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Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells al the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; This conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells., as suggested in the accompanying paper. [ABSTRACT FROM AUTHOR]

In a previous paper in this journal [Djondjurov, L., Ivanova, E. and Tsanev, R. (1979) Eur. J. Biochem. 97, 133–139], we showed that a nuclear fraction released from chromatin under a mild nuclease digestion contained an increased amount of hnRNA and the bulk of nonhistone proteins with a high metabolic rate. The present investigation has revealed that the nonhistone proteins of this fraction could be divided into three distinct metabolic groups. The first group consists of proteins with a fast turnover rate (mean half-life 30 min) which migrate into chromatin immediately after their synthesis. These proteins are predominantly acid-soluble and have relatively high molecular weights. The second group includes proteins which migrate to the nucleus more slowly and metabolize with a moderate turnover rate (mean half-life 5 h). The third group contains proteins with a more conservative metabolic behaviour. In experiments with actinomycin D it was found that the bulk of the nonhistone proteins of this fraction are not real components of the chromatin but belong to the protein moiety of heterogeneous nuclear ribonucleoprotein particles associated with chromatin. [ABSTRACT FROM AUTHOR]

In this paper evidence is provided that one of the protein components of the water-soluble fraction of the call lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr 55000 and a rather large amount of α-crystallin copurify with this polypeptide, which has a molecular weight of 42000. Highly purified 42000-Mr protein was also obtained by extraction of the water-insoluble fraction of the calf lens with 2-{(tris(hydroxyniethyl)methyl]amino}ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non-muscle actin. Furthermore the amino acid composition of the 55 000-Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin. [ABSTRACT FROM AUTHOR]

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16-S RNA, but lacking the 3′-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3′-terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16-S RNA is not involved in stable interactions with protein. [ABSTRACT FROM AUTHOR]

A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNasas Y, A and R. which produce acid-soluble products when incubated at near-neutral pH in the absence of metal ions. RNases Y and A yield products with the monoesterified phosphate at the 3′ position, through 2′,3′-(cyclic)phosphate intermediates. Oligonncleotides terminating with a 2′,3′-(cyclic)phosphate are the end-products of the action of RNase R, Double-stranded substrates are highly resistant to the action of all enzymes. On the basis of limited hydrolysis of end-labelled 5S RNA, the three enzymes differ in their preference for the susceptible phosphodiester bond. Thus, RNase Y hydrolyses preferentially the YpN bond, RNase A the ApN bond and RNase R the RpU bond where R is guanosine in most cases. The advantages and disadvantages of using homopolyribonucleotides and dephosphorylated dinucleotides and trinucleotides in determining various aspects of the specificity of RNases are discussed. [ABSTRACT FROM AUTHOR]

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields ‘extra’ bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, Le. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of ‘extra’ bands. The ‘extra’ bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weights, calculated from mobility in gels, are around 2 × 107. Double digests with two restriction endonucleases yield a new set of ‘extra’ bands, showing that the ‘extra’ bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the ‘extra’ bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 × 106, calculated from mobility in gels. We present evidence that the ‘extra’ bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 × 107, making up less than 10% of the total kDNA. 4. From these results we conclude that 3–5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 × 106 and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes. [ABSTRACT FROM AUTHOR]

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenates by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonuclease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCl to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definitive conclusion can be reached for the significance of this observation, it is shown by density equilibrium analysis that these nucleases are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction. [ABSTRACT FROM AUTHOR]

The conformational state of C-terminally truncated staphylococcal nuclease R (SNR135), with and without bound ligands, has been studied by performing limited proteolysis with a specific endoproteinase Glu-C followed by electrophoresis and mass spectrometry. Comparison of the accessibility of the cleavage sites shows that the C-terminal truncation of 14 amino-acid residues causes significant unfolding of the C-terminal part of α helix 1 and the center of α helix 2, but there is little effect on other regions of the nuclease, in particular the N-terminal subdomain, which includes the active site of the nuclease. The truncation also makes the overall conformation of the nuclease more loose and flexible. Binding of ligands makes helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state, although the conformational stability of the SNR135 complex is still much lower than that of the full-length enzyme. The results suggest that the amino-acid residues around the active site in the truncated nuclease are arranged in a similar topology to those in the full-length nuclease. The study shows that there is a clear-cut correlation between protease susceptibility and conformational stability of the protein, and the initial proteolytic events are the most critical for evaluating the conformational features of the protein. This study demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding. [ABSTRACT FROM AUTHOR]

An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA ≈ 3′AMP > RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5′dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5′dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5′dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3′ or the 5′ end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. [ABSTRACT FROM AUTHOR]

Investigates the partial purification and characterization of ribonuclease P from the organism, Dictyostelium discoideum. Sensory transduction of the organism cells; Resistance of the organism to micrococcal nuclease of the chloroplast enzyme.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition. [ABSTRACT FROM AUTHOR]

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, not in other organs. An immunohistochemical assay showed that RC-RNase was present in the regular yolk granules, but not in forming yolk granules. yolk platelets, pigment granules, mitoehondria clouds or the nucleus. The RC-RNase was restricted to the lateral amorphous area of the yolk granules, and was absent from the central area that has a vitellogenin crystal lattice. The RC-RNase was extracted from yolk granules by 0.5 M Nase and purified by dialysis and affinity chromatography. Most of the RC-RNase (94%) was found in the yolk granules, the rest RC-RNase (6%) was found in the cytosol in the form of free RNase and latent RNase. The RC-RNase extracted from yolk granules was further analyzed by immunoprecipitation and RNase activity assay on an SDS/polyacrylamide gel. Our results suggest that the RC-RNase activity is regulated by both compartmentation and inhibitor binding. [ABSTRACT FROM AUTHOR]

The chromatin structure of the macronuclear genes encoding ribosomal proteins S25 and L1 in the ciliated protozoan Tetrahymena thermophila was analyzed. Using the indirect end-labelling technique, DNase-I-hypersensitive regions were located in the promoter regions as well as in the 3' regions of the genes. The DNase-I-hypersensitive regions were present in chromatin of exponentially growing cells, where the rate of ribosomal-protein gene transcription is high, and in chromatin from starved cells, where transcription of ribosomal-protein genes is severely depressed. Micrococcalnuclease-digestion experiments revealed that the promoter regions of the S25 gene and the L1 gene are devoid of nucleosomes in exponentially growing cells. In starved cells, no nucleosomal organisation of the promoter region of the L1 gene could be detected, whereas nucleosomal structures were discernible in the promoter region of the S25 gene. A conspicuous polypurine sequence motif, AARGGGAAA, is present within or adjacent to the DNase-I-hypersensitive regions in the promoter of the S25 and the L1 gene, and interestingly, the same motif is found also in the promoter regions of the genes encoding ribosomal proteins L21 and L37. [ABSTRACT FROM AUTHOR]

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/ PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins. [ABSTRACT FROM AUTHOR]

The primary structure of nuclease P1, which cleaves both RNA and single-stranded DNA, from Penicillium citrinum was elucidated. The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter protease I of the reduced and S-aminoethylated protein and by digestion with Staphylococcus. aureus V8 protease of the reduced and S-carboxymethylated protein. Four half-cystine residues were assigned to Cys72-Cys217 and Cys80-Cys85. N-Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197. Fast-atom-bombardment and laser-ionization MS were success- fully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively. Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease Sl from Aspergillus oryzae. The His-Phe-Xaa-Asp-Ala sequence (positions 60–64) is similar to the sequence (His- Phe-Asp-Ala) involving the active-site Hisll9 of bovine pancreatic RNase A. and the Pro-Leu-His sequence (positions 124–126) is identical with the sequence involving the active-site His134 of porcine pancreatic DNase I. [ABSTRACT FROM AUTHOR]

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNAMct. Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNAMet, tDNAMet (a DNA oligomer having the sequence of tRNAMet) and the anti-tDNAMet (the complement of tDNAMet) suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double-stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2′-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation. [ABSTRACT FROM AUTHOR]

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred m the presence of 3′AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and Hisl15 were partially modified to yield a total of one mole of N′-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or Hisl15 inactivates the enzyme. [ABSTRACT FROM AUTHOR]

A method which utilises S1 nuclease to detect small length variations in cloned and genomic DNA has been evaluated. The methodology of this technique is simple and robust, permitting the rapid analysis of 104 base pairs. By employing defined sequence variants, this method is shown to have a sensitivity which should enable the detection of length variations of only a few base pairs in heterozygous individuals. [ABSTRACT FROM AUTHOR]

A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine-adenosine (Pyd-Ado) bonds in RNA. It was purified about 7000-fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity. The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa. It is called RNase M. The only other reported broadly specific endoribonuclease in E. coli is RNase I, a periplasmic enzyme. Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I. The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size.
Earlier studies had shown that mRNA from the lactose operon of E. coli is hydrolyzed in vivo primarily between Pyd-Ado bonds [Cannistraro et al. (1986) J. Mol. Biol. 192, 257-274]. We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E. coli. [ABSTRACT FROM AUTHOR]

Three ribonucleases, RNase 1, RNase II and RNase III, were purified from the 109 000 × g supernate of detergent-treated Tetrahymena pyriformis strain W. RNases I and II act optimally at pH 5.5-6.0 and are inhibited by increasing concentrations of salts of monovalent cations. RNase III acts optimally at pH 7.5 and is activated 1.5-fold by millimolar concentrations of ZnSO4 and 5-fold by 50 mM KCl. RNases II and III are activated approximately 100% in the presence of 3 M and 5 M urea respeetively. All enzymes are heat-sensitive and acidresistant. They are endonucleases forming 2',3'-cyclic products. Their base specificity, as tested against ribosomal RNAs of known sequence, is as follows: RNase I hydrolyzes preferentially YpN and secondarily GpN bonds, RNase II is highly specific for RpN bonds, though the preparation can also hydrolyze the UpU sequence. Finally the principal targets of RNase III are YpR sequences and secondarily YpY sequences. A shorthand visualization of base specificity of nucleases in the form of right isosceles triangles is presented. The triangles are constructed by subdividing each of the two perpendicular sides in as many units as the maximum number of times the most abundant dinucleotide appears in all substrates employed and plotting the frequency of hydrolysis of each dinucleotide sequence by the enzyme under study. The proximity of each dinucleotide sequence to the hypotenuse or to one of the perpendicular sides is indicative of its susceptibility or resistance to the enzyme's action. [ABSTRACT FROM AUTHOR]

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'- monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'- monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidytic acid > polyuridylic acid > polyadenylic acid ... polyguanylic acid. The enzyme also has preference for single-stranded nucleic adds. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P. H. and Ho, T.-h+ D. (1986) Plant Physiol. 82, 801 - 806]. Consistent with these results, gibberellic acid induces up to an eightfold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa. [ABSTRACT FROM AUTHOR]

A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyrihonucleotides. The enzyme has a pH optimum in the region 7–9 and relative molecular mass of about 25000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied. [ABSTRACT FROM AUTHOR]

Reports on a human myosin heavy-chain gene which was shown to code for a cardiac myosin heavy chain of the beta-type. Location of the 5' end of the 14,200-base-pair genomic DNA clone in the head region of the myosin chain; Transcription of the beta-myosin heavy-chain gene in human heart muscle as demonstrated by the S1 nuclease protection technique.

An acid ribonuclease has been purified from the insect Ceratitis capitata. The specific activity of the purified enzyme is 580 units/mg. This enzyme is a single polypeptide chain of about 35.5 kDa, containing only one disulfide bridge and no free - SH groups. The A1 cm0.1% at 280 nm is 1.90. The hydrodynamic radius of the native enzyme is 2.5 nm. The secondary structure of this RNase is composed of 10% α-helix, 31% β-structure and 59% aperiodic conformation with an average number of residues per helical segment of 10, based on circular dichroic measurements. Optimum parameters for the enzyme activity are pH 5.5, 0.15 M ionic strength and 40°C. Divalent cations are not required for the enzymic catalysis. This enzyme has been characterized as cyclizing endoribonuclease. [ABSTRACT FROM AUTHOR]

Discusses results of a study focusing on the tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase messenger RNA. Liver; Kidney; Small intestine; Primer extension; Nuclease S1 protection; Genetic transcription; Genetic code.

Reports on the modification of cytosine in nucleic acids by treatment with a mixture of bisulfite and hydrazine. Specificity of the reaction for single-stranded regions of nucleic acids with the product being N[sup 4]-aminocytosine. Use of bromopyruvate for the alkylation of protein SH groups; Labeling of the protein portion and digestion of the RNA portion with nucleases.

The micrococcal nuclease cleavage sites have been mapped in the H2A coding and flanking regions of the sea-urchin histone DNA chromatin. A hypersensitive area, centered around -- 100 base pairs from the H2A starting site, is found only in embryos actively transcribing the α-subtype histone genes. In mesenchyme blastula embryos, upon inactivation of the H2A gene, this region becomes protected while two other areas, near the transcription starting site and in the proximity of the 3′ palindromic sequence, become preferential targets for the enzyme. Analysis of the pattern of micrococcal nuclease cleavage on the same region of the histone gene cluster in sperm and late blastula chromatin and on the corresponding segment of protein-free DNA indicates that distinct nucleosomal arrangements characterize the histone genes in the two cell populations. [ABSTRACT FROM AUTHOR]

Investigates the biochemical characterization of Anabanena nuclease. Utilization of CD spectroscopy in the study; Activity of the nuclease towards high molecular mass; Kinetic parameters for the cleavage of nucleic acids.

Reports on the cleavage of rRNA by the treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium-dependent nuclease from Staphylococcus aureus. Localization of the positions of the cleaved phosphodiester bonds by primer extension and polyacrylamide gel electrophoresis; Reason for the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease; Suggestion that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the messenger RNA.

An exodeoxynuclease with single-stranded DNA specificity has been isolated from rat brain and purified to electrophoretic homogeneity. Approximately 1100-fold purification with a yield of 16% has been achieved by chromatography on DNA-agarose, hydroxyapatite and Sephadex G-200. The enzyme has a pH optimum at 8.4, requires Mg2+ or Mn2+ as a cofactor and has a molecular weight of about 60000. It hydrolyzes homologous, heterologous, synthetic and depurinated substrates at the same rate liberating nucleoside 5′-monophosphates but does not attack ultraviolet-irradiated polydeoxyribonucleotides. This DNase is localized predominantly in neuronal cell nuclei and appears to be lacking in rat liver tissue. [ABSTRACT FROM AUTHOR]

The chromosome of the Bacillus subtilis phage 2C, a linear molecule of double-stranded DNA of about 108 Da, in which thymine is completely replaced by hydroxymethyluracil, was cleaved by different endonucleases. In some cases restriction segments were much fewer than expected, suggesting a possible interference of the unusual base with the recognition mechanism of endonucleases. The physical map of 2C DNA was established by use of Sail and Hae III restriction endonucleases, which yielded a limited number of fragments. The expected number of fragments was 240 for Hae III and 23 for Sa/I; in reality, five segments were observed upon cleavage with Hae III and four with Sa/I The terminal fragments of the genome were first identified; the other fragments were ordered by hybridization and molecular weight determination of restriction fragments obtained by cleavage with the two endonucleases. In addition hybridization of restriction fragments showed the presence of homologous regions at the ends of the 2 C genome. The structure of these direct repetitive sequences was analyzed by cleavage with Rae III and hybridization with EcoRl restriction fragments. Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromosome. [ABSTRACT FROM AUTHOR]

We have been able to isolate several species of 5-S ribosomal RNA from Eschzerichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G U base pair, formed in a normally highly stable G. C-rich region. is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates laster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired. [ABSTRACT FROM AUTHOR]

A new endoribonuclease activity, RNase F, was partially purified from Escherichia coli cells. This activity can specifically cleave a precursor RNA molecule (of species 1) isolated from T4-infected cells [N. Watson & D. Apirnon (1981) Biochem. Biophys. Res. Commun. 103, 543 - 551]. The cleavage results in products which are very similar to RNA molecules found in the cell, generating a 3'-phosphate and a 5'-hydroxyl groups. The cleavage takes place between a cytosine and an adenine moiety, within a possible loop and stem structure; the cut is in the border between the double-stranded and single-stranded regions of this structure. The specificity of this enzyme could be the introduction of a cleavage near the 3' ends of tRNA molecules and other RNAs like species I which could resemble tRNA in their three-dimensional structure. [ABSTRACT FROM AUTHOR]

Partial P1 nuclease digestion of 5'-32P-labeled tRNAs, followed by polyacrylamide gel electrophoresis and autoradiography, results in a series of bands of unequal intensities. Comparison of the digestion profiles of two conformers of yeast tRNALeu3 shows that P1 nuclease is sensitive to structural features of its substrate. Examination of the digestion pattern of yeast tRNAPhe in light of its known three-dimensional conformation shows that the enzyme's activity reflects the primary, secondary and tertiary levels of tRNA structure. [ABSTRACT FROM AUTHOR]

At endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 x 104, 9 x 104, 1.55 x 105 and Sephacryl S-200 fraction at V0. Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 x 104). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA · RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant. [ABSTRACT FROM AUTHOR]

The extrachromosomal DNA coding for the ribosomal RNA precursor in Tetrahymena contains a transcribed region with a size of 6 x 10[sup3] base pairs plus non-transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific [sup32]P-labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r-chromatin are hypersensitive to DNase I. The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3[sup'] end. The hypersensitivity of the central part of r-chromatin is also found by autodigestion in isolated nucleoli. [ABSTRACT FROM AUTHOR]

A DNA-repair endonuclease has been purified 117-fold from mouse plasmacytoma cells (line MPC-11) by gel filtration, followed by ion-exchange and affinity chromatography. Its molecular weight was determined by gel filtration to be 28000 + 2000. The enzyme recognizes apurinic and apyrimidinic sites induced by acid and γ-rays in DNA, as well as another type of lesion(s) which is introduced into DNA by both ultraviolet irradiation and OsO4. Quantitative measurements of the number of nicks the purified DNA-repair endonuclease makes in DNA treated with various amounts of OsO4 and ultraviolet light suggests that the endonuclease may act on 5,6-dihydroxydihydrothymine lesions. The endonuclease activity was sensitive to the ionic strength and was most active in the presence of 100 mM KCl, whereas the presence of divalent cations did not stimulate the activity. [ABSTRACT FROM AUTHOR]

A membrane-bound endonuclease has been isolated from mitochondrial fractions of Saccharomyces cerevisiae. The enzyme is present in a stable complex and has an approximate molecular weight of 14000. It requires Mg2+ or Mn2+ for activity, and has an optimum pH of 7.0. Its activity with native DNA is five times less than with denatured DNA in 0.05 M KCl and is very low in 0.2 M KCl. The activity with RNA is 40% of that with denatured DNA; the two substrates are competitive. Its mode of action is endonucleolitic, it cuts both strands of native it DNA at the same or nearby sites. After mild digestion of DNA, analysis of 5′-end groups of the digestion products indicated a marked preference for deoxythymidylic and deoxyguanilic acid residues as the site of enzymatic cleavage. After exhaustive digestion of DNA, mononucleotides (2.4%), dinucleotides (70.5%) and trinucleotides (27%) ending in 5′-phosphate are produced. [ABSTRACT FROM AUTHOR]

Two nucleases active on alkylated-depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity. [ABSTRACT FROM AUTHOR]

1. The localization of DNAase activity in rye germ nuclei revealed 48% of the total nuclear activity in the nucleoplasm and 4% in the chromatin. 2. The endonuclease present in the nucleoplasm was purified more than 300-fold as compared with the specific activity in homogenized nuclei by means of ammonium sulphate fractionation and CM-cellulose and CM-Sephadex column chromatography. The preparation gave a single band on polyacrylamide gels. Its molecular weight is 45000, pH optimum 5.4. The preparation does not exhibit activity of non-specific phosphomonoesterase and phosphodiesterase. 3. The nuclease catalyses the hydrolysis of denatured DNA and RNA to acid-soluble 5′-phosphate-terminated products and cleaves the phosphomonoester linkage of 3′AMP. 4, Synthetic polyribonucleotides are hydrolysed at a rate decreasing in the order: poly(U) >poly(A)>poly(C)>poly(G). 5. The endonuclease shows a high preference for single-stranded nucleic acids. It does not depolymerise double-stranded poly(U) · poly(A). 6. The nuclease attacks covalently closed circular bacteriophage PM2 DNA, plasmid ColE1 and plasmid pBR322 DNA, converting it initially into the open-circular (relaxed) form and subsequently into the linear form. This reaction is strongly dependent on ionic strength. 7. The enzyme nicks the supercoiled DNA molecule of phage PM2 in only one place. At high enzyme concentrations several nicks in such a molecule are observed. [ABSTRACT FROM AUTHOR]

The interaction of the EcoRI restriction endodeoxyribonuclease with polynucleotides has been studied in a qualitative manner by an affinity adsorption technique using polynucleotides immobilized on cellulose. It is shown that EcoRI binds to single-stranded and double-stranded polyribonucleotides and polydeoxyribonucleotides. Mg2+ ions are not required for binding. In order to define differences between specific and non-specific binding we have investigated the interaction of EcoRI with d(T-A-A-A-T-G), d(T-T-A-C-A-T), d(G-A-A-T-T-C) and d(G-G-A-A-T-T-C-C). We have synthesized for this purpose d(T-A-A-A-T-G), d(T-T-A-C-A-T) and d(G-A-A-T-T-C) by the diester approach, d(G-A-A-T-T-C) and d(G-G-A-A-T-T-C-C) are self-complementary. Differential melting experiments show that the octanucleotide has a melting point of 28 °C under ionic conditions where the hexanucleotide is single-stranded even below 0 °C. Correspondingly, the octanucleotide is cleaved by EcoRI, while the hexanucleotide is not. The binding of oligonucleotides to EcoRI can be monitored by the circular dichroism of the enzyme. Titrations show that in the absence of Mg2+ ions all oligonucleotides are bound with similar magnitude: Ka ≈ 107 M-1. Complex formation is weakened with increasing temperature, corresponding to a ΔH° of -21 kJ/mol and increasing ionic strength, corresponding to an involvement of two ion-pair bonds between DNA and enzyme. Mg2+ ions have no significant influence on the binding of d(T-A-A-A-T-G), d(T-T-A-C-A-T) and d(G-A-A-T-T-C) to the enzyme. The binding of d(G-G-A-A-T-T-C-C) to EcoRI is strengthened by a factor of 50 in the presence of Mg2+ ions, as measured by cleavage experiments using d(G-A-A-T-T-C) as a competing inhibitor in the enzymatic assay. [ABSTRACT FROM AUTHOR]

The oligonucleotide 5′-triphosphoadenylyl(2′-5′)adenylyl(2′-5′)adenosine (pppA2′p5′A2′p5′A) is synthesised by reticulocyte lysates and extracts from interferon-treated cells in the presence of double-stranded RNA. It inhibits the translation of both viral and non-viral mRNAs and of poly-(rU,rC) in the micrococcal-nuclease-treated reticulocyte lysate system. The translation of poly(rU) is insensitive in this assay, however. Inhibition develops in vitro after a lag period, the length of which depends on the nature of the mRNA present and its concentration. The degradation of radioactive Mengo virus RNA or L cell cytoplasmic RNA is accelerated in reticulocyte lysates incubated with the oligonucleotide. This is due to activation of one or more endonucleases which degrade(s) RNA to large oligonucleotide fragments. Our results suggest that the endonuclease is responsible for most (if not all) of the inhibitory effects of the oligonucleotide on protein synthesis in the reticuiocyte lysate. In the presence of pppA2′p5fA2′pS′A, protein synthesis can be reactivated after it has ceased by addition of fresh mRNA. In contrast, initiation factor eIF-2 is unable to prevent inhibition by the oligonucleotide under conditions where it protects the system from the effects of the haem-controlled repressor and partially protects against inhibition by double-stranded RNA. These findings are discussed in relation to the regulation of protein synthesis by double-stranded RNA and by interferon. [ABSTRACT FROM AUTHOR]

Digestion of chromatin with micrococcal nuclease under mild conditions results in the release of a minor chromatin fraction showing an increased RNA and non-histone protein content, a fast turnover of the non-histone proteins and the presence of rapidly labelled heterogeneous nuclear RNA (hnRNA) with half-life of about 20 min. Further digestion of the chromatin leads to the elimination of about 19% of the initial chromosomal DNA, thus leaving a second chromatin fraction relatively resistant to nuclease attack. This fraction has a low protein and RNA content and contains only metabolically stable non-histone proteins. No differences in the histone complement of the two fractions was found except for a 40% deficiency of H1 in the minor fraction. [ABSTRACT FROM AUTHOR]

Haemophilus influenzae Rf 232, showing the phenomena of restriction and modification, contains an endonuclease that inactivates in vitro the biological activity of DNAs lacking the strain-specific modification. This specific restriction endonuclease has been purified to near homogeneity by a procedure that includes DNA-agarose chromatography. This highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S-adenosylmethionine. The enzyme seems to cleave DNA at well-defined sites, since it produces a specific pattern of bands upon agarose gel electrophoresis. The enzyme has no ATPase activity. A methylase activity is observed in the course of the endonucleolytic reaction, which probably protects some of the DNA sites from cleavage. [ABSTRACT FROM AUTHOR]

Incubation of the mouse L-cell-free system with a concentration of pppA2′p5′A2′p5′A [(2′-5′)An] just sufficient to inhibit protein synthesis results in formation of a high-molecular-weight, heat- labile inhibitor and enhanced ribonuclease activity and in the rapid breakdown of (2′-5′)An to ATP. The (2′-5′)An-enhanced ribonuclease activity is also unstable and in the absence of a (2′-5′)An- regenerating system inhibition of protein synthesis is transient. Although interferon treatment enhances the synthesis of(2′-5′)An. the rates of degradation of (2′-5′)An and levels of activatible nuclease are similar in extracts prepared from control or interferon-treated cells. Interestingly, the sensitivity of different cell-free systems to (2′-5′)An varies with the source of the celt-free systems and with the methods used in their preparation. There is, however, no obvious correlation between the sensitivities of the system and the rate of breakdown of (2′-5′)An. The significance of these results is discussed in relation to a possible control function for the (2′-5′)An system in both interferon- treated and control cells. [ABSTRACT FROM AUTHOR]

The organization of repetitive sequences has been studied by restriction nuclease analysis of satellite DNAs from calf thymus. Several novel structural features have been found in the 1.706-g/cm³ satellite DNA (satellite III). A long-range periodicity of 2350 base pairs detected by cleavage with restriction endonucleases Bsp or EcoRII and two short-range periodicities of approximately 22 and 11 base pairs found by digestion with restriction endonucleases Alu and Sau, respectively, are superimposed in the nucleotide sequence of this satellite DNA. About 14% of the DNA present in long-range repeats lack the short-range periodicities. The length of the long-range repeats varies by multiples of approximately 22 base pairs. The distribution of the restriction sites in the satellite DNA is non-random. The evolution of this satellite DNA is not adequately explained by saltatory amplification and random divergence alone; unequal crossing-over and additional non-random processes are discussed. In the 1.723-g/cm³ satellite DNA (satellite II) a periodicity of 45 base pairs has been found by cleavage with restriction endonuclease Alu. The cleavage pattern of this satellite DNA is compatible with a 3% random divergence of an originally homogeneous nucleotide sequence. A physical map has been established for the 1.715-g/cm³ satellite DNA (satellite I) containing the cleavage sites of restriction endonucleases Alu, Sau, Pst, and EcoRI. A periodicity of 1400 base pairs (Alu, Sau, EcoRI) and of 700 base pairs (Pst) has been observed in the nucleotide sequence of this satellite DNA. φX174 replicative form I DNA fragments of known sequence have been used to calibrate the sizes of the fragments in restriction nuclease digests of mouse, BSC-1, and the three calf satellite DNAs. The monomeric and oligomeric fragments derived from these satellite DNAs by limit or partial digestion provide overlapping sets of size standards for DNA fragments from 20 up to 20000 base pairs. [ABSTRACT FROM AUTHOR]

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, Δ..., at saturation is given by Δ... = - 109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA +dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two-site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarurn polycephalum nucleolar satellite DNA from main-band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non-transcribed sequences in this DNA have similar nucleotide compositions. [ABSTRACT FROM AUTHOR]

Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross-linkitig reaction between protein and ribosomal RNA. Under the conditions described, about 10–15% of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein-protein cross-linking was observed. The RNA-protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible. The proteins involved in the cross-linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA-protein complex. This showed that the individual ribosomal proteins were cross-linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross-linked in high yield. The formaldehyde-treated subunits were subjected to a controlled hydrolysis with ribonuclease T1, and RNA fragments of known nucleotide sequence containing the cross-linked proteins were isolated. Despite the reversibility of the cross-linking reaction, sufficient cross-linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments. In the 50-S subunit, proteins L27 and L32 were found to be cross-linked to the 18-S RNA fragment which arises from the 3′ end of 23-S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18-S RNA and the 13-S RNA fragment from the 5′ end of 23-S RNA. In the 30-S subunit, proteins S3, S5, S9 (11), S13, and to some extent S4, S7, S12 and S18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5′ end of 16-S RNA. S4, S9 (11), S13, and to a lesser extent S7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3′ end of 16-S RNA, but lacking the 3′-terminal 150 nucleotides. Protein S21 was cross-linked to 16-S RNA, but was not found in either of the two RNA regions above. [ABSTRACT FROM AUTHOR]

By the use of restriction endonucleases the organization of the α-satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed. With endo R · HindIII, endo R · AluI and with endo R · EcoRl at conditions of low salt and high pH (endo R · EcoRI*) all of the satellite was digested while only a part of the satellite was cleaved with endo R · Bsu and endo R · EcoRI under standard conditions. With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite. The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites. While the arrangement of the endo R · HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R · Bsu and endo R · EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite. Since endo R · AluI recognizes the central four nucleotide pairs of the endo R · HindIII cleavage site, the redigestion of the endo R. HindIII dimer with endo R · AluI gave information about the distribution of mutations in the satellite. The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R · HindIII and endo R · EcoRI* lend support to the hypothesis that mutations have affected all bases in the satellite evenly. The γ-satellite, another fraction of the African green monkey DNA, could be separated by Ag+/ CsSO4 density gradient centrifugation into two components. With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel clectrophoresis with some faint bands of no apparent regularity in one case. [ABSTRACT FROM AUTHOR]

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long-range periodicities in these three satellites differ rather characteristically. Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis. Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite. Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats of approximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30–40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nucleases are clustered on separate small segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA. The three guinea pig satellites are examples of more general types of satellite structures also found in other organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA. [ABSTRACT FROM AUTHOR]