RNA polymerase-dependent mechanism for the stepwise T7 phage DNA transport from the virion into E. coli.

The influence of rifampicin, streptolydigin, tetracycline and chloramphenicol on phage DNA transport from the T7 virion into the E. coli cell was studied. It has been found that the DNA transport proceeds in at least three stages. During the initial stage the phage injects into the host cell the left approximately 10 per cent of its DNA molecule. The entrance of the next 50 per cent of 17 DNA molecule is blocked by inhibitors which block transcription but not translation. Moreover, the entrance time of this part of the T7 DNA increases in the case of the T7 mutant D111 (which contains a deletion of the A2 and A3 promoters) and decreases in the case of the D53 mutant (which contains a deletion in the region of the early gene transcription terminator). It would appear, that the second stage of the phage DNA transport is tightly coupled with its transcription and that a mechanical function is carried out by RNA polymerase. The translation inhibitors completely block the entrance of the remaining 40 per cent of the 17 DNA molecule (class III genes) into the host cell. It would appear that some class I and (or) II gene product(s) are obligatory components of the final stage of 17 DNA transport. Some probable consequences of this virus DNA transport model as well as its agreement with the functional structure of T7 chromosome and with T7 development are discussed

On the promoter complex formation rate of E. coli RNA polymerases with T7 phage DNA.

Influence of ionic strength on the kinetics of the promoter complex formation between E. coli RNA polymerase and T7 phage DNA was investigated using a membrane filter assay. The enzyme-promoter association rate constant was determined. It varies from 10(9) to 3 x 10(7) M-1 sec-1 when the ionic strength is changed from zero to 0.15 M NaCl. Basing on the theoretical analysis of experimental data obtained the model for the promoter site selection assuming the enzyme sliding along the DNA is discussed

On the flexibility of the boundaries between the A-form and B-form sections in DNA molecule.

The degree of orientation of DNA in a flow has been studied within the interval of the B - A transition induced by ethanol. The orientation of the B DNA (60-65% ethanol, v/v) and that of the A DNA (80-82% ethanol) are nearly identical. This means that both conformations have similar persistence lengths and that there is no aggregation in the course of formation of the A form. Within the transition range (65-78% ethanol) the orientation attains a sharp minimum which coincides with the half-transition point (73% ethanol). The cooperative character of the B - A transition presupposes the existence of boundaries between the alternating sections of the A and B conformations that may entail an increased flexibility of the DNA molecule and a corresponding drop of orientation. Theory predicts an elliptical dependence of the number of boundaries on the proportion of the A form. The experimental degree of orientation follows the same pattern. Quantitative evaluation shows that the flexibility of a boundary is small, so that several dozen of boundaries are required to simulate free rotation

Nigellothionins from Black Cumin (Nigella sativa L.) Seeds Demonstrate Strong Antifungal and Cytotoxic Activity

High-cationic biologically active peptides of the thionins family were isolated from black cumin (Nigella sativa L.) seeds. According to their physicochemical characteristics, they were classified as representatives of the class I thionin subfamily. Novel peptides were called “Nigellothionins”, so-called because of their source plant. Thionins are described as components of plant innate immunity to environmental stress factors. Nine nigellothionins were identified in the plant in different amounts. Complete amino acid sequences were determined for three of them, and a high degree of similarity was detected. Three nigellothionins were examined for antifungal properties against collection strains. The dominant peptide, NsW2, was also examined for activity against clinical isolates of fungi. Cytotoxic activity was determined for NsW2. Nigellothionins activity against all collection strains and clinical isolates varied from absence to a value comparable to amphotericin B, which can be explained by the presence of amino acid substitutions in their sequences. Cytotoxic activity in vitro for NsW2 was detected at sub-micromolar concentrations. This has allowed us to propose an alteration of the molecular mechanism of action at different concentrations. The results obtained suggest that nigellothionins are natural compounds that can be used as antimycotic and anti-proliferative agents