Deoxyribonucleic Acid-Dependent Incorporation of Nucleotides From Nucleoside Triphosphates Into Ribonucleic Acid

Ochoa, Severo; Burma, D.P.; Kröger, Hans; Weill, J

doi:10.1073/pnas.47.5.670

Cited by 76 publications

(5 citation statements)

References 21 publications

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“…It has often been assumed that the declining rate of tumor growth is due to a general stopping of the metabolic processes of the cells, caused by a lack of nutrients providing energy and building blocks for metabolic and synthetic activities (Klein and Revesz, 1953;Patt and Straube, 1956). The present results are at variance with such a hypothesis, as it would be difficult to conceive that tumor cells may be lacking nutrients and energy for DNA synthesis but not for RNA s} nthesis, when the two processes have very much the same energy requirements (Kornberg, 1960;Ochoa et al, 1961). As noted above, on the 13th day of tumor growth, when DNA synthesis is considerably reduced, the rate of RNA synthesis, as judged by grain counts, is similar in the few cells that are also synthesizing DNA and in those that are not.…”

Section: Uptake Of H3-thymidine and Uridine-2-c 14 By Ehrlich As¢itesmentioning

confidence: 53%

A Study of Nucleic Acid Synthesis in Ascites Tumor Cells by Two-Emulsion Autoradiography

Baserga

1962

The Journal of Cell Biology

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Section: Uptake Of H3-thymidine and Uridine-2-c 14 By Ehrlich As¢itesmentioning

confidence: 53%

A Study of Nucleic Acid Synthesis in Ascites Tumor Cells by Two-Emulsion Autoradiography

Baserga

1962

The Journal of Cell Biology

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“…When phage DNA serves as a template for E. coli RNA polymerase in vitro at low ionic strength, the rate of RNA synthesis is rapid at first but reaches a plateau value after a few minutes (25)(26)(27)(28)(29)(30). This plateau corresponds to the synthesis of one chain of RNA per enzyme molecule (4, 6, 7).…”

Section: Discussionmentioning

confidence: 99%

Reinitiation of RNA Synthesis on Transcription of Chromatin DNA by Escherichia coli RNA Polymerase

Morris

Gould

1971

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

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The transcription of DNA in chromatin by Escherichia coli RNA polymerase resembles that of isolated DNA in two important respects: the release of nascent RNA and reinitiation of RNA synthesis is dependent on the salt concentration, and RNA synthesis is markedly stimulated by the addition of ribosomes.Recent experiments have established that the release of nascent RNA and reinitiation of RNA synthesis by Escherichia coli RNA polymerase with DNA templates occurs only in favorable ionic conditions (1-8). The ability of ribosomes to bind to nascent RNA (9-11) and stimulate the transcription of DNA (12)(13)(14)(15)(16)(17) in vitro is also well documented. The possibility exists that these particular factors might also govern the transcription of chromatin templates, and we show here that this is indeed the case in an in vitro system. RESULTSThe effect of variation in the KCl concentration in the assay mixture on the incorporation of labeled UTP into RNA, directed by chicken kidney chromatin, is shown in Fig. 1.The maximum rate of RNA synthesis measured in a 20-min incubation was observed at 0.25 M KCl. The optimum divalent cation concentration at 0.25 M KCl is 5-10 mM as shown in Figure 2. The rates of UTP incorporation at 0 and 0.25 M KCl are similar in the first few minutes of the incubation. Thereafter, as may be seen in Fig. 3, RNA synthesis in the absence of salt ceases, while that in the presence of salt continues for about 90 min, although at a diminishing rate. The overall stimulation of RNA synthesis in the presence of salt is generally found to be [2][3][4] fold.The following experiment shows that the stimulatory effect of KCl is accompanied by the release of nascent chains from the enzyme and the template. The synthesized RNA was fractionated by filtration on Millipore filters. Released RNA would be expected to flow through the filter, whereas RNA bound to chromatin or RNA polymerase would not (6,18,19). The results illustrated in Fig. 4 show that the RNA synthesized in the absence of salt is all retained by a Millipore filter. The excess RNA produced by the presence of salt passes through the filter.In the absence of salt, RNA polymerase cannot reinitiate RNA synthesis. This was shown with the antibiotic rifampicin, which blocks initiation but allows the synthesis of initiated chains to go to completion (6,20). Fig. 5 shows that rifampicin added 2 min after the start of incubation totally 481 represses the stimulation by KCL. The increase in RNA synthesis is thus a direct consequence of reinitiation. RNA synthesis at high, but not at low, ionic strength is further stimulated by the addition of ribosomes. At 0.25 M KCl, in the experiment shown in Fig. 6, we obtained more than a tenfold increase in RNA synthesis at the optimal ribosome concentration. Whereas the salt effect becomes increasingly more apparent as a function of incubation time, the ribosome effect is manifested early in the reaction and is relatively constant throughout the course of RNA synthesis (Fig. 7). (1, 2). Incubation was as d...

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“…C) The formation of homopolymers in the presence of he!ated DNA is catalyzed by the microbial enzymes (6,40), wherea,s the formation of a homopolymer is not detected with the maize polymerase. D) RNA polymerases isolated froim microbial cells (6,19,31,39,45) are fo-und associaited with a DNA-rich fraction of celil homogen;ates. The bulk of the RNA pollymerase from maize seedlings is fouln(d in the DNA-poor, soluble portion of tissuie homogenates (27).…”

Section: Methodsmentioning

confidence: 99%

Template Requirement of Maize RNA Polymerase

Stout

Mans

1968

Plant Physiol.

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A bstract. Maize RNA polymerase utilizes heated deoxyribonucleic acid more effectively than native deoxyribonucleic acid as a template for ribonucleic acid synthesis. A ribenucleic acid-deoxyribonucleic acid hybrid accumulates in the presence of 'heated deoxyribonucleic acid. The amount of product formed with either native or heat-denatured deoxyribonucleic acid does not exceed the amount of deoxyribonucleic acid added as template.The itemplate requirement of several RNA polymerases ('Nucleoside-Htriphosphate: RNA nudleotidyl transferase, EC 2.7.7.6), isolated and purified from microorganism's (5,7,11,41 ) anid mamtmals ( 13, 32), is best satisfied with native DNA. Heat denatured, calf thymus DNA actually inhibits the polymerase isolated from bovine lymiphosarcoma tissue (12). In characterizing the partially purified enzyme from maize seedllings we have shown that, lat relatively high levels, native or denatured DNA from several species satisfies the template requirement of the maize enzyme (42). The data presented here demonstrate thait at rate limiting concentrations, denatured DNA from maize seed1lings or calf thym'us is 8 times more effective than native DNA in satisfying the template requirement. Furthermore, an RNA-DNA hybrid, analogous to that formed by Azotobacter vinelandii RNA polymerase (44), accutmulates u'pon incuLbation of the enzyme with 'sub'stra'te and denatured DNA. The hybrid is not found w'hen native DNA is used as template. Materials and MethodsEnzyme Preparationt and Assay. Soluble RNA polymerase was isolated f'rom 5-day-old maize seedlings as described previously (42). The am'monium sullfate precipitate was desalited on Sephadex G-50 ra,ther than by di,a)lysis prior to column chromatography on diethylaminoethyl-( DEAE) cellu'lose.The enzyme had a specific activity of 1870. Specific activity is def'ined as rnLmoles of AMP incorporated in 10 minutes at 300 per milligram prote'in.

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Deoxyribonucleic Acid-Dependent Incorporation of Nucleotides From Nucleoside Triphosphates Into Ribonucleic Acid

Cited by 76 publications

References 21 publications

A Study of Nucleic Acid Synthesis in Ascites Tumor Cells by Two-Emulsion Autoradiography

A Study of Nucleic Acid Synthesis in Ascites Tumor Cells by Two-Emulsion Autoradiography

Reinitiation of RNA Synthesis on Transcription of Chromatin DNA by Escherichia coli RNA Polymerase

Template Requirement of Maize RNA Polymerase

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