Template specificity of transcription during sporulation of Bacillus subtilis

Milanesi, G.; Brevet, Jean

doi:10.1093/nar/1.3.397

Nucl Acids Res

1974

DOI: 10.1093/nar/1.3.397

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Template specificity of transcription during sporulation of Bacillus subtilis

G. Milanesi

Jean Brevet²

Abstract: Partially purified extracts from sporulating Bacillus subtilis cultures transcribed different natural DNAs with different efficiencies. This template specificity results in an increased or a decreased synthetic activity with respect to extracts from vegetative cells, depending on the template used. With SPP1 DNA a decrease in activity occurs, whereas with T7 DNA an increased activity was observed, which is due to a higher efficiency of initiation. This is not an intrinsic property of RNA polymerase, but is due… Show more

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Cited by 4 publications

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References 17 publications

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“…RNA, preparations were digested with electrophoretically purified DNase (Merck) that had been treated with iodoacetate (24). After phenol/chloroform extraction and ethanol-precipitation, RNA was incubated at 700 in 0.3 M NaCI/30 mM Na citrate and RNase resistance was determined as described (25).…”

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Minichromosome from BK virus as a template for transcription in vitro.

Meneguzzi¹,

Pignatti²,

Barbanti‐Brodano³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA. In recent years, a number of studies have been reported on the structural and biological properties of human papovavirus BK (1-5). Originally isolated from the urine of an immunosuppressed patient (6), this virus has been shown recently to induce malignant transformation in hamster cells (7-9). Interest in this infectious agent has further increased since it was shown that antibodies to BK virus (BKV) are widespread in the human population (10-12) and since BKV DNA was found in human tumors (13 DNA Purification. BKV DNA was extracted from purified virions as follows. Five milliliters of virus suspension containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% sodium dodecyl sulfate (NaDodSO4), and 50 jg of proteinase K per ml (Merck) was heated at 500 for 30 min and then incubated at 370 for 3 hr. CsCl (5.3 g) and ethidium bromide (2 mg) were then added and the density was adjusted to 1.600 g/cm3. After centrifugation in a Spinco Ti5O rotor at 43,000 rpm and 100 for [50][51][52][53][54][55][56][57][58][59][60] hr, fractions containing the superhelical form (F I) of DNA were pooled, extracted with isopropanol, and dialyzed against 10 mM Tris-HCl (pH 7.5)/1 mM EDTA.Transcription Assay. Fractions from the sucrose gradient used to isolate the nucleoprotein complex (see above) were assayed for template activity in an in vitro transcription system with E. coli RNA polymerase. Assays were performed by adding 50 ,ul of each fraction to 50 ,ul containing 80 mM Tris-HC1 (pH 7.9), 20 mM Mg9l2, 200 mM NaCl, 0.8 mM EDTA, 8 mM dithiothreitol, 0.8 mM each ATP, GTP, and CTP, and 0.4 mM [a-32P]UTP (specific activity 8 X 104 cpm/nmol). Final NaCl concentration was 150 mM. Reactions were started by addition of 5 ,ul of E. coli RNA polymerase (0.5 unit, purification procedure to be described elsewhere). After a 30-min incubation at 370, reactions were precipitated with 5% trichloroacetic acid/1% Na pyrophosphate and filtered on GF/C filters. Radioactivity was then determined. In all experiments, Abbreviations: BKV, BK virus; SV40, simian virus 40; NaDodSO4, sodium dodecyl sulfate. 1126The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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confidence: 99%

Minichromosome from BK virus as a template for transcription in vitro.

Meneguzzi¹,

Pignatti²,

Barbanti‐Brodano³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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“…These putative factors might be lost during the course of purification of RNA polymerase. One such case was previously described (13): a protein fraction that stimulated transcription of coliphage T7 deoxyribonucleic acid (DNA) preferentially was demonstrated in I Present address: Division of Clinical Pharmacology, Department of Medicine, Stanford University Medical School, Stanford, Calif. 94305. a t2 extract and separated from the holoenzyme by diethylaminoethyl-cellulose chromatography.…”

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Evolution of the transcription complex during sporulation of Bacillus subtilis

Brevet¹

1976

J Bacteriol

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Ribonucleic acid polymerase activity in partially purified extract of cells of Bacillus subtilis harvested at different times (t 1, to, t,, and t2) was studied by zone centrifugation. During the course of sporulation, vegetative sigma-factor activity decreased and the transcription complex lost some of its affinity for active sigma factor. The complex underwent a two-stage change in sedimentation value, from 14.5S in vegetative growth phase to a 13S species very early in sporulation to a 16S species at later times. Two Spo0 mutants have been studied by zone centrifugation. One strain, a rifampin-resistant (RfmR) mutant, failed to show any modification of the transcription complex, whereas the other, a Rfms strain, underwent a partial evolution of the transcription complex after to. M Tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.9; 0.01 M MgCl,; 0.2 M KCl; 0.1 mM 74

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“…It is generally accepted that, in bacteria, the ribonucleic acid (RNA) polymerase determines strategies of gene expression either by interacting with other proteins or through a modification of its own structure; the enzyme-mediated regulations become particularly evident under certain physiological conditions or during phage infection. Accordingly, changes in the pattern of transcription have been shown to occur during sporulation and germination in Bacillus subtilis (2,5,17,22,30,31,34). Early reports of modification of one of the subunits of RNA polymerase during sporulation ofB.…”

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Bacillus subtilis mutant temperature sensitive in the synthesis of ribonucleic acid

Riva¹,

Villani²,

Mastromei³

et al. 1976

J Bacteriol

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A Bacillus subtilis temperature-sensitive mutant (PB1653) has been isolated in which the rate of ribonucleic acid (RNA) synthesis sharply decreases after shift to 45 degrees C. Both stable and unstable RNAs are affected by the mutation. The possibility that the block of transcription at high temperature could be due to a "stringent" effect, mediated by an increase in the concentration of "magic spot" nucleotides, has been ruled out. Treatment with chloramphenicol (or streptomycin) rapidly restores the rate of RNA synthesis at 45 degrees C. The synthesis of RNA in the mutant during the early phases of spore germination is not temperature sensitive. The phage-specific transcription during infection with SPP1 phage, at high temperature, is less affected than that of the bacterial chromosome. In vitro experiments indicate that, in the mutant at high temperature, RNA polymerase undergoes a change in template specificity. The rna-53 mutation has been located on the B. subtilis genetic map near the hisA locus.

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Template specificity of transcription during sporulation of Bacillus subtilis

Cited by 4 publications

References 17 publications

Minichromosome from BK virus as a template for transcription in vitro.

Minichromosome from BK virus as a template for transcription in vitro.

Evolution of the transcription complex during sporulation of Bacillus subtilis

Bacillus subtilis mutant temperature sensitive in the synthesis of ribonucleic acid

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