Biomedical subjects

F A Shotkoski

Publications and source records attributed to F A Shotkoski.

6 recordsLinked to original sources

PubMed1999

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.

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↗

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

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↗

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↗

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.

Aedes↗