Retrieval of flanking DNA using a PCR-based approach with restriction enzyme-digested genomic DNA template.
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Animals↗
Biomedical subjects
A M Fallon
Publications and source records attributed to A M Fallon.
Peptide sequence of an antibiotic cecropin from the vector mosquito, Aedes albopictus.
We have identified a 35-amino acid antibiotic cecropin secreted by an established mosquito cell line. C7-10 cells from the vector mosquito, Aedes albopictus, were incubated with heat-killed Escherichia coli, and materials secreted into the cell culture supernatant were recovered by acid precipitation. Following batch elution from Sep-Pak C18 cartridges and further purification by reverse phase high performance liquid chromatography (RP-HPLC) a predominant peak of antibacterial activity was characterized by mass spectrometry, amino acid composition analysis, and Edman degradation, yielding the sequence GGLKKLGKKLEGVGKRVFKASEKALPVAVGIKALG. Unlike other cecropins, the peptide was not amidated at the C-terminus. Aedes albopictus Cecropin A (AalCecA) is the first cecropin to be described from a mosquito vector of human disease. Consistent with the classification of mosquitoes among the Dipteran suborder Nematocera, AalCecA shares only 36% amino acid identity with cecropins from Drosophila melanogaster and other Cyclorrhaphid flies, whose mature cecropins share 80% to 100% amino acid identity.
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Effect of nutrient deprivation on ribosomal RNA and ribosomal protein mRNA in cultured mosquito cells.
The effect of nutrient deprivation on RNA and protein synthesis in cultured Aedes albopictus mosquito cells was investigated by replacing the culture medium with phosphate-buffered saline. After a 2 h starvation treatment, incorporation of radiolabeled precursor into total RNA was inhibited by 50%, and after 4 h, incorporation of amino acids into protein was inhibited by 50%. To investigate directly the effects of starvation on rRNA synthesis, ribosomal subunits were prepared from treated cells by sucrose density gradient centrifugation. After 4 h in saline, incorporation of [3H]uridine into ribosomal subunits had declined to baseline levels. Even after 8 h starvation, however, the effect was reversed by refeeding with complete medium, in which cells resumed rRNA synthesis and ribosomal subunit assembly. During 8 h starvation, total amounts of rRNA detected by northern blot remained stable. Ribosomal protein mRNA abundance was measured on northern blots, using probes corresponding to L8 and L31 ribosomal protein genes. The content of these ribosomal protein mRNAs was unchanged during starvation, or during treatment with actinomycin D. These results suggest that ribosomal protein mRNAs belong to a long-lived mRNA population, and suggest that post-transcriptional regulation of ribosomal protein synthesis is an important regulatory mechanism in growing mosquito cells.
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Culture of mosquito cells in Eagle's medium.
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Aedes↗
Similarities to a LINE element shared by Anopheline and Culicine mosquitos map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells.
To extend our understanding of amplicon structure in methotrexate-resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3'-end of the Type 1 dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an approximately 0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.
Animals↗
Analysis of a ribosomal DNA intergenic spacer region from the yellow fever mosquito, Aedes aegypti.
We have sequenced the 1.8 kb intergenic spacer (IGS) region from an Aedes aegypti ribosomal DNA repeat and have identified conserved functional motifs shared with the related mosquito, Aedes albopictus. Despite the shorter length and greater homogeneity of the Ae. aegypti IGS region, the sequences of two potential RNA polymerase I core promoters and closely associated terminator elements were highly conserved. Primer extension analysis indicated that the predominant transcription initiation site in the Ae. aegypti rDNA repeat unit region lay at or near the A residue at nucleotide position 1003 in the 'upstream' RNA polymerase I promoter. This observation was supported by the higher sequence identity between the upstream promoters in Ae. aegypti and Ae. albopictus, relative to the downstream promoters. In contrast to strong similarities among proximal regulatory elements, the Ae. aegypti IGS sequence upstream of the transcription initiation site lacked the ordered array of contiguous approximately 200 nucleotide subrepeats previously found in the IGS of Ae. albopictus. In Ae. aegypti, only 4 approximately 50 nucleotide R subrepeats separated by unique sequences, followed by 2 approximately 50 nucleotide E subrepeats, occurred upstream of the transcription initiation site. Despite their differences in size and sequence, however, the four Ae. aegypti R subrepeats shared an internal structural organization that included a conserved core with 'spacer' promoters and recombinogenic elements similar to those in the longer Ae. albopictus subrepeats. These observations provide an important basis for further characterization of transcription specificity among mosquito RNA polymerase I promoters and associated regulatory elements, and contribute towards the eventual use of these elements in transgenic applications.
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The mosquito dihydrofolate reductase amplicon contains a truncated synaptic vesicle protein gene.
When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopicfus mosquito cells amplify an 200 kb region of DNA containing the dihydrofolate reductase gene. To determine whether the amplicon contained additional coding regions, Southern blots of cosmid clones containing amplicon DNA were probed separately with reverse-transcribed mRNA from methotrexate-sensitive and methotrexate-resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridized with cDNA from resistant cells. Of these, fragments B and F also hybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild-type cells. Fragment G, which appeared to encode a low copy number gene in wild-type cells that subsequently became part of the dihydrofolate reductase amplicon in methotrexate-resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots containing RNA from resistant cells. Fragment G contained a 1203 bp open reading frame, encoding 401 amino acids homologous to synaptic vesicle protein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N-terminal transmembrane domains, an internal cytoplasmic loop, a seventh transmembrane domain, and most of an intravesicular loop. This partial sequence, which appears to correspond to a truncated gene generated during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.
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Mosquito transferrin, an acute-phase protein that is up-regulated upon infection.
When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3' end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312-321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.
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Evaluation of a heterologous metallothionein gene promoter in transfected mosquito cells.
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.
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Structural mapping of the dihydrofolate reductase amplicon in mosquito cells resistant to methotrexate.
A cosmid library containing genomic DNA from mosquito cells in which the dihydrofolate reductase gene had become amplified in response to methotrexate selection was used to define the structure of the amplified DNA region (amplicon). A series of overlapping cosmids identified more than 200 kb of amplicon DNA, which was mapped relative to BssHII fragments from genomic DNA separated by transverse alternating field electrophoresis. These analyses indicated that, in Mtx-5011-256 mosquito cells, dhfr genes occur in at least two types of amplicon. The predominant Type I amplicon measures at least 215 kb and contains most of the dhfr genes. In addition, a small proportion of dhfr genes reside in a Type II amplicon, which is arranged in head-to-tail tandem repeats measuring 162 kb. Both amplicons share at least 70 kb of DNA sequence at their 5' ends. Approximately 20 fragments containing repeated sequence elements have been identified in the cloned amplicon DNA. Hybridization of amplicon DNA fragments containing repeated sequences to genomic DNA detected polymorphisms between wild type and methotrexate-resistant cells, suggesting that recombination may generate the divergence observed at the 3'-ends of Type I and Type II amplicons. This first detailed analysis of an insect dhfr amplicon provides an essential basis for ongoing investigation of repeated sequences, transcribed units and replication origins within the amplicon DNA.
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Synchronization of Aedes albopictus mosquito cells using hydroxyurea.
We have established conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, to synchronize the division cycle of a continuous cell line from the mosquito, Aedes albopictus. In the range of 0.15-0.25 mM hydroxyurea, an 18 h treatment, followed by removal of the drug, results in effective synchronization. When combined with the partial synchronization that occurs within 10 h of dilution and plating, more than 80% of cells treated with hydroxyurea could be recovered in the synthesis (S) phase of the cell cycle during the 4 h period after removal of the drug. The degree of synchrony was enhanced when cells were exposed to two consecutive hydroxyurea treatments spaced 10 h apart. Synchronized cells expressed maximal levels of a reporter gene when transfected immediately after removal of hydroxyurea. This is the first description of effective chemical synchronization of an insect cell line using hydroxyurea.
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Transgenic insect cells: mosquito cell mutants and the dihydrofolate reductase gene.
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Cell cycle parameters in Aedes albopictus mosquito cells.
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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Changes in ribosomal protein rpL8 mRNA during the reproductive cycle of the mosquito, Aedes aegypti.
Changes in the abundance of ribosomal protein rpL8 mRNA were compared with 18S rRNA and vitellogenin mRNA in fat body from adult female Aedes aegypti during the reproductive cycle. Levels of rpL8 mRNA began to increase within 2 h after adult eclosion, peaked at about 24 h post-eclosion, and remained high throughout the next 48 h. During this same period, rRNA abundance increased about 2-fold. After the bloodmeal, levels of rpL8 mRNA gradually decreased over the next 48 h, while rRNA levels increased about 4-fold within 16-24 h post-bloodfeeding and eventually returned to previtellogenic levels. Vitellogenin mRNA was induced only after the bloodmeal, and disappeared by 48 h after feeding. After oviposition, rpL8 mRNA levels again increased to pre-bloodfeeding levels. These results indicate that rpL8 mRNA transcription in mosquito fat body is independent of rRNA transcription during the previtellogenic acquisition of competence and also during the post-bloodmeal ovarian cycle. Moreover, unlike the vitellogenin gene, the rpL8 gene is under post-transcriptional regulation in blood-fed females.
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Evidence for a DNA homologous pairing activity in nuclear extracts from mosquito cells.
Using a sensitive homologous pairing/DNA strand transfer assay, we detected formation of joint molecules in the presence of nuclear extract from cultured mosquito C7-10 cells in a reaction containing single stranded circular m13 DNA and a linear, double stranded DNA 5'-end-labeled on the strand complementary to a portion of the single-stranded substrate. Joint molecules were detected by the reduced electrophoretic mobility of labeled probe on agarose gels, which indicated that the 5'-end labeled strand of the linear duplex had formed a hybrid with the single-stranded substrate. Characterization of the activity detected initially in crude nuclear extracts provided a basis for a 5-fold enrichment of activity after a two-step KCl elution from heparin-Sepharose. Further purification by preparative electrophoresis yielded a band at approximately 35 kDa, which, when transferred to Immobilon P membrane, specifically bound the labeled, complementary strand probe. Optimal activity of the electroeluted enzyme required both magnesium and ATP and was sensitive to the ratio of single-stranded and double-stranded DNA substrate and to the amount of protein. This homologous pairing activity from mosquito cells is the first such activity to be described from an insect other than Drosophila melanogaster.
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Promoter utilization in a mosquito ribosomal DNA cistron.
In the mosquito Aedes albopictus, two potential RNA polymerase I promoters that map 531 and 143 nucleotides upstream of the 18S rRNA gene have been defined on the basis of sequence homology with rRNA promoters from other species. Using the polymerase chain reaction, we confirmed that a 717 nucleotide region spanning the upstream (-531) and downstream (-143) promoters is homogeneous in genomic DNA and in cloned DNA. DNA probes representing each of these promoters, as well as upstream "spacer" promoters, exhibited protein-binding activity, and each unlabeled probe was an effective competitor of protein binding with the other probes, suggesting that these potential regulatory sequences interact with a common protein(s). Analysis of precursor ribosomal RNAs accumulated during temperature shock indicated that transcription is initiated primarily at the upstream (-531) promoter. RNAse protection and primer extension analyses confirmed the predominant use of this promoter, both in cultured cells and in mosquito life stages.
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Evidence for DNA endonuclease activity in nuclear extracts from mosquito cells.
We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3' OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg2+, Ca2+, Mn2+, Ba2+). In the presence of Mg2+, Zn2+, Hg2+ and Cu2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18-35 degrees C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.
Adenosine Triphosphate↗
Evaluation of a viral thymidine kinase gene for suicide selection in transfected mosquito cells.
An Aedes albopictus cell line previously shown to be deficient in thymidine kinase activity was transfected with a thymidine kinase gene (tk) from Herpes simplex virus. Survival of the transfected lines in a 'TK+ selective medium' indicated that the viral gene was actively expressed at a level sufficient to rescue the TK-deficient phenotype of the parent line. Unlike the parental cells, TK+ transformants (TK6:hsv cells) were sensitive to 5-bromodeoxyuridine, and contained DNA corresponding to the constructs introduced by transfection. This TK selection system will facilitate recovery of other cotransfected, non-selectable mosquito genes in cultured cells. Transformed cells were treated with several antiviral drugs to define conditions for a 'suicide selection' system, in which cells expressing the viral thymidine kinase enzyme (TK) under the control of an inducible promoter would be selectively destroyed, whereas cells expressing the endogenous mosquito enzyme would remain relatively unaffected. The anti-herpetic drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) showed greater cytotoxicity against transformed cells expressing the viral enzyme, and less toxicity to wild-type mosquito cells. This cell culture system provides a model for initial evaluation of suicide selection systems that may ultimately be adapted to the mosquito using sex- or tissue-specific promoters to drive expression of heterologous genes.
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