Den'ichi Mizuno

Conservation of ribosomal fidelity during ageing.

The function of ribosomes prepared from the liver of young (2--5 months old) and senescent (15--26 months old) mice were compared in vitro. The conclusions that can be drawn from the liver of senescent mice is 10 to 40% lower than that of young mice; (2) the fidelity of translation does not change grossly with age; (3) the thermosensitivity of translational activity of ribosomes and the activity of ribosome-associated ribonuclease do not change with age; (4) there is an age-dependent accumulation of free 40S ribosomal subunits in the cytoplasm of mouse liver. The relation of age-dependent changes in the structure and function of ribosomes is discussed.

The turnover of ribosomal RNA of Escherichia coli in a magnesium-deficient stage

Abstract 1. 1. Escherichia coli was grown in a magnesium-deficient medium. The ribosomal RNA was labeled, and followed during separation by chromatography on methylated bovine serum albumin. Marked degradation of ribosomal RNA was observed, although the total amount remained constant. This means that there was a rapid turnover of ribosomal RNA during growth. 2. The degradation products of ribosomal RNA during magnesium deficiency first appeared in the region of soluble RNA on methylated bovine serum albumin chromatograms, but were later released into the culture medium. 3. When pulse labeled with 32 P in the magnesium-deficient stage, no DNA-type RNA was obtained, but heterogeneous RNAs appeared with nucleotide compositions of the ribosomal RNA type. These RNAs were presumed to be precursors of ribosomal RNA.

Increase in the ratio of 18S RNA to 28S RNA in the cytoplasm of mouse tissues during aging

The possibility of alterations in cytoplasmic RNA in mouse liver, kidney and brain during aging was investigated. The cytoplasmic RNAs in these organs gave similar profiles of optical density at 260 nm with three major peaks at 28S, 18S and 4S on sucrose gradient centrifugation. However, the ratio of the amounts of 18S and 28S RNA increased significantly with age in the brain and liver. The polyacrylamide gel electrophoretic patterns of extracts of the three tissues under both native and denaturing conditions were nearly identical regardless of the age of the animals. Since most of the minor components separated on gels were probably in vivo degradation products of ribosomal RNA, these results suggest that the extent of apparent and hidden breaks in ribosomal RNA does not change during aging.

The participation of ribonuclease in the degradation of Escherichia coli ribosomal ribonucleic acid as revealed by oligonucleotides accumulation in the phosphorus-deficient stage

Abstract 1. 1. Escherichia coli ribosomal RNA degradation during the phosphorus-deficient stage 1,2 was investigated by analysis of its degradation products. 2. 2. After exhaustion of the phosphorus in the medium, 32 P in the 50 % ethanolsoluble fraction of the bacterial cell increased greatly in parallel with the degradation of ribosomal RNA, while trichloroacetic acid-soluble 32 P did not. 3. 3. Soon after phosphorus exhaustion, most of the ethanol-soluble 32 P consisted of characteristic oligonucleotides. Later, inorganic phosphate increased gradually parallel with a decrease in oligonucleotides in ethanol extracts. 4. 4. The differences in the base ratios, the terminal structures and other properties of these oligonucleotides suggest that the breakdown of ribosomal RNA was caused mainly by ribosomal latent ribonuclease (EC 2.7.7.17) 3 . These oligonucleotides, in their turn, were found to be finally converted to inorganic phosphate and nucleosides by the cooperation of alkaline phosphomonoesterase (EC 3.1.3.1) 2,4 and cyclic phosphodiesterase 5,6 .

Ribosome degradation and the degradation products in starved Escherichia coli. I. Comparison of the degradation rate and of the nucleotide pool between Escherichia coli B and Q-13 strains in phosphate deficiency.

Abstract The differences in ribosomal degradation and the fate of degradation products between Escherichia coli wild-type (B) and a ribonuclease less mutant (Q-13) during phosphate starvation were compared. The degradation rate of 32P-labeled ribosomes was found to be of the same order at the early stage but to differ at the later stage of deficiency between Escherichia coli B strain and Q-13 strain. The degradation in Q-13 nearly stopped after 6-h starvation, while it continued for more than 10 h in B strain, resulting in more than 80% degradation of 32P-prelabeled ribosomes. In the acid-soluble 32P pool of Q-13 strain, there was an increase in the nucleoside monophosphates but no increase and a continuous decrease in nucleoside di-and triphosphates, respectively. Q-13 contained no nucleoside 2′,3′-cyclic phosphate or nucleoside 3′-phosphate and little oligonucleotide, all of which had been detected in B strain during this stage. From these facts it was suggested that ribosomal RNA degradation in Escherichia coli Q-13 is mainly caused by ribonuclease II in vivo.

Possible heterogeneity of the distribution of lysosomal marker enzymes among “lysosomal particles” of rat liver

Abstract The postnuclear supernatant and the crude lysosomal fraction were layered onto Ficoll gradients and centrifuged. After centrifugation ten fractions were ontained and each fraction was analyzed for protein, cytochrome oxidase, D -alanine oxidase and three to six acid hydrolases known as lysosomal enzymes. Latent and total activity were measured in the case of the acid hydrolases. Six marker lysosomal enzymes were distributed in broad patterns from the bottom to the top of the gradient on centrifugation of the postnuclear supernatant, though the markers of other particles were recovered in one or two fractions. After similar centrifugation of the crude lysosome fraction the ratios of the activity of three acid hydrolases differed, depending on their position on the gradient. These results indicate the presence of lysosomes heterogeneous in their content of acid hydrolases.

In vitro reassembly of functionally active 50 s ribosomal particles from ribosomal proteins and RNA's of Escherichia coli

Abstract Ribosomal RNA of NH 4 Cl-treated unfolded Escherichia coli ribosomes (25 s and 15 s particles) is completely hydrolysed to acid-soluble materials by RNase II from E. coli , while their proteins remain intact. The ribosomal proteins released during the complete digestion of ribosomal RNA re-formed the typical ribosomal subunits (50 s and 30 s particles) in vitro upon the addition of ribosomal RNAs (23, 16 and 5 s) prepared by phenol-ethanol extraction. In the reassembly process, ribosomal proteins of 50 s subunits origin were incorporated only into the re-formed 50 s particles and not into the 30 s particles, while those from 30 s were only found in the re-formed 30 s particles, as shown by 35 S-labelling of either 50 s or 30 s proteins. The reconstituted ribosomal particles were active in polypeptide synthesis directed by polyuridylic acid and f2 phage RNA. The 5 s RNA is required for the reassembly of 50 s subunits. The reassembly of 50 s subunits depends either on the presence of 30 s particles or on the simultaneous reassembly of 30 s subunits from 16 s RNA and proteins.

Mechanism of colicin E2-induced DNA degradation in Escherichia coli

Abstract During colicin E2-induced degradation of DNA in Escherichia coli W3110, no activation of known deoxyribonucleases or change in the subcellular locations of these enzymes was observed. In the initial stage of DNA degradation, when only a small percentage of the total DNA had been degraded to acid-soluble materials, endonucleolytic breakage of DNA strands could be detected by centrifugal analysis in alkaline and neutral sucrose density gradients, even in Escherichia coli K-12, Strain 1100, which lacks endonuclease I. In this colicin-induced breakage, both strands of DNA rather than just one were broken since the molecular weight of broken DNA observed in alkaline sucrose gradients was half that in neutral gradients. The resulting broken strands of DNA separated in an alkaline sucrose gradient could not be repaired even when degradation of DNA was stopped in the initial stage by digestion of colicin with trypsin. This initial breakage of DNA strands appeared to be the primary lethal action of colicin E2 in Escherichia coli because the extent of breakage was inversely proportional to the survival of the bacteria.

Turnover of ribosomal RNA in a ribonuclease I-less mutant of Escherichia coli, Q-13, which was found to possess polynucleotide phosphorylase.

Abstract 1. 1. A completely synthetic medium was found for Escherichia coli Q-13 (ribonuclease I-less mutant), containing methionine, tryptophan, tyrosine and leucine. 2. 2. Ribosomal RNA degradation was observed in Q-13 in phosphate- and magnesium-deficient states. All the patterns coincided well with those in the wild type of E. coli. 3. 3. When pulse-labelled with [14C]uracil, RNA gave [14C] UDP and [14C]UMP as degradation products in the presence of Na2HPO4. When labelling with [14C]adenine was used, a very small amount of [14C]ADP was detected among the degradation products. 4. 4. E. coli Q-13 contains higher nucleoside diphosphate pyrophosphatase activity than the wild strain. Evidence is given that it masked the polynucleotide phosphorylase (EC 2.7.7.8) actually present in E. coli Q-13. 5. 5. It is proposed that the enzymes responsible for the degradation of ribosomal RNA and messenger RNA are polynucleotide phosphorylase and ribonuclease II (EC 3.1.4.1.) in both the wild and mutant strains used.

Unfolding of Escherichia coli ribosomes by phosphate ion in the presence of oligonucleotides.

Two discrete changes in shape of Escherichia coli ribosomes were observed when they were incubated for short periods with various concentrations of Na2HPO4 and oligonucleotides (oligonucleotides alone had been shown to stop the degradation of ribosomes completely). A temperature of 37 °C and phosphate ion were essential for this reaction. No significant loss of protein or degradation of RNA was observed in this reaction, suggesting that the ribosomes unfolded on addition of phosphate ion. The change was not reversible on dialysis against magnesium buffer and the particles were more sensitive than normal ribosomes to pancreatic RNase and RNase II purified from Escherichia coli Q13. n nIt is suggested that a conformational change of the ribosomes occurs prior to the ribosome degradations observed in vivo in various bacteriostatic conditions.