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Biomedical subjects

L A Luchkina

Publications and source records attributed to L A Luchkina.

18 recordsLinked to original sources
PubMed2000

PCR hot start using primers with the structure of molecular beacons (hairpin-like structure).

A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.

Base Pairing↗
PubMed1999

[Hot start of the polymerase chain reaction using DNA helicase].

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.

DNA Helicases↗
PubMed1992

[Polymerase chain reaction with universal primers for studying genomes].

Universal primer ability of generating conservative and variable UP-PCR (universally primed polymerase chain reaction) species-specific patterns was analysed on bacteria to serve as an example. Also, two important properties of the UP-PCR patterns (species/primer DNA hybridization specificity) are characterized.

Base Sequence↗
PubMed1986

[Apurinic-apyrimidinic DNA-endonuclease activity of cytochrome c and pancreatic RNAse].

Cationic proteins--cytochrome c and pancreatic RNAase--possess the apurinic-apyrimidinic DNA-endonuclease activity. The affinity of these proteins for DNA-apurinic sites does not differ from that of specific apurinic DNA-endonucleases described in literature. The main features of the apurinic activity of cationic proteins are as follows: low specific activity, high temperature optimum of the reaction, absence of primer-stimulated activity. The feasibility of participation of cationic proteins and some other nucleophilic compounds in single-stranded breaks production in apurinic DNA is discussed.

Animals↗
PubMed1985

Uracil-DNA glycosylase of thermophilic Thermothrix thiopara.

An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria. The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a native molecular weight of 26,000 and existed as a monomer protein in water solution. The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100. It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide. The purified enzyme did not contain any nuclease that acted on native or depurinated DNA. The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C. The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C. Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation. One T. thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C.

Bacteria↗
PubMed1985

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.

Ammonium Sulfate↗
PubMed1984

Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae.

Cell-free extracts from wild-type yeast (RAD+) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4 -1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD37 (about 10(4) PD per haploid genome).

DNA, Fungal↗
PubMed1982

[Radiosensitive lines of Drosophila. V. The sensitivity of mutant mus(2) 201G1 to methyl methanesulfonate and UV radiation and disordered DNA repair in UV-irradiated cells].

The characteristics of the mus(2)201G1 mutant controlling the mutagensensitivity of Drosophila were studied. The data obtained on the survival of flies affected by MMS of UV light during the larval stage of the development show that this mutation determines the extremally high sensitivity of early and late larvae of Drosophila to the lethal action of both mutagens. The primary embryonic cell cultures were used to estimate the influence of mus(2)201G1 locus on the repair of radiation-induced genetic damages. The rate of excision of UV-induced pyrimidine dimers in the DNA was examined and shown to be significantly lower in mutant cells, as compared with those of the while type files. Incomplete elimination of photodimers is, apparently, due to the defect of the UV-specific endonuclease, since the extracts of mutant cells reveal the 4-5 fold decrease in the corresponding enzymatic activity.

Animals↗
PubMed1981

Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus.

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.

Cations↗
PubMedDate not supplied

[Method for producing spheroplasts from yeast cells].

A method for producing spheroplasts from yeast cells has been developed. The method involves: 1) prefreezing and thawing of cells in the minimal nutrient medium; 2) treatment with the mixture of 86 mM 2-mercaptoethanol, 5% sodium dodecylsulphate and 6% Triton X-100; 3) enzymic digestion by the digestive juice of grape helix. The formation of spheroplasts was controlled by microscopy and measurement of the optic density of the spheroplast suspension in the incubation medium and in 1% sodium dodecylsulphate. The method has been successfully tested on some bakery yeast: Saccharomyces cerevisiae, Saccharomyces paradoxus, Schizosaccharomyces pombe, Saccharomyces globosus, Saccharomyces carlsbergensis.

Ascomycota↗