Keiichi Nozu

Mechanism of post-irradiation degradation of deoxyribonucleic acid in a radiosensitive Escherchia coli (NG30) irradiated with ultraviolet light☆

The post-irradiation degradation of DNA in a radiosensitive mutant, NG30, of Escherchia coli, was investigated. An extensive degradation of DNA corresponding to the ultraviolet dose was observed. The post-irradiation degradation of DNA having no replicating points is not initiated from a specific point in the DNA, but does occur simultaneously at many sites of the irradiated DNA. The proposal that the initial cause for DNA degradation is the excision of pyrimidine dimers was supported by the following experimental results: (1) an immediate fragmentation of cellular DNA was observed in the cells shortly after ultraviolet irradiation by an alkaline sucrose-gradient analysis, (2) pyrimidine dimers were detected in the acid-soluble fraction from the cells incubated for only five minutes after ultraviolet irradiation, (3) post-irradiation degradation of DNA was markedly suppressed in a double mutant of E. coli, NG30uvrC, and (4) caffeine significantly inhibited the post-irradiation degradation of DNA. It is also shown that the extensive DNA degradation which follows, must result from the unsuccessful repair of gaps in the DNA strands produced by excision of pyrimidine dimers.

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.

Ultraviolet effects on biological function of RNA phage MS2.

Abstract Ultraviolet effects on the biological function of MS2 phage were investigated. The adsorption of MS2 phage to the host cells is not affected by ultraviolet irradiation of the phage, but the penetration of phage RNA is reduced. Hydrated uridine was detected in the RNA molecules of ultraviolet-irradiated MS2 phage. The sedimentation patterns of the parental phage RNA molecules extracted from Escherichia coli at 12 min after infection with ultraviolet-irradiated phage are heterogeneous in size. However, ribonuclease-resistant RNA duplex (RF) detectable in cells infected with non-irradiated MS2 phage is not found in these cells. The phage RNA in the cells infected by ultraviolet-irradiated MS2 phage is fragmented into small size molecules even when the infected cells were incubated with chloramphenicol; no degradation of parental phage RNA is observed in cells infected with non-irradiated MS2 phage under the same conditions. Ultraviolet-irradiated MS2 phage also might be able to induce an RNA replicating enzyme (RNA replicase) in the host cells, although the enzymic activity seems to be lower than that induced in the cells infected with non-irradiated MS2 phage.

On the ribosomal subparticles formed in Escherichia coli Bs-1 irradiated with ultraviolet light: Formation and biological function of small particles

Abstract The formation and biological function of small ribosomal subparticles in Escherichia coli B s-1 irradiated with ultraviolet light were investigated. The experimental results are as follows: 1. 1. Ultraviolet irradiation inhibited both the formation of 50-S ribosomal subunits in E. coli B s-1 and the synthesis of 23-S rRNA. 2. 2. No recovery of the synthesis of either 50-S ribosomal subunits or 23-S rRNA was found in ultraviolet-irradiated E. coli B s-1 even when the bacteria were incubated for 2 h after the irradiation. 3. 3. Synthesis of 16-S rRNA was resistant to ultraviolet irradiation, and synthesis of ribonucleoprotein particles did not correspond to the smaller counterpart in 70-S ribosomes. However, the ribonucleoprotein particles formed were always smaller than 30 S. Higher ultraviolet doses formed smaller particles; the smallest particles observed were about 20 S. 4. 4. The sedimentation coefficient of the RNA component of these small particles was 16 S in all cases. The base composition of the 16-S RNA of small particles did not differ from that of normal 16-S rRNA which was the RNA component of 30-S ribosomal subunits. The base sequences of both 16-S RNA and 16-S rRNA were identical. 5. 5. The small particles were not able to form complexes with 50-S ribosomal subunits and had no ability to bind poly U and T2 phage-specific mRNA. 6. 6. The amount of protein component in the ribosomal particles was less in the small particles than in normal 30-S ribosomal subunits.

Base complementarity between rapidly labeled RNA in Escherichia coli B infected with ultraviolet-irradiated T2 phages and T2-DNA

Abstract The amounts of hybrid between the rapidly labeled RNA formed in Escherichia coli B after infection with ultraviolet-irradiated T 2 phage and the denatured T 2 -DNA were less than those between the RNA specific for the non-irradiated T 2 phage and the T 2 -DNA. The amount of hybrid in the former couple decreased with the increase of ultraviolet dose on T 2 phages. The results of saturation and competition experiments on hybrid formation made it clear that the base sequence of the rapidly labeled RNA in the bacteria infected with ultraviolet-irradiated T 2 phages was different from that of normal T 2 messenger RNA. The reduced formation of hybrid between the T 2 -DNA and the UV-T 2 -RNA was caused by a decreased base complementarity to the T 2 -DNA in large molecules of UV-T 2 -RNA.

Ribosome-bound RNA polymerase of Escherichia coli

Abstract DNA-dependent RNA polymerase (nucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) is associated with conventional ribosomes prepared from Escherichia coli K-10 in the presence of deoxyribonuclease (EC 3.1.4.5). The enzyme is not released from the ribosomes by repeated washing with 10 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl 2 , nor by dissociation of the ribosomes into two sub-units in the presence of 0.1 mM MgCl 2 . The enzyme can be released from the ribosomes in the presence of 0.5 M NaCl. However, removal of NaCl causes a re-association of the released enzyme with the ribosomes. Association between the enzyme and the ribosomes takes place in the absence of NaCl even when the ribosomes have been washed with NaCl. The enzymic properties of the ribosome-bound RNA polymerase are indistinguishable from those of the purified DNA-dependent RNA polymerase prepared from the whole cells by the procedure of Chamberlin and Berg . Since the enzyme of Chamberlin and Berg also associates with the ribosomes, and since the ribosomes prepared from the cells in the absence of deoxyribonuclease contain only a small amount of RNA polymerase, it appears that the presence of RNA polymerase on the ribosomes has resulted from the disruption of the cells in the presence of deoxyribonuclease.