Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures.

A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37-80 kb subpopulation, which was predominant in whole plants, to 18-34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5-10 kb significantly increased in suspension culture cells. In addition, a new peak (10-12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4-2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.

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.

A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase.

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.

Control of bacteriophage phi X 174 gene expression by the transcription termination factor rho in vitro.

The effect of rho factor on bacteriophage phi X 174 gene expression was studied in a cell-free, DNA-dependent, RNA-directed protein synthesizing system (transcription-translation coupled system) using S30 extracts of wild-type and rho-mutant Escherichia coli cells. It has been found that in the presence of a functionally active and endogenous or added rho factor, only a limited number of the viral genes (A, B, and D) are expressed effectively, whereas the expression of genes, F, G, and H, which encode capsid proteins, is strongly inhibited. Mutational or thermal inactivation of rho factor results in a considerable activation of capsid protein synthesis and, at the same time, in some depression of the synthesis of gene A, B, and D products. From the results of this study it is suggested that phage phi X 174 has two classes of gene, late and early, which are separated on the chromosome by a rho-dependent transcription terminator. We propose that the expression of these classes is regulated by a termination-antitermination mechanism.

Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene.

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images

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.

Properties of Bacillus subtilis ATP-dependent deoxyribonuclease.

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.