[Immunological similarity of diphtheria toxin and EGF receptor].

Cardiomyopathy and neuropathy are the two commonly observed complications in diphtheria patients and in, some instances, individuals vaccinated against diphtheria. The nature of these complications remains not well understood. It was suggested that autoimmunity may play a role in the development of these afflictions. Based on functional similarities between diphtheria toxin (DT) and epidermal growth factor receptor (EGFR), which both can bind to the heparin-binding EGF-like growth factor (HB-EGF) precursors, we suggested that antibodies developed against DT can cross react with EGFR. Here, using serum from healthy donors (n = 10) and diphtheria patients (n = 15), we demonstrated that B-subunit of DT has the antigenic epitopes similar to those of EGFR. Diphtheria toxin as well as EGFR could be recognized by antibodies raised against EGFR and by serum antibodies from diphtheria patients. Moreover serum of diphtheria patients competitively inhibits binding of anti-EGFR antibodies to the receptor. The truncated diphtheria toxin without B-subunit could be detected by serum antibodies of diphtheria patients, but not by anti-EGFR antibodies. Collectively, these studies demonstrate cross-reactivity of antibodies raised against B-subunit of DT and extracellular domain of EGFR and suggest that clinically observed post-diphtheria complications may result from autoimmune inhibition of EGFR function and possible destruction of receptor-positive tissues.

Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells.

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.

Camptothecin enhances random integration of transfected DNA into the genome of mammalian cells.

In order to study the involvement of DNA topoisomerase I (top1) in recombination, we examined the effect of the anti-neoplastic drug camptothecin, which selectively poisons top1 by trapping top1-cleavable complexes on integration of exogenic vector into the genome of mammalian cells. We transfected mouse F9 teratocarcinoma cells as well as Chinese hamster V79 cells with a plasmid carrying a selectable neo gene treated with camptothecin, and determined the frequency of neo+ (G418(R)) colonies. We found that treatment with camptothecin for as short a time as 4 h after electroporation resulted in a 4- to 33-fold stimulation of plasmid integration into the recipient genome via non-homologous recombination. These results imply that top1-cleavable complexes trapped by camptothecin could be potentially recombinogenic structures and could stimulate non-homologous recombination in vivo, promoting the integration of transfected plasmids into mammalian genome.

[Hot start of the polymerase chain reaction using DNA helicase]. / Goriachii start polimeraznoi tsepnoi reaktsii pri pomoshchi DNK-helikaz.

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.

[Effect of inhibitors of topoisomerases and poly(ADP-ribosylation) on homologous and non-homologous integration of exogenous DNA in genes of mammalian somatic cells]. / Vliianie ingibitorov topoizomeraz i pol(ADF-ribozilirovaniia) na na gomologichnuiu i negomologichnuiu integratsiiu ékzogennoi DNK v genom somaticheskikh kletok mlekopitaiushchikh.

A study was made of the influence of inhibitors of poly(ADP-ribose)polymerase, topoisomerase I and topoisomerase II on the frequency of gene targeting of hprt gene as well as on the frequency of random integration of targeting vector pRV9.1 into genome of mouse F9 teratocarcinoma cells. We found that the treatment of cells with the inhibitor of poly(ADP-ribose)polymerase 3-aminobenzamide after electroporation resulted in 3-4-times increase of homologous integration of exogenic vector into chromosomal DNA, and did not affect the frequency of random insertion of transfected DNA. The treatment of cells after electroporation with inhibitors of topoisomerases VP-16, ICRF-193 enhanced random integration of transfected DNA but exerted no effect on the frequency of gene targeting in this experimental system.

[The production of clones of "man x Chinese hamster" hybrid cells containing different parts of the human genome]. / Poluchenie klonov gibridnykh kletok "chelovek x kitaiskii khomiachok", soderzhashchikh raznye chasti genoma cheloveka.

Some approach has been described to create hybrid cell lines (human x Chinese hamster) which contain different parts of human genome, and then efficiently to reveal and isolate the human DNA from these. This method involves the introduction of a selective marker in different sites of the human cell genome, by transfecting them with plasmid SV2neo, and the use of flow cytometry and DNA polymerase chain reaction with primers specific only for human DNA.