Sequence of a mosquito ribonucleotide reductase cDNA and evidence for gene amplification in hydroxyurea-resistant cells.

Ribonucleotide reductase is essential for production of the deoxyribonucleotide substrates required for DNA synthesis in all eukaryotic cells. The full-length cDNA encoding a mosquito ribonucleotide reductase R2 subunit was obtained from Aedes albopictus cells using a polymerase chain reaction (PCR)-based approach. The cDNA contained 1197 nucleotides, and encoded a 398 amino acid R2 protein. Overall, mosquito R2 shared approximately 70% amino acid sequence identity with R2 proteins from vertebrates, but conservation at the N-terminus of the protein was relatively low. The sequence of R2 cDNA was identical in hydroxyurea-resistant mosquito cells and in wild-type cells, while the R2 gene copy number was increased ten to twentyfold in resistant cells.

The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.

Stem cell gene therapy, position effects and chromatin insulators.

Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.

Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi.

Two well-described elements of the immune response of insects include encapsulation of metazoan parasites (blood-cell-mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co-regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitoid Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal-related immunity factor (Dif), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.

Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.

A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.

Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines.

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selected marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chlorine ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the generic transformation of non-Drosophilid insects.

Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti.

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homomultimeric expression of a gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus-infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100-fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.

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.

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.

Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila.

In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a gamma-aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non-Drosophilids is discussed.

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.

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.

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.