Transcription of bacteriophage φX174 in vitro: Selective initiation with oligonucleotides

Axelrod, Nancy

doi:10.1016/s0022-2836(76)80115-5

Cited by 49 publications

(23 citation statements)

References 19 publications

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“…Thus, in the eucaryotic system, this part of the genome codes for the synthesis of three different proteins in two different reading frames. This fi nding is similar in principle, but more complex, than that found for the ¢X 174 phage (57,69). Another unexpected finding came from the comparison of the oligonu cleotides in the late 16S mRNA with those expected in a transcript of the SV40 DNA coding for the VPl protein (90).…”

Section: Application Of the Chemical Methods Fo R Sequence Analysissupporting

confidence: 53%

“…Robertson et al isolated four different CPX 174 DNA fragments protected by ribosomes, and determined their sequence after synthesizing complementary RNA in vitro(63)(64)(65)(66). Sinsheimer et al(67,68) and Axel rod(69) reported the sequences of the 5' ends of three mRNA species synthesized in vitro and located them on the restriction enzyme map of cpX 174 DNA.Recently, Sanger et al(57) reported the provisional DNA sequence for the entire genome of cpX 174, comprising 5,375 nucleotides. This impressive work served to link up or confirm all other sequences determined earlier.…”

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confidence: 99%

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DNA Sequence Analysis

Wü¹

1978

Annu. Rev. Biochem.

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Section: Application Of the Chemical Methods Fo R Sequence Analysissupporting

confidence: 53%

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confidence: 99%

DNA Sequence Analysis

Wü¹

1978

Annu. Rev. Biochem.

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“…DNA-Bound Protein Effects RNA Polymerase. Studies on the in vitro transcription of fX174 DNA (20) and T7 DNA (21) indicated that in transcription reactions performed with excess polymerase and rifampicin, termination events occur at promoter sites, and are attributed to blockage of elongation by downstream RNA polymerase-rifampicin complexes incapable of initiating transcription. Our experiments show that the slow start-time for initiation from the lac UV5-polymerase complex may also result in the blockage of elongation of another polymerase molecule.…”

Section: Promoter Functionmentioning

confidence: 99%

Regulation of transcription from tandem and convergent promoters

Horowitz

Platt

1982

Nucl Acids Res

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ABSTRACrWe have examined transcription on templates containing the trp and lac UV5 promoters arranged in tandem or opposing orientations. These studies have revealed that the strengths of the two promoters are comparable, though the lac UV5 promoter is much more sensitive to the level of initiating purine present. Kinetic experiments have shown that a polymerase molecule poised at the lac promoter, or a lac repressor molecule bound to the lac operator, can temporarily block a polymerase molecule initiated from the trp promoter, though transcription eventually continues through. In the convergent construct, transcription from the lac promoter is hindered only when initiation is suboptimal due to low purine concentrations.

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“…Since then, several authors have used this system to initiate transcription at distinct promoters of viral DNA [39,42,43]. Taking advantage of this finding the synthesis of T4 transcripts was studied in vitro.…”

Section: Specific Initiation With Dinucleotidesmentioning

confidence: 99%

“…It was shown by Downey and So [41] using d(A-T),, as a template that the formation of a single phosphodiester bond between a dinucleotide, such as A-U, and a ribonucleoside triphosphate, such as ATP, rendered the RNA polymerase resistant to rifampicin and to dissociation by high salt. Since then, several authors have used this system to initiate transcription at distinct promoters of viral DNA [39,42,43]. Taking advantage of this finding the synthesis of T4 transcripts was studied in vitro.…”

Section: Specific Initiation With Dinucleotidesmentioning

confidence: 99%

Transcription of Bacteriophage T4 DNA in vitro: Selective Initiation with Dinucleotides

Rüger¹

1978

Eur J Biochem

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The transcription products of phage T4 DNA in vitro are separated on polyacrylamide gels. The influence of salt, polymerase, triphosphate concentration and glucosylation on the RNA synthesis are shown. Individual transcripts are initiated selectively with dinucleotides and a single triphosphate. This technique allows the prediction of the initiation sequences of several T4 transcripts.The linear and double-stranded DNA of bacteriophage T4 has a molecular weight of 1.19 x lo8 [l], corresponding to approximately 160 000 base pairs. About 100 genes of T4 are identified and localized on a circular map [2,3]. The sequence of the genes is circularly permuted [4,5] and the genome shows 2-3 % redundancy [6]. In place of cytosine T4 DNA contains 5-hydroxymethylcytosine [7] to which glucose is attached in c1 or fi linkage [8,9], rendering the DNA resistant against host restriction enzymes [lo]. In addition 0.35 % of the adenine residues are modified toThroughout phage development T4-specific RNA synthesis remains sensitive to rifampicin [13,14]. Therefore it is assumed that the host enzyme participates in transcription of all T4 mRNA. In vitro, however, the only promoters recognized efficiently by the Escherichiu coli RNA polymerase are those of the immediate early functions [15,16] the RNA of which is found in vivo at about 1.5 min after infection. A modification of the host RNA polymerase and the binding of phage-coded proteins to it then seem to be required in vivo for the transcription of the other classes of T4 mRNA. Among the T4-coded proteins involved in the transcription of the later This paper is dedicated to R. W. Kaplan on the occasion of his 65th birthday.Abbreviations. Abbreviations for nucleotides follows CBN recommendations, Eur. J. Biochem. 15,203-208 (1970); C-T4 DNA, cytosine-containing T4 DNA.Enzymes. RNA polymerase or DNA-dependent RNA polymerase (EC 2.7.7.6); RNase 111 or double-stranded-ribonucleate 3'-oligonucleotidohydrolase (EC 3.1.4.24); EcoRI or endodeoxyribonuclease EcoRI (EC 3.1.4.32).classes of mRNA are the products of the T4 genes mot, 45, 55, 33 and alc [20-251. To study T4 transcription in vitro it is desirable to separate clearly single transcription products. It will be shown in this paper that electrophoresis of T4 transcripts on a polyacrylamide gel results in a pattern of defined RNA bands. The chain lengths of the transcripts range between about 10000 and 450 nucleotides. The influence of template DNA, salt, enzyme and ribonucleoside triphosphate concentration will be investigated. Initiation of T4 transcription with dinucleotides allows the synthesis of single transcripts to be selected for. The sequences of some initiation sites will be predicted by alignment of overlapping trinucleotides stimulating the synthesis of unique transcripts. MATERIALS AND METHODS MaterialsUnlabeled nucleoside triphosphate and polyinosinic acid, (rI)" were purchased from Boehringer

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Transcription of bacteriophage φX174 in vitro: Selective initiation with oligonucleotides

Cited by 49 publications

References 19 publications

DNA Sequence Analysis

DNA Sequence Analysis

Regulation of transcription from tandem and convergent promoters

Transcription of Bacteriophage T4 DNA in vitro: Selective Initiation with Dinucleotides

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