Mercurated polynucleotides. New probes for hybridization and selective polymer fractionation

Dale, R.M.K.; Ward, David C.

doi:10.1021/bi00682a028

Cited by 164 publications

(64 citation statements)

References 29 publications

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“…This RNA, transcribed in vivo, is clearly a potential competitor with any in vitro transcript and is usually present in excess [4]. The facility with which UTP can be mercurated and the apparent lack of effect of this derivative on the utilisation of this nucleoside triphosphate as a substrate for RNA synthesis and on the properties of polymers containing it [5,6], suggested a method by which RNA synthesised in vitro could be resolved from any endogenous material. The following results confirm that the purified eucaryotic form A RNA polymerase will utilise Hg-UTP as a substrate analogue.…”

Section: Hg-utpmentioning

confidence: 99%

“…The RNA products synthesised by the endogenous nucleolar form A RNA polymerase in the presence of Hg-UTP are indistinguishable from those made with UTP and they may be used in hybridisation studies after demercuration. 6 B was from Pharmacia (Uppsala, Sweden). Hg-UTP was prepared by Mr John Jenkins in this laboratory, using the method described by Dale et al [5] and, by analysis on sulphydryl-Sepharose, was shown to be free of unmercurat-ed UTP.…”

Section: Hg-utpmentioning

confidence: 99%

“…RNA-containing fractions were again pooled, made 3 M with respect to 2-mercaptoethanol and allowed to stand overnight at room temperature. This procedure demercurates RNA [6]. Products made with unmercurated UTP were processed similarly.…”

Section: Purification Of R N Amentioning

confidence: 99%

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The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

Beebee¹,

Butterworth²

1976

European Journal of Biochemistry

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1. Purified form A RNA polymerase and the endogenous, nuclear form A RNA polymerase are shown to incorporate 5-mercuri-uridine 5'-triphosphate (Hg-UTP) into RNA in vitro. The K , for both Hg-UTP and UTP are in the region of 10 pM for the purified enzyme.2. The RNA products formed in nucleoli by endogenous RNA polymerase A have similar base compositions (G + C-rich) whether UTP or Hg-UTP is provided as the substrate in vitro.3. Sulphydryl-Sepharose chromatography of RNA synthesised in vitro by nucleoli allows separation of this material from the endogenous RNA, when the former is synthesised in the presence of 4. In-vitro-synthesised nucleolar RNA hybridises with cot profiles similar to 28-S ribosomal RNA, when made with either Hg-UTP or UTP.5. Hybridisation studies using DNA excess suggest that little competition occurs between the in vitro transcripts and the endogenous nucleolar RNA.6. Size analysis of in vitro transcripts show that although some degradation occurs during isolation, purification and hybridisation of the RNA species, most of the RNA remains larger than 5 S throughout.Hg-UTP.

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Section: Hg-utpmentioning

confidence: 99%

Section: Hg-utpmentioning

confidence: 99%

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The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

Beebee¹,

Butterworth²

1976

European Journal of Biochemistry

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“…Hybridization to radiolabeled cDNA would not be a suitable detection method because it does not distinguish in vitro synthesized from preexisting mRNA molecules. To overcome these difficulties, we used two different approaches, both exploiting the affinity of mercurated polynucleotides for sulfhydrylagarose (SH-agarose) (9,10). Using either method we could show that ovalbumin mRNA sequences have been synthesized by RNA polymerase B in a highly selective manner in isolated hen oviduct nuclei.…”

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confidence: 99%

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

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

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The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydrylagarose and hybridized to radioactive ovalbumin cDNA. (ii) (3HJUTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of a-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vivo RNA synthesis is retained in isolated nuclei.The synthesis of the egg white proteins in the tubular gland cell of the chicken oviduct is primarily related to the cellular accumulation of the corresponding mRNAs (1-7). During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2, 4, 5-7). Similar results have been found for the induction of ovomucoid and lysozyme mRNA (7). The steroid-mediated induction of specific mRNA molecules in the chicken oviduct may result from altered rates of synthesis and/or degradation. We therefore studied the synthesis rate of the egg white protein mRNAs. Because it was not possible to measure the rate of specific mRNA synthesis in the animals, we chose isolated oviduct nuclei as the cell-free system that might best reflect the transcriptional activity of the intact cell (8). The 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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“…For efficient binding of mercurated polynucleotides to SH-containing sorbents, one mercury atom/100 -200 nucleotides is sufficient [7]. Hence, for isolation of mercurated tRNAs by means of such sorbents, one mercury atom/tRNA molecule is adequate.…”

Section: Resultsmentioning

confidence: 99%

Preparation and properties of monomercurated tRNA

Abdurashidova

Nargisyan

Budowsky

1983

European Journal of Biochemistry

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Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated t RNA under non-denaturing conditions. The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions. Monomercurated tRNA quantitatively binds to thiopropyl-Sepharose and can be completely desorbed by mercaptoethanol-containing solutions. Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor-dependent polypeptide synthesis in vitro.One of the approaches to studying the functional topography of tRNA is determination of nucleoside residues involved in direct interactions with ribosomal proteins at different stages of translation. Here ultraviolet-induced tRNA-protein crosslinks could be used [l -51. For identification of proteins crosslinked to a definite tRNA molecule inside ribosomal complexes, as well as for location of cross-linked nucleoside residues in a tRNA sequence, it is necessary to isolate crosslinked nucleoproteins containing only this tRNA. This is possible if the tRNA molecule contains a specific affinity group, for instance, a mercury atom.Polynucleotides mercurated at C5 of pyrimidine bases are known to be firmly bound to sorbents containing an SH group [6 -81. Subsequent action of mercaptoethanol or similar compounds results in complete desorption of mercurated polynucleotides [6-81. Since ribosomal complexes contain several polynucleotides, a mercury atom in the respective tRNA must be introduced before complex formation. Therefore mercurated tRNAs should be functionally similar to the unmodified preparations and cause no side effects by the formation, ultraviolet irradiation and subsequent dissociation of complexes.Although mercuration of tRNA had been described earlier [9,10], the properties of mercurated tRNA mentioned above were not studied. In this paper we describe a method for preparation of monomercurated tRNA, which is able to bind reversibly to thiopropyl-Sepharose, but does not differ from the unmodified preparation in functional properties. MATERIALS AND METHODS MaterialsIndividual '4C-labelled amino acids (specific activity 25 -500 Ci/mol) were obtained from Amersham International; [203Hg](OA~), (specific activity 26 Ci/mol) from Isotop, USSR; individual tRNAPhe of Escherichia coli from Serva; elongation factors EF-Tu and EF-G from Biokhimreactiv, USSR: thiopropyl-Sepharose and DEAE-cellulose from Pharmacia; poly(U) and Fixion plates from Reanal, Hungary. Other reagents were of chemically pure grade.Labelled and unlabelled Hg(0Ac)z were prepared from [203Hg](N03)2 and HgClz ; total E. coli tRNA preparation was obtained according to [Ill; those deficient in tRNAPhe according to [12] ; total aminoacyl-tRNA synthetase according to [13]; 705 ribosomes of E. coli [activity in poly(U)-dependent binding of Phe-tRNAPhe = 80-90%] according to [14]; factor-dependent synthesis of poly(Phe) was ...

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Mercurated polynucleotides. New probes for hybridization and selective polymer fractionation

Cited by 164 publications

References 29 publications

The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Preparation and properties of monomercurated tRNA

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