycosylase activity that participate in DNA repair. It has been suggested that these activities couple DNA repair to the translational machinery. To establish a basis for participation of RpS3 in DNA repair in mosquitoes, we cloned RpS3 cDNAs from Aedes aegypti and Aedes albopictus mosquito cell lines. The sequence data were used to reconstruct the homologous gene from the Anopheles gambiae database. Mosquito RpS3 is a single copy gene, which in Aedes albopictus, lacks introns in the amino acid coding region. Although RpS3 proteins are well-conserved among eukaryotes, a critical glutamine residue, Q59, essential to robust DNA repair activity in the Drosophila protein, is replaced by an asparagine (N) in all three mosquito RpS3 proteins. In this respect, the mosquito protein resembles human RpS3, which has relatively modest DNA repair activity. None of the insect RpS3 proteins available in the database, other than those from Drosophila, contain glutamine at position 59. However, in the Lepidoptera, N59 is consistently replaced by serine (S), and the putative interactive site at position 134 is replaced by arginine (R). These data suggest that in the case of RpS3, the Drosophila protein may be uniquely unusual in having robust DNA repair activities that are unlikely to be common to RpS3 from other insects.
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Folsomia candida (Collembola: Isotomidae), a springtail that occurs commonly in soils throughout the world, is widely used in soil pollution and ecotoxicological studies. F. candida typically repro...
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Page 1. In Vitro Cell. Dev. Biol.--Animal 34:629-630, September 1998 © 1998 Society for In Vitro Biology 1071-2690/98 $05.00+0.00 Letter to the Editor CULTURE OF MOSQUITO CELLS IN EAGLE'S MEDIUM Dear Editor: Aedes ...
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Transient expression of a heat-shock protein-chloramphenicol acetyltransferase (hsp-CAT) recombinant plasmid was used to define the parameters that influence transfection of cultured mosquito cells using DNA-calcium phosphate... more
Transient expression of a heat-shock protein-chloramphenicol acetyltransferase (hsp-CAT) recombinant plasmid was used to define the parameters that influence transfection of cultured mosquito cells using DNA-calcium phosphate coprecipitates. The efficiency of the calcium phosphate procedure was strongly influenced by the growth state of recipient cells, and by the temperature at which the coprecipitate was prepared. Under optimal conditions, which included formation of the DNA-calcium phosphate coprecipitate at 50 degrees C, transfection frequencies were up to tenfold higher than those obtained using the previously described polybrene procedure.
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We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with... more
We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with Drosophila melanogaster cells, treatment of C7-10 cells with 20E inhibits proliferation. In the presence of 10(-6) M 20E, a gradual decline in cell number is typically apparent at 24 h. Media components such as phenol red and the potential presence of endogenous steroids in serum have no effect on the response to 20E. Pre-treating the cells with 10(-8) M 20E, with or without an intervening hormone-free period, did not alter the response to 10(-6) M 20E. However, replenishment of the medium appeared to synchronize the response to 10(-6) M 20E, causing an abrupt and complete cessation of cell division by 48 h. Flow cytometry over a 20 h period showed a decrease in the proportion of cells in S within 4-6 h after exposure to 20E. By 6-10 h, a transient increase in G2 was followed by the accumulation of more than 70% of the cells in G1. These data suggest that after treatment with 20E, cells complete the ongoing cycle before arresting in G1. Consistent with the decrease in the proportion of cells in S and G2, western blots show that levels of cyclin A, which is required during the S phase of the cycle, decreased in 20E-treated cells.
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The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes,... more
The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5'-end of the operon, is weakly expressed. The 3'-end of the operon encodes an intact wspB, which ...
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Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC... more
Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second p...
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We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were... more
We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.
Research Interests: Biology, Medicine, DNA, Cell line, Animals, and 14 moreDrug Resistance, European, Introns, Molecular cloning, Methotrexate, Amino Acid Sequence, Base Sequence, Aedes, Biochemistry and cell biology, Dihydrofolate Reductase, Molecular Sequence Data, Restriction Mapping, gene amplification, and Medical biochemistry and metabolomics
Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass... more
Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae. albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila S6, however, Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins.
Research Interests: Biology, Drosophila melanogaster, Medicine, Animals, Phosphorylation, and 10 moreRibosomal proteins, Aedes albopictus, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, European, Autoradiography, Molecular weight, Aedes, Ribosomal Protein, Biochemistry and cell biology, and Medical biochemistry and metabolomics
The eclosion hormone triggers a stereotyped preprogrammed pattern of behavior in silk moths. The effects of the hormone were duplicated by the injection of dibutyryl adenosine 3', 5'-monophosphate, adenosine 3',... more
The eclosion hormone triggers a stereotyped preprogrammed pattern of behavior in silk moths. The effects of the hormone were duplicated by the injection of dibutyryl adenosine 3', 5'-monophosphate, adenosine 3', 5'-monophosphate (cyclic AMP), or guanosine 3', 5'-monophosphate (cyclic GMP) into theophylline-treated pharate moths. Treatment with theophylline reduced the latency of the response to a low dose of hormone, presumably by blocking phosphodiesterase. Endogenous levels of cyclic AMP, but not cyclic GMP, increased significantly in the central nervous system within 10 minutes after hormone injection. We conclude that an early step leading to the release of the eclosion motor program is an increase in cyclic AMP in target neurons of the central nervous system.
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Research Interests: Evolutionary Biology, Genetics, Molecular Evolution, Biology, Medicine, and 15 morePhylogeny, Animals, Aedes albopictus, Structure Analysis, Phylogenetic Diversity, ribosomal RNA, Secondary Structure, Species Specificity, Base Sequence, Ribosomal DNA, Nucleic Acid Conformation, Aedes, Biochemistry and cell biology, Molecular Sequence Data, and Higher order
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Guanylate cyclase (E.C. 4.6.1.2.) was investigated in the accessory reproductive gland of the male house cricket, Acheta domesticus, which is known to accumulate exceptionally high levels of guanosine 3′,5′-cyclic monophosphate (cyclic... more
Guanylate cyclase (E.C. 4.6.1.2.) was investigated in the accessory reproductive gland of the male house cricket, Acheta domesticus, which is known to accumulate exceptionally high levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Accessory gland guanylate cyclase activity was linear with time for at least one hour, and with enzyme concentration to about 5 mg soluble protein per ml. Activity was dependent on Mn2+ and was maximal at pH 7.3 to 8.0. Sodium fluoride had no effect on activity, but sodium azide was slightly stimulatory. About 80% of the activity was sedimentable at 16,000 g, and both soluble and particulate activities were increased slightly in the presence of Triton X-100. Kinetic analysis indicated half-maximal velocity at 85 μM GTP in the presence of excess Mn2+, and reciprocal plots were concave upward. Changes in activity during maturation of the gland were small, and did not provide evidence for a regulatory role of guanylate cyclase in the accumulation of accessory gland cyclic GMP. The regulation and role of cyclic GMP in the accessory gland are discussed.
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An important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an... more
An important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an internal region of 25 amino acids from a 52 kDa protein that was differentially expressed in 20-hydroxyecdysone-treated Aedes albopictus cells in culture. Within the GenBank non-mouse and non-human expressed sequence tag (EST) database, this "Aedes peptide" uncovered a putative homology to hypothetical translation products from Anopheles gambiae, Caenorhabditis elegans and Drosophila melanogaster. The hypothetical translation product from D. melanogaster, which included 462 amino acids, uncovered five expressed sequence tags (ESTs) from the malaria vector, Anopheles gambiae. When the Anopheles ESTs were aligned against the hypothetical Drosophila protein, we found that in aggregate they covered 324 amino acids, with gaps measuring 19, 30, and 87 amino acids. To approximate the complete amino acid sequence, gaps between translation products from Anopheles ESTs were replaced with corresponding amino acids from Drosophila to arrive at a calculated mass of 51 104 and a pI of 5.84 for the mosquito protein, consistent with the position of the Ae. albopictus protein on two-dimensional polyacrylamide gels. Finally, tandem mass spectrometry of a tryptic digest of the 52 kDa Ae. albopictus protein revealed 33 peptides with masses within 1 Dalton of those predicted from an in silico digestion of the reconstructed Anophleles protein. In addition to providing the first direct evidence that a hypothetical protein in Drosophila is in fact translated, this analysis provides a general approach for maximizing recovery, from existing databases, of information that can facilitate prioritization of efforts among several candidate proteins.
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We have sequenced cDNAs encoding proliferating cell nuclear antigen (PCNA) from Aedes albopictus cells and from Aedes aegypti mosquitoes. The mosquito cDNAs contained an open reading frame encoding a 260 amino acid protein with a... more
We have sequenced cDNAs encoding proliferating cell nuclear antigen (PCNA) from Aedes albopictus cells and from Aedes aegypti mosquitoes. The mosquito cDNAs contained an open reading frame encoding a 260 amino acid protein with a calculated mass of 29.0 kDa and a pI of 4.46. There was a single amino acid difference between PCNA proteins from Ae. albopictus and Ae. aegypti. In An. gambiae, the PCNA homolog contained 260 residues, and the pcna gene was interrupted by a single 67 nucleotide intron in the betaC2 region of the protein. A phylogenetic comparison grouped known Dipteran PCNA sequences into two clusters, representing the Nematocera and the Cyclorrhapha. PCNA transcripts measured 1.1 kb, and were stable, as was PCNA protein. Mosquito PCNA was efficiently recognized by a commercially available mouse anti-PCNA monoclonal antibody, which coprecipitated 29 kDa and 35 kDa proteins from mosquito cells representing different growth states. These results support the feasibility of recovering mosquito cell cycle inhibitory proteins by virtue of their interaction with PCNA.
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The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus... more
The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.
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An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15... more
An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15 days in the presence of selective medium containing 1 microM methotrexate. The transformed clones contained an estimated 100-500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.