scholarly journals Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength

1978 ◽  
Vol 175 (1) ◽  
pp. 1-13 ◽  
Author(s):  
E Pays
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Chain Elongation ◽  
Rna Sequences ◽  
Double Stranded Dna ◽  
Low Concentrations ◽  
Dna 2 ◽  
Low Ionic Strength ◽  
Dna Ends

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable ‘hairpins’ can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable ‘hairpins’ in RNA is independent of the polymerase/DNA ratio.

scholarly journals Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin

1977 ◽  
Vol 165 (2) ◽  
pp. 237-245 ◽  
Author(s):  
E Pays
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rat Liver ◽  
Rna Sequences ◽  
Complementary Sequences ◽  
Exogenous Rna ◽  
Liver Chromatin ◽  
Low Ionic Strength

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes

1993 ◽  
Vol 13 (7) ◽  
pp. 4214-4222
Author(s):  
Y Chen ◽  
J Weeks ◽  
M A Mortin ◽  
A L Greenleaf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Chain Elongation ◽  
Developmental Defect ◽  
Genes Encoding ◽  
Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro

FEBS Letters ◽  
1992 ◽  
Vol 300 (1) ◽  
pp. 89-92 ◽  
Author(s):  
R.M. Sánchez ◽  
M. Viñals ◽  
M. Alegret ◽  
M. Vázquez ◽  
T. Adzet ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Chain Elongation ◽  
Fatty Acid Chain

scholarly journals Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

1993 ◽  
Vol 13 (7) ◽  
pp. 4214-4222 ◽  
Author(s):  
Y Chen ◽  
J Weeks ◽  
M A Mortin ◽  
A L Greenleaf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Chain Elongation ◽  
Developmental Defect ◽  
Genes Encoding ◽  
Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

scholarly journals Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation

1976 ◽  
Vol 156 (1) ◽  
pp. 7-13 ◽  
Author(s):  
S Sperti ◽  
L Montanaro ◽  
A Mattioli ◽  
G Testoni ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Elongation Factor ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Gtp Hydrolysis ◽  
Elongation Factor 2 ◽  
The Individual ◽  
Liver Ribosomes

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2-GDP-ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.

scholarly journals Inhibition by α-amanitin of ribonucleic acid polymerase solubilized from rat liver nuclei

1970 ◽  
Vol 116 (2) ◽  
pp. 177-180 ◽  
Author(s):  
F. Novello ◽  
L. Fiume ◽  
F. Stirpe
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Isolated Rat

1. α-Amanitin inhibits in vitro the RNA polymerase solubilized from isolated rat liver nuclei. 2. In contrast with previous observations with whole nuclei, the inhibition occurs approximately to the same extent in the presence and in the absence of ammonium sulphate. 3. Evidence is presented that the toxin acts by interacting with the enzyme itself and not with DNA or other components.

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

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