Site-directed mutagenesis in the uracil-repair system. Preparation of mutated forms of human alpha 2 interferon

The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system. To intensify the process the procedure of the uracil-containing DNA template preparation is modified. It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency. It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations.     

Kemantane pharmacokinetics in rats

Pharmacokinetics of novel immunostimulating drug kamantane was studied by using gas-liquid chromatography in experiments on rats. It was found that kemantane biotransformed rapidly after oral administration with the forming of active metabolite. Kemantane and its metabolites are distributed rapidly from the blood to organs. The drug is eliminated from the organism of rats as metabolite.     

Regulation of apolipoprotein expression]

Recent data and concepts on the structure and functioning of apolipoprotein genes as well as on the role of hereditary factors in pathogenesis of atherosclerosis are reviewed. The most important variants of inherited disorders in the system of apolipoproteins promoting the development of atherosclerosis are considered. Special attention is paid to the RFLP of apolipoprotein genes which serves as a peculiar genetic background, increasing probability of the atherosclerosis development in certain individuals.     

A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.     

Neurospecific and neuron-specific enolase levels in human nerve tissue at early stages of embryogenesis

Neurospecific and neuro-nonspecific isozymes of glycolytic enzyme enolase known to be molecular markers of neuronal and glial cells respectively were isolated from human brain. Using immunoenzyme assay, the content of these proteins was measured in the brain of 7-13-week human embryos and adult subjects. The content of both isoenzymes gradually increases attaining constant level in 10-13-week embryos. Relative content of neuro-nonspecific enolase in the brain of 10-13-week embryos is 1 1/2 times higher than that in adults, whereas the content of neurospecific enolase amounts only to 2% of its level in the adult brain.     

Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.