Detection of RNAase inhibitor from different species and organs

Human placental alkaline RNAase inhibitor was purified to homogeneity. Activity was measured after each purification step. The final identification of the purified protein was done by two-dimensional polyacrylamide gel electrophoresis and by immunoblotting. Antibodies were prepared by immunization of rabbits with the highly purified inhibitor. The availability of the antiserum directed against the human inhibitor enabled the detection of RNAase inhibitor from various other organs and species. This procedure has the advantage over the usual activity test in that the inhibitor can be found even if its activity has been lost.     

Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.     

Purification and characterization of a human urine ribonuclease (RNAase 1) showing genetic polymorphism

A ribonuclease (RNAase) was isolated and purified from the urine of a 45-year-old man by column chromatographies on DEAE-Sepharose CL-6B, cellulose phosphate and CM-cellulose followed by gel filtrations on Bio-Gel P-100 and Sephadex G-75, and finally to a homogeneous state by SDS-polyacrylamide gel electrophoresis. The enzyme was designated RNAase 1. It was possible to detect RNAase 1 isozymes in urine and serum without difficulty using isoelectric focusing electrophoresis followed by immunoblotting with a rabbit antibody specific to RNAase 1. The existence of genetic polymorphism of RNAase 1 was detected in human serum utilizing this technique (Yasuda, T. et al. (1988) Am. J. Hum. Genet., in press). RNAase 1 in serum and urine seemed to exist in multiple forms with regard to molecular weight and pI value. Genetically polymorphic RNAase 1 was a glycoprotein, containing three mannose, one fucose, four glucosamine and no sialic acid residues per molecule, with a molecular weight of 16,000 and 17,500 determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was most active at pH 7.0 on yeast RNA substrate and inhibited remarkably by Cu2+, Hg2+ and Zn2+. It also showed definite substrate preference for poly(C) and poly(U), but much less activity against poly(A) and poly(G). Thus, the enzyme is a pyrimidine-specific RNAase.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.     

Activation of a latent RNAase from yeast by nucleoside triphosphates

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5′-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The K_m for ATP is 10 μM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.     

E. coli RNAase P has a required RNA component

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Bull semen RNAase revisited

A new RNAase, RNAase SPL, was discovered (Reddy et al., 1979), which constituted most of bull semen RNAase activity; it was reminiscent in many of its properties of the bovine seminal RNAase we have studied for many years (see References), but different from it in other respects. When the procedure devised by those authors for its isolation was repeated, we found that an RNAase SPL such as that described in the above-mentioned paper is not to be found in bovine seminal plasma.     

A simple method for elimination of RNAase contamination from DNAase preparations

RNAase which usually contaminates commercial pancreatic DNAase preparations can be removed by affinity chromatography on agarose-coupled anti-RNAase antibodies. RNA treated with purified DNAase can be re-isolated intact, as determined by polyacrylamide gel electrophoresis under denaturing conditions. This method might be applicable to purification of other preparations which are used in RNA research, such as PNPase (polynucleotide phosphorylase) and specific antibodies for polysome immunoprecipitation. The non-specific binding of DNAase in our system is less than 5% and the loss of specific activity of DNAase I is less than 1%.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

The transfer RNA processing defect in Escherichia coli strains BN and CAN is not due to a mutation in RNAase D or RNAase II

Escherichia coli strains BN and CAN are unable to support the growth of bacteriophage T4 psu₁⁺-amber double mutants. For strain BN, this phenotype has been attributed to a defect in 3′ processing of the precursor to psu₁⁺ tRNA^Ser. Since RNAase D and RNAase II are the only well-characterized 3′ exoribonucleases to be implicated in tRNA processing, the status of these activities and their genes in the mutant strains was investigated. Although extracts of strains BN and CAN were defective for hydrolysis of the artificial tRNA precursor, tRNA-C-U, these strains contained normal levels of RNAase D and RNAase II, and purified RNAase D or RNAase II could only partially complement the mutant extracts. Introduction of the wild-type RNAase D gene into strains BN and CAN did not correct the mutant phenotype. Likewise, strains defective in RNAase D and/or RNAase II plated T4psu₁⁺-amber phage normally. These results indicate that the tRNA processing defect in strains BN and CAN is not due to a mutation in either RNAase U or RNAase II. The possibility that the mutation in these strains affects another exoribonuclease or a factor influencing the activity and specificity of RNAase D or RNAase II is discussed.     

Extracellular RNAase Pch2 of Penicillium chrysogenum 152A: purification, specificity and physico-chemical properties

Acid RNAase Pch2 was isolated from a filtrate of the cultural fluid of the fungus Penicillium chrysogenum 152A and purified to homogeneity. An analysis of RNAase Pch2 action on RNA and synthetic substrates showed that the enzyme can be attributed to non-specific true ribonucleases (ribonucleate-3'-oligo-nucleotide hydrolase, EC 3.1.4.23). The maximal effect of the enzyme on RNA is observe at pH 4.5 and 55 degree. The RNAase Pch2 is not activated by bivalent metal ions, p-chloromercurybenzoate or beta-mercaptoethanol and is reversibly inactivated by 8 M urea. The enzyme molecule consists of 332 amino acid residues; its molecular weight is 36160, the isoelectric point lies at 5.2.     

Reconstitution of RNAase P activity using inactive subunits from E. coli and HeLa cells

HeLa cell RNAase P activity found in the flow-through of anti-Sm affinity columns can be separated into inactive RNA and protein components. These components can be used to reconstitute active hybrid enzyme complexes with purified subunits from E. coli RNAase P. The RNA in the HeLa cell fractions employed is enriched for species between 85 and 115 nucleotides long. This reconstitution assay is a convenient means of purifying the functional RNA and protein of HeLa cell RNAase P. Probes derived from the genes for the subunits of E. coli RNAase P hybridize to genomic DNA of gram-negative prokaryotic organisms, but no positive signals are seen with genomic DNA from a variety of eukaryotic organisms.     

Regional differences in ribonuclease content of rat and mouse kidney

Kidney cortex, red medulla and white medulla were separated into nuclei, mitochondria, microsomal and 105000g supernatant fractions. Assay of RNAase (ribonuclease) activity at pH7.8 revealed that, for each subcellular fraction, activity was much greater in cortex than in red or white medulla; this was true for both free RNAase and total (free plus latent) RNAase. For example, the free RNAase activity in the 105000g supernatant of cortex was 5 and 8 times higher than in red and white medulla respectively. No latent RNAase activity was found in any particulate fraction. Latent supernatant RNAase activities (suggesting presence of bound RNAase inhibitor) were similar in cortex and medulla. The cortex supernatant contained minimal free RNAase inhibitor, whereas that of the red and white medulla showed about one-third and one-tenth respectively of the inhibitor activity measured in liver. Adrenalectomy did not change RNAase activity in any fraction nor the content of free RNAase inhibitor in the kidney supernatant, but did decrease the liver RNAase inhibitor content by 40%. In supernatants from mouse kidney, both free and total RNAase activities of both cortex and red medulla were similar to those of rat red medulla. Mouse cortex contained appreciably higher amounts of free RNAase inhibitor than rat cortex. The difference between the rat and mouse cortical RNAase activity and inhibitor content may help explain the relative ease with which satisfactory renal polyribosome profiles were obtained from mouse kidneys. Our results, as well as those of Kline & Liberti [(1973) Biochem. Biophys. Res. Commun. 52, 1271–1277], showing that renal red and white medulla are more active than cortex in protein synthesis, are consistent with the hypothesis that the RNAase–RNAase-inhibitor system may participate in the regulation of protein synthesis.     

A rapid method for the isolation of ribonuclease from yeast (Saccharomyces carlsbergensis).

A ribonuclease (RNAase; EC 3.1.14.1) from brewer's yeast was purified 90-fold. Crude RNAase was initially separated from other proteins by precipitation at pH 4.0 after incubation of the mechanically disrupted yeast cells at pH 6.0 and 52 degrees C for 30 min. The RNAase was purified from the supernatant by ultrafiltration with a PM-30 membrane and adsorption chromatography on hydroxyapatite. RNAase preparation was free of phosphatase, deoxyribonuclease and phosphodiesterase activities. It showed maximum activity at pH 6.0 and a temperature optimum of 52 degrees C with yeast RNA as substrate. This RNAase hydrolysed yeast RNA to nucleoside 3'-phosphates and showed no evidence of base specificity.     

Site selection by Xenopus laevis RNAase P

Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one. In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites.     

Activation of latent ribonuclease in the fat-body of fleshfly (Sarcophaga peregrina) larvae on pupation.

A crude extract of the fat-bodies of third-instar larvae of Sarcophaga peregrina (fleshfly) was found to contain latent RNAase (ribonuclease) consisting of RNAase and inhibitor protein that is sensitive to p-chloromercuribenzoic acid. The RNAase activity in the crude extract of fat-bodies became detectable with time after puparium formation, indicating that the inhibitor is selectively inactivated and RNAase is released from the RNAase-inhibitor complex during metamorphosis.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.