The SV4O transcription complex. I. Effect of viral chromatin proteins on endogenous RNA polymerase activity

Brooks, Timothy L.; Green, Melvin H.

doi:10.1093/nar/4.12.4261

Cited by 35 publications

(18 citation statements)

References 34 publications

(24 reference statements)

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“…This requires the use of a rather high salt concentration, but not so high as to promote the dissociation of protein from the SV40 chromatin (24). It is the purpose of this report to demonstrate that protein does not dissociate from SV40 DNA molecules during the process of transcription.…”

Section: Introductionmentioning

confidence: 89%

“…Except for the following procedures, all materials and methods are as described in the preceding paper (24). a.…”

Section: Methodsmentioning

confidence: 99%

“…2). After the synthesis of 3H-RNA, samples were made 0.1% in sodium dodecyl sulfate (SDS), then centrifuged through Sephadex G-25 equilibrated with 0.1% SDS (24). SV40 14C-DNA form I was added as marker, and the samples were adjusted to a density of 1.75 g/cc by the addition of saturated CsCl and buffer (10 mM Tris, pH 7.4; 10 mM EDTA; 0.25% Triton X-100) in a final volume of 5 ml.…”

Section: Methodsmentioning

confidence: 99%

“…Purified SV40 transcription complex (23) was used to synthesize 3H-RNA in vitro under conditions which do not cause the dissociation of protein from the bulk of the viral DNA (24). Reactions were carried out for either 15 min with a rate-limiting concentration of 3H-UTP (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…, either in the presence of a limiting amount of 3H-UTP (7 iM) for 15 min (part A) or in excess 3H-UTP (43 pM) for 120 min (part B). 3H-RNA was prepared and sedimented through sucrose gradients as described previously (24). Fractions were analyzed for 3H-RNA (-) and 21S SV40 14C-DNA form I (---).…”

Section: Methodsmentioning

confidence: 99%

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The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

Green

Brooks

1977

Nucl Acids Res

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ABSTRACT.A small fraction of the SV40 chromatin isolated from infected monkey cell cultures by the Triton method contains active RNA polymerase which had initiated transcription in vivo. This viral transcription complex (VTC) was utilized to answer the question of whether proteins dissociate from chromatin during transcription in vitro. 3H-RNA was synthesized by the VTC under conditions such that over half the label was in transcripts which were longer than half the length of the SV40 genome. Virtually all of the 3H-RNA remained associated with the SV40 chromatin, causing an increase in sedimentation rate from 55S to 78S. The density of the VTC-3H-RNA complex indicated that less than 5% of the original protein dissociated from the SV40 DNA which served as a template for transcription. We conclude that SV40 chromatin can be transcribed while the proteins remain associated with the DNA.INTRODUCTION.

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Section: Introductionmentioning

confidence: 89%

“…Except for the following procedures, all materials and methods are as described in the preceding paper (24). a.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

Green

Brooks

1977

Nucl Acids Res

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Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Luchnik¹,

Bakayev²,

Zbarsky³

et al. 1982

The EMBO Journal

135

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After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2‐5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2‐5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS‐minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

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Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: Retention of in vivo sensitivity to 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)

Holst

Egyházi

1985

J of Cellular Biochemistry

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The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42-, 33-, and 30-kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42-, 33-, and 30-kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.

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The SV4O transcription complex. I. Effect of viral chromatin proteins on endogenous RNA polymerase activity

Cited by 35 publications

References 34 publications

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: Retention of in vivo sensitivity to 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)

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