Synthesis, cloning and expression of genes for antibacterial peptides: Cecropin, magainin and bombinin

Summary Three DNA sequences encoding the antimicrobial peptides bombinin, cecropin and magainin were synthesised. DNA fragments were cloned into pET-21d plasmid under T7 promoter for expression in vivo and in vitro and into pRIT-2T plasmid for expression as a fusion product with protein A. The polypeptides synthesised in both systems possess antibacterial activity.     

The flexible region of protein L12 from bacterial ribosomes studied by proton nuclear magnetic resonance

The dimeric protein L7/L12 from bacterial ribosomes has a highly elongated and flexible structure. We have, using 1H NMR methods, analyzed the extent of the flexible region and also the size of the organized structures of the molecule. A number of mutants of the protein as well as monomeric and dimeric forms of the protein and a COOH-terminal fragment have been used for the identification of certain resonances. Thus, residues 37-50 were found to be highly mobile whereas the amino-terminal and COOH-terminal regions are organized into folded domains. The flexibility between the domains and its relation to functional properties of the protein are discussed.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

Variability of human rRNA genes: inheritance and nonrandom chromosomal distribution of structural variants of nontranscribed spacer sequences

Summary Human rRNA genes contain variable regions, one of which is located in nontranscribed spacers (NTSs) closely downstream from the 3-end of the transcribed region. This polymorphism may be detected by means of blot hybridization analysis as a set of distinct restriction fragments corresponding to this part of the rRNA genes. We have analyzed DNA of 51 individuals and found eight structural NTS variants of this region; two of these were common to all individuals analyzed, and six others were found in different combinations and with different frequencies. The copy number of each variant also differed but was not less than 10–20 copies per cell. The analysis of DNA isolated from leukocytes of the members of 11 families indicated that some of the structural variants (of the NTS region) are inherited as a single Mendelian locus. We propose that rRNA genes that belong to one particular structural variant form clusters on separate chromosomes. To test this proposition, we developed a combined method, including AgNO₃-staining of chromosomes, in situ hybridization, and DNA analysis with methylation-sensitive restrictases, and used it for study of persons who had methylated rRNA genes located on AgNO₃-negative nucleolar organizers. It was found that in three of four cases methylated genes really belonged to one structural variant. This approach may be used for detailed localization of separate classes of NTS structural variants of human rRNA genes.     

Comparison of the physical properties of ribosomal proteins from Escherichia coli 50S subparticles isolated by different methods

Physical properties of ribosomal proteins obtained with or without denaturing agents were compared. CD measurements and PMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturing agents. CD and PMR data of L1, L6, L11, L23, L25 and L29 obtained by us under denaturing conditions practically coincide with the data for these proteins obtained in "mild" conditions and published in the literature. These findings indicate that the differences of physical properties reported in the literature can be due to different procedures of protein renaturation rather than to the methods of their isolation.     

The N-terminal sequence protein of L7/L 12 is responsible for its dimerization.

Ultracentrifuge studies of intact protein L7/L12, of its fragments 27--120, 1--74 and 74--120 and of protein L7/L12 with oxidized methionine residues, indicate that the N-terminal sequence of the protein L7/L12 is responsible for its dimerization. The symmetry model of the dimer is discussed.     

Oxygen effect in heat-induced DNA damage

The major kinds of heat-induced damage to DNA (depurination, guanine oxidation to 8-oxoguanine, cytosine deamination to uracil) were shown to depend in their extent on the oxygen content in solution. Formation of hydrogen peroxide in water upon heating was enhanced in the presence of D₂O and decreased by various scavengers of singlet oxygen, corroborating the involvement of ¹O₂ in the thermal generation of reactive oxygen species. The aggregate data indicate that all kinds of heat-induced DNA damage in solution arise through this common mechanism.