Magnesium ion-independent ribonucleic acid depolymerases in bacteria.
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ABSTRACT:
SUBMITTER: Wade HE
PROVIDER: S-EPMC1270130 | biostudies-other | 1966 Nov
REPOSITORIES: biostudies-other
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Project description:1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.
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Project description:1. Crude extracts of the extreme halophile Halobacterium cutirubrum contain separable DNA-dependent and RNA-dependent RNA polymerases. 2. The RNA-dependent enzyme has been purified about 2800-fold. 3. It requires RNA, preferably of high molecular weight, and all four ribonucleoside triphosphates to incorporate (14)C-labelled nucleoside triphosphate into an acid-insoluble, ribonuclease-sensitive product. 4. Both the stability and activity of the RNA polymerase are relatively insensitive to changes in potassium chloride or sodium chloride concentration, but incorporation is stimulated by both Mg(2+) and Mn(2+). 5. The molecular weight of the enzyme is about 17000-18000.
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Project description:Noncoding RNA (ncRNA) is a kind of RNA that plays an important role in many biological processes, diseases, and cancers, while cannot translate into proteins. With the development of next-generation sequence technology, thousands of novel RNAs with long open reading frames (ORFs, longest ORF length > 303 nt) and short ORFs (longest ORF length ? 303 nt) have been discovered in a short time. How to identify ncRNAs more precisely from novel unannotated RNAs is an important step for RNA functional analysis, RNA regulation, etc. However, most previous methods only utilize the information of sequence features. Meanwhile, most of them have focused on long-ORF RNA sequences, but not adapted to short-ORF RNA sequences. In this paper, we propose a new reliable method called NCResNet. NCResNet employs 57 hybrid features of four categories as inputs, including sequence, protein, RNA structure, and RNA physicochemical properties, and introduces feature enhancement and deep feature learning policies in a neural net model to adapt to this problem. The experiments on benchmark datasets of 8 species shows NCResNet has higher accuracy and higher Matthews correlation coefficient (MCC) compared with other state-of-the-art methods. Particularly, on four short-ORF RNA sequence datasets, specifically mouse, Saccharomyces cerevisiae, zebrafish, and cow, NCResNet achieves greater than 10 and 15% improvements over other state-of-the-art methods in terms of accuracy and MCC. Meanwhile, for long-ORF RNA sequence datasets, NCResNet also has better accuracy and MCC than other state-of-the-art methods on most test datasets. Codes and data are available at https://github.com/abcair/NCResNet.
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Project description:1. Nucleic acids were released from Escherichia coli by lysing with tri-iso-propylnaphthalene sulphonate and 4-aminosalicylate and then extracting with a phenol-cresol mixture. 2. Nucleic acids were similarly released from Bacillus subtilis after initial treatment with lysozyme. 3. DNA was sedimented after careful precipitation with m-cresol or 2-butoxyethanol (0.1-0.12vol.) in the presence of 20% sodium benzoate. 4. Contaminating ribosomal RNA was removed by precipitation in the presence of 4m-sodium chloride or by extracting DNA with an acetate-butyrate mixture, in which RNA is insoluble. 5. The DNA from B. subtilis has a transforming ability of 0.3-0.6% for the tryptophan marker. 6. Ribosomal RNA was then precipitated with rapidly labelled RNA by the addition of an equal volume of 2-butoxyethanol. 7. There was good separation of the nucleic acids from protein and polysaccharides.
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Project description:1. A simple method for the preparation of ribonuclease-free ribosomal RNA is described in which ribonuclease-deficient bacteria are treated with acetone and the RNA is extracted with phenol and purified by precipitating it with potassium acetate. The treatment with acetone appears to render the cell wall permeable to RNA but not to DNA during the extraction with phenol. The method thus avoids the need to disrupt the bacteria and greatly simplifies the subsequent purification. 2. The method has been used successfully with ribonuclease-deficient strains of Escherichia coli, Pseudomonas fluorescens and Staphylococcus epidermidis. The recovered purified RNA accounts for about 70% of the total ribosomal RNA and shows the normal sedimentation pattern of the 16s and 23s components in the analytical centrifuge.
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Project description:Background and aimSupplementation of AKBISprob (developed in a previous study) in feed can improve production efficiency and poultry health, especially laying hens. In addition, it can also increase cellulolytic lactic acid bacteria (LAB) in chicken intestines, but these bacteria are still unknown; thus, they need to be identified. This study aimed to identify cellulolytic LAB in the intestines of laying hens administered AKBISprob based on 16S ribosomal ribonucleic acid (16S rRNA) gene analysis.Materials and methodsThe samples used in this study were 13 LAB isolates from the intestines of laying hens that were given AKBISprob 4%. Cellulolytic LAB DNA was isolated and 16S rRNA gene was amplified by polymerase chain reaction, followed by sequencing, bioinformatics analysis, and phylogenetic tree construction.ResultsFrom 10 cellulolytic LAB isolates with a clear zone of >6 mm, four were selected and their DNA was amplified with BaCF and UniB primers ~1500 bp DNA fragments. Of these, the P31H62 isolate was genetically close to Enterococcus hirae strain 1-1X-16 with 92.90% maximum identity, the P33S52 isolate had homology with Enterococcus mundtii strain ZU 26 with 96.76% maximum identity, and the P33S62 isolate was closely related to E. hirae strain SJ3 with 72.96% maximum identity. The phylogenetic tree revealed that the cellulolytic LAB isolates P31H62 and P33S52 were in one cluster closely related to the genus Enterococcus.ConclusionThis study suggests that the isolates P31H62, P33S62, and P33S52 from the intestines of laying hens administered 4% AKBISprob are cellulolytic LAB belonging to the genus Enterococcus.
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Project description:1. The effect of removing Mg(2+) from a purified high-molecular-weight (1.07x10(6)) fraction of Escherichia coli ribosomal RNA was examined by ultracentrifugation, thermal denaturation and optical rotation. 2. At moderate I (0.1m-sodium chloride), EDTA at 2-50mm has little effect on RNA; at low I, 0.01-0.04 (with tris as counter-ion), two boundaries appear. 3. The leading boundary, S(20,w) about 20s, is identified with the original material with counter-ion Mg(2+) (;ionic atmosphere') removed, leading to an expanded form. 4. The slow boundary, 15-16s, is associated with a further loss of Mg(2+) and a further expansion, sensitive to EDTA concentration: it is proposed that this Mg(2+) is localized on the polynucleotide chain, i.e. ;site-bound'. 5. I is important and the EDTA effect at low I is reversible if Na(+) is added immediately after the EDTA: this Na(+) reversibility is lost on standing at 0 degrees . It is suggested that changes in the tertiary structure may be associated with this loss of reversibility. 6. Thermal-denaturation studies show that there is no loss of secondary structure associated with these changes: change in the optical-rotatory-dispersion spectrum in the region of the Cotton effect may be associated with this change in tertiary structure.
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Project description:1. Experiments were performed to investigate two hypotheses about the function of long-lived messenger RNA in bacteria. After RNA synthesis had been stopped by the addition of actinomycin, continuing protein synthesis was used as a measure of persistent messenger RNA. 2. The hypothesis that messenger RNA responsible for the synthesis of membrane protein is exceptionally long-lived was tested in experiments with protoplasts of Bacillus megaterium. However, this messenger RNA proved to be of approximately average stability. 3. The hypothesis that long-lived messenger RNA is responsible for the synthesis of constitutive proteins was tested by comparing the synthesis of penicillinase in an inducible and a constitutive strain of Bacillus licheniformis. After the addition of actinomycin, penicillinase synthesis continued for far longer in the constitutive than in the inducible strain. This difference is attributed to a difference in stability of the penicillinase-messenger RNA in the two strains, which does not extend to all messenger RNA indiscriminately. 4. A model is tentatively proposed to account for the altered stability of messenger RNA in the constitutive mutant.
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Project description:Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.
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