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A M Fallon

Publications and source records attributed to A M Fallon.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mosquito ribosomal protein rpL31 resembles rat rpL34: cDNA and deduced amino acid sequence.

An Aedes albopictus ribosomal protein rpL31 cDNA was sequenced, and found to encode a protein with homology to rat rpL34. The 544 bp mosquito cDNA contained an ATG start site, three in-frame termination codons, and an AATAAA polyadenylation signal. Mosquito rpL31 had a mass of 15,137 Da, a pI of 12.39 and contained 14.5% Arg and 14.5% Lys. PCR analyses with genomic DNA suggested the presence of a large intron near the 5'-end of the gene.

Aedes↗
PubMed1994

Mosquito large subunit ribosomal RNA: simultaneous alignment of primary and secondary structure.

We report the sequence and propose a secondary structure for the cytoplasmic large subunit (5.8S and 28S) ribosomal RNA of the mosquito, Aedes albopictus, in an aligned format that incorporates secondary structure comparisons with Homo sapiens, Drosophila melanogaster, and Escherichia coli ribosomal RNAs. This format facilitates comparison of subtle differences between models, allowing nucleotide by nucleotide analysis at each position of discrepancy. Comparison of the A. albopictus large subunit ribosomal RNA gene with those from other species revealed new compensatory base changes. The aligned format focuses attention to the specific contribution of the A. albopictus sequence by facilitating comparison with the sequence of another dipteran, D. melanogaster. This is the second report of a complete large subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a member of the lower Diptera (Nematocera).

Aedes↗
PubMed1994

Expression of an antisense dihydrofolate reductase transcript in transfected mosquito cells: effects on growth and plating efficiency.

Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.

Aedes↗
PubMed1994

Secretion of an inducible cecropin-like activity by cultured mosquito cells.

Characterization of activities that provide potential targets for genetic manipulation of pathogen development, maintenance, and transmission in transgenic insects has applications to the eventual control of malaria and other arthropod-transmitted diseases. We have identified inducible activities from cultured mosquito (Aedes albopictus) cells, including one that shares the antimicrobial properties of cecropins from other insects. The cecropin-like activity can be induced by treatment with heat-killed Escherichia coli, is secreted into the cell culture medium, and can be detected after electrophoresis of acid-precipitable proteins on polyacrylamide gels at pH 4.3. Cecropin lacks the amino acids methionine and cysteine. Other proteins secreted in response to bacterial induction measure 111, 66, 53, and 32 kD; these proteins incorporate sulfur-containing amino acids and were detected on denaturing polyacrylamide gels. The synthesis of antimicrobial proteins by mosquito cells in culture will contribute to an understanding of the diversity of molecules that participate in insect immunity and to the use of continuous cell lines and their inducible products to explore and manipulate regulation of physiological processes relevant to vector biology.

Aedes↗
PubMed1993

Cultured Aedes albopictus mosquito cells synthesize hormone-inducible proteins.

To provide a framework for biochemical investigation of ecdysteroid action in Aedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10(-6) M 20-hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In both Aedes aegypti and A. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified in A. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in some Drosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 cells. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfected A. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on a D. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.

Aedes↗
PubMed1993

Transcripts from a mosquito dihydrofolate reductase gene: evidence for heterogeneity at the 5' end.

Heterogeneity among transcripts from the mosquito (Aedes albopictus) dihydrofolate reductase (DHFR) gene in wild-type C7-12 cells and in methotrexate-resistant Mtx-5011-128 cells has been analyzed by Northern blotting, RNAase mapping, and primer extension. In both sensitive and resistant cells, a major transcription initiation site mapped c. 11 nucleotides downstream of the TATAA box, near position -66 relative to the AUG codon. Two other major transcription initiation sites mapped approx. eight and 45 nucleotides, respectively, upstream of the TATAA box. In addition, at least six minor sites were detected, four of which mapped within TATA-like sequences. Within the AT-rich region flanking the 5'-end of the mosquito DHFR gene were four T-rich motifs and three "GTTTGTG" repeats. Additional "GTTTGTG" repeats occurred in the first exon and in the single 56 nucleotide intron of the mosquito DHFR gene. In contrast to the heterogeneity at the 5'-ends of mosquito DHFR transcripts, the 3'-end terminated at a single position, c. 22 nucleotides downstream of the polyadenylation signal.

Aedes↗
PubMed1993

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells.

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.

Aedes↗
PubMed1993

Plasmid modifications in a tick-borne pathogen, Borrelia burgdorferi, cocultured with tick cells.

We describe an in vitro system that will facilitate molecular analysis of the association between Lyme disease spirochetes and vector cells. We cocultured Borrelia burgdorferi continuously with two tick cell lines, RAE25 (from Rhipicephalus appendiculatus) and IDE8 (from Ixodes scapularis). A clone isolated after twenty-two passages with RAE25 cells had lost the largest (49 kb) plasmid, and probes containing information normally encoded on it, including genes for two surface proteins, hybridized to smaller plasmids. Spirochetes maintained with IDE 8 cells showed a new 43 kb plasmid that hybridized to a probe made from the 49 kb plasmid. After reisolation from hamsters, these spirochetes carried a large plasmid (100 kb) that hybridized with the 49 kb plasmid. These changes may illustrate a plasticity that enables B. burgdorferi to adapt to different environments.

Animals↗
PubMed1993

An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution.

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.

Aedes↗
PubMed1992

Is higher-order structure conserved in eukaryotic ribosomal DNA intergenic spacers?

Computer-based structural analysis of the ribosomal DNA intergenic spacer (IGS) from the mosquito Aedes albopictus revealed a potential to form strong and extensive secondary structures throughout a 4.7-kilobase (kb) region. The predicted stability of secondary structures was particularly high within a 3.15-kb region containing 17 tandem 201 base-pair subrepeats. Similarly strong secondary structure potential was also found when IGS subrepeats were analyzed from 17 phylogenetically diverse eukaryotes, including vertebrates, invertebrates, and plants. Conservation of higher-order structure potential in the IGS region of ribosomal DNA may reflect evolutionary and functional constraints on chromatin organization, transcriptional regulation of the ribosomal RNA genes, and/or transcript processing and stability.

Aedes↗
PubMed1992

Thymidine kinase-deficient mutants of Aedes albopictus mosquito cells.

Aedes albopictus mosquito cells resistant to the thymidine analog 5-bromodeoxyuridine (BrdU) were obtained using a single-step selection procedure. The resistant cells were characterized with respect to growth in the presence of BrdU, incorporation of [3H]uridine and [3H]thymidine, and thymidine kinase activity in crude extracts. The LC50 for TK-6 cells was 95 micrograms BrdU/ml, and for TK-8 cells was 45 micrograms/ml. Both clones incorporated [3H]uridine at levels corresponding to those in wild-type cells. TK-6 and TK-8 cells did not incorporate [3H]thymidine into acid-precipitable material, nor did they contain measurable thymidine kinase activity. Thymidine kinase activity in crude extracts from wild-type cells had a Km of 2 microM and a Vmax of 10 pmol.min-1.mg-1 protein.

Aedes↗
PubMed1992

Sequence analysis of a mosquito ribosomal protein rpL8 gene and its upstream regulatory region.

The gene encoding Aedes albopictus ribosomal protein L8 was isolated using a cDNA probe. Based on the deduced amino acid sequence, rpL8 has a mass of 28,605 Da, a pI of 11.97, and contains 9.6% Arg and 11.9% Lys. The rpL8 gene spans 1229 nucleotides, and contains three exons measuring 73, 150, and 648 nucleotides. The first intron is 293 nucleotides long and interrupts an 85-nucleotide untranslated leader sequence. The AUG codon is located 12 nucleotides downstream of the 5'-end of the second exon. Separating the second and third exon is a 65-nucleotide intron. The major transcription initiation site, identified by primer extension and polymerase stop reactions, mapped 378 nucleotides upstream from the AUG start codon; minor initiation sites were also detected. The DNA sequence upstream of the rpL8 gene was T-rich, but conventional TATA and CAAT boxes were absent. This is the first molecular analysis of a mosquito ribosomal protein gene.

Aedes↗
PubMed1991

An amplified insect dihydrofolate reductase gene contains a single intron.

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.

Aedes↗
PubMed1991

Nucleotide sequence of a mosquito 18S ribosomal RNA gene.

We have sequenced an 18S ribosomal RNA gene from the mosquito, Aedes albopictus. Computer alignment of the 1950 nucleotide coding region (56% A + T) with 18S rRNA sequences from two insect and three vertebrate species revealed greater sequence divergence among the insects than among the vertebrates. Sequence alignments showed that variable region V4, which has been considered to be the most poorly conserved domain in the 18S rRNA gene, was better conserved among insects and vertebrates than was the V6 domain.

Aedes↗
PubMed1991

Efficient transfection of mosquito cells is influenced by the temperature at which DNA-calcium phosphate coprecipitates are prepared.

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.

Aedes↗
PubMed1991

Ribosomal DNA structure in Aedes mosquitoes (Diptera: Culicidae) and their cell lines.

The molecular organization of the ribosomal DNA repeat unit in laboratory colonies of Aedes albopictus (Skuse) and Aedes aegypti (L.) and in continuous cell lines derived from these species was examined using restriction enzyme mapping and Southern blotting techniques. In the rRNA coding regions, restriction enzyme sites were conserved in Ae. aegypti and Ae. albopictus. In particular, sites that had been mapped in rDNA cloned from the Mauritius strain of Ae. albopictus mosquitoes were confirmed in cloned DNA from cultured Ae. alboptictus cells. The intergenic spacer region of the ribosomal DNA repeat unit in Ae. albopictus was longer than that in Ae. aegypti; contained multiple recognition sites for AluI, SstI, PvuI, and XhoI; and was more heterogeneous in DNA from cultured cells than in DNA from inbred laboratory mosquitoes. The rDNA copy number was 3- to 4-fold lower in cell lines from both Ae. albopictus and Ae. aegypti than in the corresponding adult mosquitoes.

Aedes↗
PubMed1990

Genetic changes in methotrexate-resistant mosquito cells.

A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.

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