An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels

Ledeboer, A.M.; Hille, Jacques; Schilperoort, R. A.

doi:10.1016/0005-2787(78)90135-1

Cited by 9 publications

(2 citation statements)

References 17 publications

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“…The fragments of 4x174 RF I J)NA (New England Biolabs GmbH, Schwalbach) were obldined by digestion with commercially available restriction endonucleases and isolated according to Lederboer et al [7]. with enzyme hybrid) were incubated h r 30 min at 37 "C. The concentration of glycerol in the incubation mixture was less than 1 "/, [8].…”

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confidence: 99%

Species Specificity of Promoter Recognition by RNA Polymerase and Its Transfer by the Sigma Factor

Ernst

Hartmann

Domdey

1982

European Journal of Biochemistry

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RNA polymerase holoenzyme from Micrococcus luteus synthesizes in vitro a run-off transcript of 85 nucleotides from a D N A fragment containing part of gene E of bacteriophage 4x174. This KNA starts with GTP as the 5' terminus 18 nucleotides downstream from the start of gene E on the viral (+ )strand. Transcription does not occur when the fragment is cleaved 36 nucleotides upstream of the initiation site. No transcript is obtained with RNA polymerase core or holoenzyme from Escschrrichia coli. Other DNA fragments containing the three major E. coli promoters of 4 x 1 7 4 are transcribed by both enzymes although much less efficiently by M . luteus RNA polymerase. When subunit sigma in E. coli RNA polymerase is replaced by sigma from M . luteus the resulting hybrid enzyme actively transcribes the DNA fragment containing the inner region of gene D with formation of the same run-off transcript which is obtained with M . luteus holoenzyme. In the presence of sigma from E. coli this R N A is not synthesized. The hybrid enzyme also transcribes it DNA fragment containing the geneA promoter of 4x174 with even higher efficiency than RNA polymera4e holoenzyme from E. coli:RNA polymerase holoenzymes from bacteria recognize specific sites on double-stranded D N A from which they start to transcribe the template [I]. Upon comparison of the lranscription products obtained with T7 bacteriophage DNA and RNA polymerases originating from a wide range of microorganisms it was found that all these enzymes recognize the strong promoters on the left end of the phage genome [2]. This implies that the recognition of a promoter sequence is independent of the eubacterial species.To check the general validity of this conclusion we have compared R N A polymerase from Micrococcus luteus [3] with that from Escherichiu coli with regard to the specificity of transcription initiation on various double-stranded D N A fragments derived from the DNA of bacteriophage 4 x 1 7 4 whose sequence is known [4,5]. We found that RNA polymerase from M . luteus is responsible for high-rate synthesis of a transcript on a DNA fragment which is not transcribed by either E. coli holoenzyme or core enzyme. The same transcript is produced upon addition of subunit sigma from M . luteus RNA polymerase to core enzyme from E. coli. MATERIALS A N D METHODS Enzymes and Nucleic _____tively. The fragments of 4x174 RF I J)NA (New England Biolabs GmbH, Schwalbach) were obldined by digestion with commercially available restriction endonucleases and isolated according to Lederboer et al. [7]. The reaction products were precipitated with ethanol and dissolved by heating to 90°C for 2 inin in 36 mM Tris/ phosphate pH 7.7 containing 10 niM EDTA, 8 M urea, 0.5 sodium dodecylsulfate and 10% glycerol [lo].

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confidence: 99%

Species Specificity of Promoter Recognition by RNA Polymerase and Its Transfer by the Sigma Factor

Ernst

Hartmann

Domdey

1982

European Journal of Biochemistry

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“…Both enzymes terminate synthesis of RNA I at exactly the same position on the template, indicating that the termination might not be species-specific. [16]. The size markers 5s rRNA.…”

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Synthesis of RNA I by the RNA polymerase from Micrococcus luteus on the Escherichia coli plasmid pBR 322

1984

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Incubation of DNA-dependent RNA polymerase from Micrococcus luteus (gram-positive) with the plasmid pBR322 under transcription conditions in vitro leads to the formation of a rather short-chained RNA. This transcript is initiated at the same site on pBR322 as RNA I, a defined Escherichia coli RNA polymerase product. M. luteus RNA polymerase initiates transcription at the RNA I site much more efficiently than the E. colienzyme, using either a plasmid preparation of pBR322 or an appropriate linear restriction fragment as template. In the latter case, cleavage of the restriction fragment 24 or 71 nucleotides (but not 91 nucleotides) upstream of the initiation site destroys template activity. By sequence analysis it was determined that the 3' terminus of the M . luteus RNA polymerase transcription product is identical with that known for RNA 1. Moreover, in agreement with synthesis of RNA I by E. coli RNA polymerase in uitro, termination by the M . luteus enzyme is also a stutter process characterized by the same dependence on the available UTP concentration. These observations lead to the hypothesis that termination by eubacterial RNA polymerases might not be species-specific.During the process of RNA synthesis in viuo and in citro, characteristic oligodeoxynucleotide sequences contained on the DNA template are recognized by eubacterial RNA polymerases as signals for the initiation and termination of transcription [l -31. In the case of the initiation signal, it has been shown that the efficiency of promoter recognition is not only a function of the DNA sequence but also depends on the RNA polymerase. Several different forms of RNA polymerase from Bacillus subtilis are known to exist. These are characterized by thcir respective sigma subunits of which seven distinct species have been detected so far. The oligonucleotide sequcnces recognized as promoters by these RNA polymerase forms are also quite different and depend on the subunit sigma present [2]. This finding is an important indication for the decisive role of the subunit sigma in signal recognition. The promoter recognition ability of hybrid enzymes made by combining subunits from different bacterial species is also directed by their respective sigma subunits [3-51. RNA polymerase from the gram-positive organism Micrococcus luteus synthesizes a specific 85-nucleotide run-off RNA when assayed with restriction fragment ffuelII/7 of Escherichia coli bacteriophage 4x1 74 under in ziitro transcription conditions. Detectable quantities of this transcript are not formed by the RNA polymerase from E. coli (gram-negative). Replacement of the subunit sigma of the E. coli RNA polymerase with that of M . luteus leads to a chimaeric enzyme which synthesizes the 85-nucleotide run-off RNA with a high yield [4]. Obviously, recognition of promoter sequences may vary, depending on the bacterial species from which the RNA polymerase originates. However. the question arises whether this is also true for termination of transcription. A prerequisite for the experimental investigat...

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Recognition of Promoter Sequences by RNA Polymerases from Different Sources

Leib

Ernst

Hartmann

1980

Chemical Recognition in Biology

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An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels

Cited by 9 publications

References 17 publications

Species Specificity of Promoter Recognition by RNA Polymerase and Its Transfer by the Sigma Factor

Species Specificity of Promoter Recognition by RNA Polymerase and Its Transfer by the Sigma Factor

Synthesis of RNA I by the RNA polymerase from Micrococcus luteus on the Escherichia coli plasmid pBR 322

Recognition of Promoter Sequences by RNA Polymerases from Different Sources

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