Double-Stranded Viral and Cellular DNA’s as Templates for Purified Mammalian DNA-Dependent RNA Polymerases

Chambon, Pierre; Mandel, Jean‐Louis; Gissinger, F.; Kédinger, Claude; Gross-Bellard, M.; Hossenlopp, Paul

doi:10.1007/978-3-642-65725-2_7

Cited by 4 publications

(1 citation statement)

References 41 publications

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“…coli holoenzyme and bacteriophage DNAs [3,4]. I n this respect, as well as by their incapacity to transcribe intact linear doublestranded DNAs [30,31], the purified mammalian RNA polymerases resemble E . coli core enzyme.…”

Section: Resultsmentioning

confidence: 99%

Animal DNA‐Dependent RNA Polymerases

Hossenlopp

Oudet

Chambon

1974

European Journal of Biochemistry

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The binding of purified mammalian RNA polymerases AI and B to Simian virus 40 (SV40) DNA was studied under various conditions (ionic strength, divalent cation, temperature) by two methods, electron microscopy and retention of enzyme * DNA complexes on nitrocellulose filters. Our studies demonstrate that there are multiple binding sites for mammalian RNA polymerases on SV40 DNA form I and that highly stable enzyme -DNA-form-I complexes are formed. However, in contrast to the bacterial RNA polymerase holoenzyme, the purified mammalian enzymes are unable to form a highly stable complex with a linear double-stranded DNA (SV40 DNA form 111). Our results strongly suggest that the formation of highly stable complexes between mammalian RNA polymerases and SV 40 DNA form I is due to their binding to the unpaired regions which are present in the superhelical DNA.Results presented in the accompanying papers have revealed that initiation of RNA synthesis by mammalian RNA polymerases on Simian virus 40 (SV40) DNA is markedly dependent on the configuration of the template and on the nature of the divalent cation present in the reaction medium. These results have also shown that superhelical SV40 DNA-FI contains multiple initiation sites for mammalian RNA polymerases and that both strands of the template are transcribed. On the other hand, Herzberg and Winocour [i] have reported that superhelical SV40 DNA-FI possesses only one single binding site for the mammalian RNApolymerase B. In an attempt to resolve this discrepancy and to get a better insight into the mechanisms regulating the initiation of RNA synthesis by the mammalian enzymes on SV40 DNA-FI, we have studied the binding of purified mammalian RNA polymerases to this DNA. Since such studies can only be adequately performed with highly purified enzymes, we have mainly used purified calf thymus enzymes AI or B. Most of the experiments were carried out with calf thymus enzymes B This paper is part 8 of a series. The binding of mammalian RNA polymerases to SV40 DNA was studied by two complementary methods : electron microscopy and retention of enzyme DNA complexes on nitrocellulose filters. This latter method is very convenient, since it is rapid and since it gives a semi-quantitative picture of the binding of RNA polymerase to DNA and of the effect of some parameters on this binding reaction. Using this method, Hinckle and Chamberlin 113-51 have shown that site selection occurs during binding of Escherichia coli RNA polymerase holoenzyme to T7 bacteriophage DNA and involves the formation of a highly stable holoenzyme * T7 DNA complex at, or near, the T7 promoter site. Formation of this complex requires the cr subunit and most likely involves the opening of the double helix near the promoter site. In contrast, binding of E. coli core RNA polymerase to intact T7 DNA does not lead to the formation of a, highly stable complex.Eur. J. Biochem. 41 (1974)

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Section: Resultsmentioning

confidence: 99%

Animal DNA‐Dependent RNA Polymerases

Hossenlopp

Oudet

Chambon

1974

European Journal of Biochemistry

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Isolation and some properties of a mammalian ribosomal DNA

1976

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The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2% Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9 X 10(6) daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9 X 10(6) dalton fragment, while both the 16.4 and 4.9 X 10(6) dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3 X 10(6) daltons which includes a nontranscribed spacer of about 12.5 X 10(6) daltons.

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9. Eucaryotic RNA Polymerases

Chambon¹

1974

Protein Synthesis DNA Synthesis and Repair RNA Synthesis Energy-Linked ATPases Synthetases

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Double-Stranded Viral and Cellular DNA’s as Templates for Purified Mammalian DNA-Dependent RNA Polymerases

Cited by 4 publications

References 41 publications

Animal DNA‐Dependent RNA Polymerases

Animal DNA‐Dependent RNA Polymerases

Isolation and some properties of a mammalian ribosomal DNA

9. Eucaryotic RNA Polymerases

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