Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.     

Purification of a calcium dependent ribonuclease from Xenopus laevis.

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.     

The inhibition of ribonuclease activity and the isolation of polysomes from leaves of the french bean, Phaseolus vulgaris

The effect of inhibitors on the ribonuclease activity of soluble and microsomal fractions of bean leaves has been examined. The soluble ribonuclease activity could be completely inhibited by Zn²⁺, Cu²⁺, bentonite, and diethylpyrocarbonate, although these inhibitors had little effect on the microsomal ribonuclease activity. Ribonuclease activity in the soluble fraction was completely inhibited by guanosine 2′(3′)-monophosphate, which was the first nucleotide to accumulate on degradation of yeast RNA. Adenosine 2′(3′)-monophosphate, the first nucleotide to accumulate on degradation of yeast RNA by the microsomal preparations, completely inhibited the ribonuclease activity of the microsomal fraction.The ribonuclease activity of both enzyme preparations was completely inhibited by an analog of the transition state of the ribonuclease reaction, a complex of guanosine and vanadyl sulfate. Inclusion of this complex in homogenization media markedly increased the proportion of polysomes isolated from bean leaves.     

Ribonucleic Acid Dependent Ribonucleic Acid Replicase in the Immune Response

An immune ribonucleic acid (iRNA) preparation was made using phenol extracts of spleens of mice previously immunized with Salmonella tennessee flagella. An enzyme, also prepared from the spleens of these mice, induced the incorporation of ³H-UTP into the acid-insoluble fraction in a cell-free system in the presence of this RNA. The enzyme activity could be demonstrated from the spleens of immunized mice but not from normal ones, and this activity was also inhibited by two derivatives of rifamycin. Treatment with ribonuclease or heating at alkaline pH resulted in a loss of activity in added RNA. The ³H-uridine-labeled product was found resistant to ribonuclease treatment but became sensitive when the product was subjected to heat treatment. However, actinomycin D, mitomycin C or bleomycin A₂ did not inactivate the enzyme activity. These results suggest that this enzyme induces the incorporation of UTP into the acid-insoluble fraction using iRNA as a template and the product may be a newly synthesized RNA which forms a hybrid with iRNA. This enzyme activity may play a role in the antibody formation process, and may account for the in vivo replication of iRNA by this enzyme, viz., probably an RNA-dependent RNA replicase.     

Ribonucleic Acid Polymerase Induced in Cells Infected with Sendai Virus

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.     

Studies on the chromatin of barley leaves during senescence

1. The activity of soluble ribonuclease and deoxyribonuclease first declined during senescence, but later increased during advanced stages of senescence. 2. Young leaves had very low ribonuclease or deoxyribonuclease activity associated with the chromatin, but the activity of these enzymes increased progressively during senescence until the leaves died. 3. No significant changes in the composition of chromatin from first seedling leaves of barley plants during aging (from 7 to 25 days) were noted. 4. The amount of RNA synthesized by chromatin in vitro declined as the leaf aged. However, if the loss of RNA due to chromatin-associated ribonuclease was taken into account, the RNA-synthesizing activity of chromatin from senescing (15-16-day-old) leaves appeared to be somewhat higher than that of chromatin from young (7-8-day-old) leaves. In leaves at the terminal stages of senescence (23 days old) the estimates of RNA synthesis by chromatin could not be made owing to complications created by high nuclease activities. 5. It is suggested that senescence may be triggered by a decline in some hormonal factor in leaves, and that the resulting production of chromatin-associated deoxyribonuclease and ribonuclease in increasing proportions may progressively cause increased degradation of DNA and newly synthesized RNA, so that ultimately the cellular functions are impaired and the cells die.     

Ribosomal RNA turnover and the level of ribonuclease activity in the liver of the pregnant rat.

Studies were undertaken on the turnover of ribosomal RNA and on ribonuclease activity in the liver of the pregnant rat in an attempt to explain the accumulation of liver RNA which occurs during the latter half of pregnancy. Between the 15th and 20th day of gestation the rate constant of degradation, biological half-life and daily rate of synthesis of ribosomal RNA were calculated to be 0.0887, 7.81 days and 6.21 mg per liver per 100g body weight respectively. Corresponding values in non-pregnant rats were 0.123, 5.68 days and 3.47 mg per liver per 100g body weight. The increase in RNA was therefore associated with an increase in its rate of synthesis and a decrease in its rate of breakdown. From the 14th day of pregnancy there was a decrease in alkaline ribonuclease activity and a marked increase in the level of alkaline ribonuclease inhibitor. The activity of acid ribonuclease was found to increase and that of acid phosphatase to decrease during this period.     

The subcellular localization of the changes in ribonuclease and phosphatase activities of the mouse kidney produced by castration and testosterone

Male mice were castrated at 2 months of age and a pellet of testosterone propionate was implanted subcutaneously ten days later. After 14 days, normal, castrated and androgen treated mice were autopsied simultaneously. The kidneys of each group were homogenized, pooled and fractionated by centrifugation into the various subcellular components. The alkaline ribonuclease and phosphatase activities were localized in the microsomal fraction and changed in inverse relationship to the previously observed changes in the rate of protein biosynthesis and the concentration of microsomal and polysomal RNA induced by castration and androgen administration. Esterase activity also was localized in the microsomal fraction and its specific activity decreased after castration and was restored to normal by testosterone. The activities of acid ribonuclease, acid phosphatase and the several marker enzymes (glucose-6-phosphatase, nucleotidase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase) changed in direct proportion to the changes in weight of the kidney after castration and androgen stimulation.     

Ribonuclease H: a Ubiquitous Activity in Virions of Ribonucleic Acid Tumor Viruses

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.     

In vitro synthesis of type IV procollagen

Total RNA was isolated from parietal endoderm cells of 131/2-day mouse embryos that synthesize large amounts of type IV procollagen. In vitro translation of this RNA in the reticulocyte lysate supplemented with a ribonuclease inhibitor yielded two equally prominent polypeptides of Mr = 165,000 and 168,000, immunoprecipitable with anti-mouse type IV collagen serum. The Mr = 165,000 polypeptide was shown by one-dimensional peptide mapping to represent an unmodified chain of type IV procollagen. The Mr = 168,000 polypeptide, the in vitro synthesis of which was barely detectable in the absence of a ribonuclease inhibitor, most likely represents the other genetically distinct chain of type IV procollagen. Similar results to those described were also obtained using poly(A) + RNA prepared from murine F9 embryonal carcinoma cells induced to differentiate in vitro into parietal endoderm.     

The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.     

Porcine thyroid cytosolic, latent alkaline ribonuclease: resistance to protein denaturants.

1. A ribonuclease isolated from porcine thyroid cytosol using phenol: sodium dodecylsulfate treatment was associated with RNA and identical to latent alkaline ribonuclease. 2. Distribution of activity between aqueous and phenolic phases depended on pH, RNA, and ribonuclease inhibitor. 3. The ribonuclease was totally resistant to urea, guanidinium: HCl, chloroform:isoamyl alcohol, ethanol, heating at 100 degrees C for 10 min or at 80 degrees C plus 100 mM NaCl. It was highly resistant to hydrolysis by proteinase K except in the presence of detergent. 4. The extreme stability and other properties of latent alkaline ribonuclease could be the result of its association with RNA.     

Characterization of ribonuclease P isolated from rat liver cytosol.

Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.     

An Escherichia coli ribonuclease which removes an extra nucleotide from a biosynthetic intermediate of bacteriophage T4 proline transfer RNA.

The biosynthesis of bacteriophage T4 tRNAPro, tRNASer, and tRNAIle requires enzymatic removal of extra nucleotides from the 3' terminus of the respective precursor RNAs. A ribonuclease activity capable of catalyzing such reactions has been partially purified from uninfected Escherichia coli using an artificial precursor RNA as substrate. A number of ribonuclease activities were resolved during purification. Use of E. coli strain BN, a mutant known to be deficient in the relevant ribonuclease activity, permitted us to identify it in wild-type cells. This activity was designated the BN ribonuclease. BN ribonuclease had an apparent molecular weight of 35,000 as measured by Sephadex gel filtration. Mg2+ was required for activity, which was optimal at [Mg2+] of 2mM. Activity did not require monovalent cations K+ or Na+. BN ribonuclease was less efficient at removing extra residues in the biosynthesis of tRNASer and tRNAIle than in the biosynthesis of tRNAPro.     

Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I.

It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49, the putative yeast equivalent of bovine ribonuclease H IIb. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H IIb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H IIb epitopes. This observation suggests that a protein displaying ribonuclease H IIb antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.     

Protease and ribonuclease activities in bovine pituitary lobes

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin-Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3-9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a ;cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.     

Changes in Ribonuclease Activity and Content of RNA during Dormancy and Growth of Bird-cherry Buds

Ribonuclease has been extracted from buds of bird-cherry (Prunus padus L.) by a three-step method. In step 1 ribonuclease was extracted from the homogenized tissue at p^H 6. In step 2 the tissue residue from (1) was extracted at p^H 7. In step 3 the tissue residue from (2) was extracted with a buffer of p^H 8.5 and containing 0.7 M KCl. The ribonuclease from step 1 and 2 is named soluble and that from step 3 bound ribonuclease. The activity of the soluble ribonuclease was very low in October and increased slowly until March. When the buds sprouted in April the activity of this fraction rose rapidly. The activity of the bound ribonuclease was rather high in October and increased continuously from autumn to spring. RNA accumulated slowly between autumn and spring. The change in activity of the two ribonuclease fractions is discussed in relation to development.     

Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Mycobacterium tuberculosis

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis. J. Bacteriol. 91:2146-2154. 1966.-The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenediaminetetraacetic acid (EDTA) alone, EDTA and pancreatic ribonuclease, or with trypsin and ribonuclease. After each of these treatments, the ribosomal fractions were injected intraperitoneally into male CF-1 mice to test their capacity to produce an immune response to infection with virulent tubercle bacilli, strain H37Rv. Removal of protein with trypsin left the immunogenicity unchanged; EDTA alone reduced immunogenicity in the smaller vaccinating doses; EDTA plus ribonuclease reduced the immunogenicity by approximately 50% in the highest (1.0 mg) vaccinating dose; ribonuclease alone, after treatment with trypsin, reduced immunogenicity also approximately 50%. A crude mycobacterial ribonucleic acid (RNA) was prepared by extraction of the ribosomal fraction with alcohol. This RNA preparation was as effective in producing an immune response as the ribosomal fraction from which it was prepared, unless the RNA was partially or completely degraded during the preparation. The effect of ribonuclease on the immunogenicity of the RNA was similar to that obtained with the ribosomal fractions, except that ribonuclease completely destroyed the immunogenicity of a partially degraded RNA. RNA appears to be an essential part of an immunizing substance in attenuated tubercle bacilli, which produces a high degree of immunity in mice; 50 mug (dry weight) will protect approximately 80% of the mice, and as little as 0.5 mug will protect approximately 30% of the mice. Mycobacterial RNA not incorporated in Freund's incomplete adjuvant was nonimmunogenic. Yeast RNA incorporated in Freund's incomplete adjuvant was not immunogenic.     

Properties of a ribonuclease from Aedes aegypti larvae

1. The properties of a soluble ribonuclease from Aedes aegypti larvae have been compared with ribonuclease activity in adult female tissue. 2. In larval extracts ribonuclease activity was maximal at 40-45 degrees C whereas activity in tissue from adult females was highest at 50 degrees C. 3. Ribonuclease activity that was recovered in a 20-60% ammonium sulfate precipitate was further purified by batch elution from DEAE-Sephacel and from carboxymethylcellulose. 4. Ribonuclease activity in the partially purified fraction was sensitive to EDTA, stimulated by magnesium, had a pH optimum at 9.0 and a Mr of 45,000. 5. Agarose gels containing yeast RNA substrate were used to monitor partial purification of the larval ribonuclease.