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Results for “DNA-Directed RNA Polymerases”

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PubMed1977

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.

Adenosine Diphosphate↗
PubMed1979

Genetic analysis of Staphylococcus aureus RNA polymerase mutants.

Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.

Aminoglycosides↗
PubMed1979

Enhanced transformability with heterospecific deoxyribonucleic acid upon removal of nascent ribonucleic acid from the Streptococcus sanguis genome.

Treatment of Streptococcus sanguis recipient cells with rifampin (RIF) at the time of deoxyribonucleic acid (DNA) addition was an effective means of reducing discrimination, that is, of causing an increase in the number of transformants induced by irreversibly bound heterospecific DNA without significantly changing the number induced by bound homospecific DNA. RIF was unable to reduce discrimination when the recipient cells were RIF resistant due to an altered ribonucleic acid (RNA) polymerase. When recipient cells were treated at the time of DNA addition with concentrations of streptolydigin (STG) as inhibitory of RNA synthesis as RIF, discrimination was not reduced. The kinetics of RNA synthesis inhibition with these inhibitors indicated that, as reported for other bacterial species, RIF inhibited the initiation of transcription by RNA polymerase, whereas STG inhibited the progression of RNA polymerase at any point. Pulse-labeling of RNA immediately before STG addition showed that, if cells were incubated under STG inhibition for 10 to 15 min, their nascent RNA was degraded. Genome-bound RNA polymerase was not released under these conditions. When recipient cells were incubated with STG until nascent RNA was degraded and then exposed to transforming DNA, STG was as effective as RIF in reducing discrimination. The presence of nascent RNA was thereby implicated in the transforming inefficiency of incompletely homologous DNA.

Aminoglycosides↗
PubMed1975

Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus.

In search of an anti-transcriptase, antibody was raised in rabbits to partially purified, soluble NS protein present in cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis (VSInd) virus. This antiserum gave specific reactions of identity by agar immunodiffusion with both cytoplasmic and virion NS protein. NS antiserum also preferentially precipitated NS 3-H-labeled protein from infected cytoplasmic extracts, whereas anti-whole VSInd virion serum also precipitated N 3-H-labeled protein from extracts both of infected cytoplasm and virion nucleocapsids. Transcriptase activity of VSInd cytoplasmic or virion-derived nucleocapsids was effectively inhibited by ribonuclease-free immunoglubulin prepared from homologous NSInd antiserum or from anti-whole vesicular stomatitis virus serum. Transcriptase activity of heterologous New Jersey serotype (VSNJ) nucleocapsids and virions was not appreciably affected by anti-NSInd or by anti-whole VSInd virion gamma globulin. Anti-NS gamma glubulin immediately switched off RNA synthesis by actively transcribing VSInd nucleocapsids, a finding which suggests that NS antibody inhibits RNA chain elongation.

Animals↗
PubMed1977

Transcriptase activity associated with rabies virion.

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.

DNA-Directed RNA Polymerases↗
PubMed2026

Genomic insights into low-level rifampicin resistance mediated by borderline rpoB mutations in Mycobacterium tuberculosis: prevalence and phylogeny in Northeast China.

The emergence of low-level rifampicin (RIF) resistance in Mycobacterium tuberculosis poses a challenge to tuberculosis (TB) control, as it often leads to discordance between genotypic resistance detected by molecular assays (e.g., Xpert MTB/RIF) and phenotypic susceptibility in conventional drug susceptibility testing (DST). In this study, we performed whole-genome sequencing (WGS) on 17 clinical isolates from Changchun, Northeast China, which exhibited such discordance. All isolates harbored functional borderline mutations in the rpoB RRDR region, predominantly Leu452Pro and Leu430Pro (29% each), followed by His445Asn (18%). RIF minimum inhibitory concentration (MIC) values ranged from ≤0.25 to 1.0 mg/L, confirming low-level resistance. Notably, 53% (9/17) of the isolates were co-resistant to fluoroquinolones and 24% (4/17) to isoniazid (INH). According to WHO classification, 59% (10/17) were pre-extensively drug-resistant TB (Pre-XDR-TB) or multidrug-resistant TB (MDR-TB). Phylogenetic analysis revealed that 94% (16/17) belonged to the East Asian Beijing lineage (Lineage 2.2.1), with no evidence of recent local transmission. These findings underscore the complexity of low-level RIF resistance and its frequent association with broader drug resistance in a dominant lineage, highlighting the need for integrating MIC and WGS into diagnostic algorithms to guide appropriate treatment and surveillance.IMPORTANCEThe accurate detection of RIF resistance is critical for the management of TB, yet standard phenotypic methods often fail to identify strains with low-level resistance conferred by borderline rpoB mutations. This study provides the first genomic characterization of such discordant isolates in Northeast China, revealing a high prevalence of co-resistance to other key drugs and a strong association with the locally dominant Beijing lineage. The findings emphasize that reliance on phenotypic DST alone may lead to underestimation of drug resistance and inappropriate treatment, potentially contributing to the emergence and spread of Pre-XDR-TB and MDR-TB. Incorporating MIC determination and WGS into routine diagnostics could enhance detection, inform tailored therapy, and improve surveillance of these clinically significant strains.

Mycobacterium tuberculosis↗
PubMed2026

Genetic diversity and drug resistance profiles of Mycobacterium tuberculosis among Ethiopian children as determined by whole-genome sequencing.

UNLABELLED: Ethiopia ranks 30th among the tuberculosis (TB) burden countries, with children representing a significant yet understudied population group. This study aims to investigate the genetic diversity and drug-resistant profile among Ethiopian children. We included children under 15 years of age diagnosed with culture-confirmed pulmonary TB/drug-resistant TB between January 2017 and June 2023. Phenotypic drug susceptibility testing and whole-genome sequencing were conducted for 85 Mycobacterium tuberculosis (MTB) isolates. Demographic data were combined with genomic information. Lineage 4 was the most dominant (77.6%), while lineage 2 was less common (1%). Within lineage 4, several sub-lineages were identified, with lineage 4.2.2.2 being notably the most predominant (48%). Most of these cases were from Oromia (58%), including the hotspot areas for lineage 4 that were identified at a 99% confidence level. Among 17 MDR/pre-XDR-TB isolates, lineages 3 and 4.2.2.2 were the dominantly observed lineages/sub-lineages, with proportions of 29% and 65%, respectively. Of the 85 cases, 30.5% were drug-resistant TB to at least one of the five first-line anti-TB drugs tested by phenotypic drug susceptibility testing. Of these 26 drug-resistant TB cases, 23 were concordant with whole-genome sequencing characterization. The most frequent resistance mutations to rifampicin were found in the rpoB gene, specifically p.Ser450Leu (88%), followed by isoniazid in the katG gene, p.Ser315Thr (86%). Multidrug-resistant TB was strongly associated with MTB lineages (P = 0.007). This study identified high genetic diversity of M. tuberculosis and related drug-resistance mutations, with a strong concordance between whole-genome sequencing-based predictions and phenotypic drug susceptibility testing. IMPORTANCE: Our findings revealed a high genetic diversity of Mycobacterium tuberculosis among Ethiopian children, with the most common lineage being lineage 4, specifically lineage 4.2.2.2, in which a higher frequency of multidrug-resistant tuberculosis (TB) was observed. Additionally, we identified regional hotspots, suggesting ongoing community transmission. Moreover, whole-genome sequencing demonstrated high concordance with phenotypic drug susceptibility testing and identified mutation genes associated with first- and second-line anti-TB drugs, highlighting its usefulness in providing comprehensive results for resistance detection in children. Thus, it is essential for integrating genomic surveillance into childhood TB and drug resistance control.

Humans↗
PubMed1977

Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum.

Three, two major and one minor, distinct RNA polymerases have been isolated and partially purified from heterotrophically grown Rhodospirillum rubrum, a facultative photosynethetic bacterium. Associated with each of these three enzymes is a distinct polyadenylic acid polyemrase. All of these enzyme activities are dependent on DNA templates and are resistant to rifampicin and streptovaricin. The structural subunit composition, the response to various chemical compounds and DNA templates, and the properties of the products of these enzymes are studied in detail and compared with those of similar enzyme activities from other bacterial systems. Several unique features have been observed in the R. rubrum enzyme systems, such as an uneven incorporation of purine and pyrimidine nucleotides by the RNA polymerases, and the presence of a lag period in the polyadenylic acid polymerase activities.

Animals↗
PubMed1978

DNA-dependent RNA and polyadenylic acid polymerase from phototrophically grown Rhodospirillum rubrum.

DNA-dependent RNA and polyadenylic acid polymerases have been purified from phototrophic Rhodospirillum rubrum. Their properties have been found to be very similar to those of the previously reported heterotrophic R. rubrum enzymes. However, several important differences do exist between the enzymes from the phototrophic and the heterotrophic cells, such as the lack of response to added polyadenylic acid for poly A synthesis and the presence of the sigma subunit in the phototrophic enzymes. Furthermore, additional purification steps were necessary for preparation of phototrophic enzyme fractions with high DNA-dependence.

DNA-Directed RNA Polymerases↗
PubMed2026

Pathogenic POLRMT variants in mice impair mtDNA transcription and affect perinatal survival.

Mitochondrial gene expression is essential for oxidative phosphorylation that generates the bulk of the cellular ATP, and mitochondrial dysfunction is a common cause of human metabolic diseases. Recently, the first pathogenic variants in the only known mitochondrial RNA polymerase (POLRMT) were described in patients presenting with a wide variety of clinical manifestations, including hypotonia, short stature, and developmental delay. Here, we modeled two human pathogenic POLRMT variants by creating the corresponding substitutions in mice: the dominant S582F and the recessive R984C variant. Mice homozygous for the R984C variant showed perinatal lethality without apparent embryonic developmental defects, a finding consistent with a failure to adapt to the metabolic transition to oxidative metabolism at birth. Mice carrying the S582F variant were viable and exhibited decreased mitochondrial transcript levels due to impaired de novo transcription. However, mtDNA levels and in organello mtDNA replication remained normal, which recapitulates the molecular phenotypes observed in patients. Altogether, our findings indicate that the conserved arginine near the active site is essential for POLRMT function, while the serine in the intercalating hairpin of the N-terminal domain is required for near-genome length transcription but not primase activity. This study highlights genotype-phenotype differences and provides new insights into POLRMT function.

Animals↗
PubMed2026

Development and optimization of T-ARMS PCR assays for detection of lethal haplotypes of TADA2A, UR1B, and PORL1B in pigs in Vietnam.

Marker-assisted selection has increasingly relied on single-nucleotide polymorphisms (SNPs) as robust genetic markers, particularly in livestock breeding programs. In pig farming, embryonic mortality significantly affects litter size, and SNPs in reference genes have been implicated as potential causal factors. We developed and optimized a tetra-primer amplification refractory mutation system (T-ARMS) PCR assay for rapid, cost-effective detection of SNPs in 3 candidate genes-TADA2A, PORL1B, URB1-that are associated with embryonic lethality and reproductive performance. Primer sets were designed based on known mutation sites and validated using synthetic gene constructs and porcine genomic DNA from pigs of Duroc and Landrace breeds. Optimization of annealing temperatures and primer concentration ratios yielded distinct and reproducible allele-specific amplicon patterns that were corroborated by PCR-RFLP and Sanger sequencing. Our T-ARMS PCR protocol, which requires minimal equipment and reduces processing time to <3&#x2009;h, had high specificity and efficiency in differentiating wild-type, heterozygous, and homozygous mutant genotypes in 20 Duroc and 20 Landrace pigs. Our Tetra-ARMS PCR assay is a robust and economically viable tool for SNP genotyping in pig breeding programs, potentially contributing to the reduction of embryonic lethality and the improvement of overall reproductive outcomes.

Sus scrofa↗
PubMed1977

Nucleic acid biosynthesis in rat embryo cells infected with X14 or H-1 parvovirus.

Nucleic acid biosynthesis was studied in rat embryo cell (REC) cultures 48 hours after infection with X14 or H-1 parvovirus. The incorporation of 14C-formate and [6-(14C]-orotic acid into purines and pyrimidines of various was lowered after infection with these parvoviruses. 14C-Formate incorporation into acid-soluble thymine was greatly inhibited in H-1 virus-infected cells whereas it was slightly inhibited in X14 virus-infected cells. These results suggest that X14 virus-infected cells can carry out the biosynthesis of thymidylic acid utilizing some endogenous pyrimidine nucleotide (e.g. deoxycytidylic acid, via deoxyuridylic acid). In the infected cells, the nucleoplasmic RNA polymerase activity was strongly inhibited. This results suggests an interference by the two viruses with hosts RNA synthesis.

Adenine↗
PubMed1977

[Role of the functional groups of the sibiromycin molecule in DNA binding].

Biological activity of 2 derivatives of sibiromycin, an antibiotic close by its chemical structure to antramycin and their capacity for formation of complexes with DNA was studied. Anhydrosibiromycin like sibiromycin formed a complex with DNA. The antibiotic increased the DNA melting point but to a less extent than sibiromycin. Anhydrosibiromycin had a low activity in the system of DNA-dependent RNA-polymerase. The low biological activity of anhydrosibiromycin must be due to instability of the antibiotic complex with DNA. Methyl ether of sibiromycin by the phenol hydroxyl, the other derivative of sibiromycin had no biological activity and did not interact with DNA. On the basis of experimental data it was suggested that definite functional groups of the sibiromycin participated in DNA binding.

Antibiotics, Antineoplastic↗
PubMed1979

Characterization of RNA polymerases from Rous sarcoma virus-induced mouse ascites sarcoma cells.

RNA polymerase was extracted from the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV)-induced C3H/He mouse ascites sarcoma cells (SR-C3H). RNA polymerase was separated into RNA polymerases I and II by DEAE-Sephadex chromatography. RNA polymerase I was separated into Ia and Ib fractions by phospho-cellulose chromatography. In SR-C3H cells RNA polymerase Ib was the main component of RNA polymerase I. At 0.05--0.1 M ammonium sulphate RNA polymerase I transcribed native DNA most actively, and RNA polymerase II transcribed denatured DNA most actively. Partial digestion of DNA by DNAase I enhanced RNA synthesis by RNA polymerases I and II. At ionic strength over 0.2 M ammonium sulphate, the initiation reaction of RNA polymerases I and II was inhibited. The initiation complexes of RNA polymerases I and II with native DNA were more stable against high salt concentration than with denatured DNA.

Animals↗
PubMed1975

Tyrocidine and the linear gramicidin. Do these peptide antibiotics play an antagonistic regulative role in sporulation?

1. The cyclic peptide antibiotic tyrocidine, synthesized by Bacillus brevis (ATCC 8185), inhibits RNA synthesis in an in vitro transcriptional system by forming a complex with the DNA. 2. The linear peptide antibiotic gramicidin, synthesized by the same strain, reverses at least partly this inhibition. The molecular mechanism of this reactivation is unknown. Gramicidin by itself inhibits transcription in vitro. This inhibition is not due to a complex formation between DNA and the peptide. 4. A possible regulative role of the two peptides in sporulation is discussed.

Bacillus↗