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F M DeFilippes

Publications and source records attributed to F M DeFilippes.

11 recordsLinked to original sources
PubMed1994

A biological method for examining the effect of codon changes in a conserved region of DNA polymerase.

The vaccinia virus genome encodes a DNA polymerase that is similar to other DNA polymerases. A mutation in the polymerase gene at a site that is adjacent to conserved residues allows viral replication in the presence of aphidicolin. Since wild-type virus is converted to aphidicolin-resistance by site-directed mutagenesis, it was feasible that active virus with substituted conserved residues could be detected by linking alterations to the aphidicolin-resistance mutation. Altered DNA, from a PCR, was introduced into virus by a marker transfer procedure. DNA from plaques of drug-resistant virus was amplified, and the product was sequenced to check for the conserved residue alteration. An alteration that introduced a Bg1I site was designed to facilitate the selection of drug-resistant virus containing substituted residues. One positive result was the replacement of two amino acids, tyrosine and alanine, by tryptophan and threonine. The failure to substitute aspartic acid for tyrosine indicates that drastic changes of the conserved sequence are not tolerated. Although the limitations associated with negative results apply, the method provides an in vivo assay for selecting a polymerase with conserved residue changes.

Amino Acid Sequence↗
PubMed1991

Decontaminating the polymerase chain reaction.

Template DNA, which was added to a modified polymerase chain reaction mixture to simulate contamination, was inactivated by restriction enzyme digestion. After inactivation of the restriction enzymes, additional template, buffer and Taq polymerase were added to the reaction and the polymerase chain reaction proceeded. Results are presented using several different restriction enzymes with four base recognition sequences. The limitations of the method are discussed to indicate the tactics to be used for other DNA sequences.

DNA↗
PubMed1989

Site of the base change in the vaccinia virus DNA polymerase gene which confers aphidicolin resistance.

An aphidicolin-resistant mutant of vaccinia virus has been shown to encode an altered viral DNA polymerase that is more resistant to aphidicolin. Marker transfer experiments with the DNA from the resistant virus localized the mutation site to an RsaI segment within the portion of the HindIII-E segment which has been shown to contain the viral DNA polymerase gene. Nucleotide sequence analysis of the mutant DNA showed a single GC to AT transition at position 2430, which indicates a leucine-to-methionine change at residue 645 in the protein.

Antiviral Agents↗
PubMed1984

Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.

Animals↗
PubMed1982

Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.

Base Sequence↗
PubMed1981

Characterization of bacteriophage N3 DNA.

The DNA from Haemophilus influenzae temperate phage N3 was characterized by centrifugation and by electrophoresis after nuclease digestion. The double-stranded DNA, with a mass of 25.8 X 10(6) daltons, had single-strand cohesive ends. Strand association through cohesion was reduced by heat and removed by S1 nuclease digestion. N3 DNA contained five EcoRI, one KpnI, two SacI, six XbaI, and four XhoI cleavage sites. The cohesive end segments were identified by heating the digests before electrophoresis. This was the first step in the construction of the physical maps of this DNA.

Bacteriophages↗
PubMed1965

Ribosomes: analysis by cesium sulfate gradient centrifugation.

Ribosomes from the cytoplasm of a mammalian tissue, HeLa cells, were partially purified and then concentrated into a zone by prolonged centrifugation in Cs(2)SO(4) solution (density, 1.43 grams per milliliter). Ribosomal preparations treated with bentonite prior to density-gradient centrifugation showed single peaks after centrifugation. Material recovered from the peaks represented aggregates of the original 74S particle.

Aluminum Silicates↗