Initiation of transcription of ribosomal deoxyribonucleic acid sequences in isolated nuclei of Physarum polycephalum: studies using nucleoside 5'-[.gamma.-S]triphosphates and labeled precursors

Sun, I Y; Johnson, Edward M.; Allfrey, Vincent G.

doi:10.1021/bi00588a018

Cited by 31 publications

(16 citation statements)

References 38 publications

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“…Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recover selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [ The method has been employed to study the initiation of 5S RNA in myeloma nuclei (S), ofpreribosomal RNAs in Physarum polycephalum (6) and Xenopus laevis oocyte nuclei (7), and of pre-tRNAs in yeast nuclei (8).Thio-derivatized deoxynucleoside triphosphates substituted in the a position have been employed as precursors for the in vitro synthesis ofviral DNAs (9, 10), and they permit an analysis of the stereochemistry of the DNA polymerase reaction (11). Some interesting recent applications include the synthesis of a 'S-labeled cDNA of rabbit globin mRNA (12) and the use of dATP [a-35S] for the nick-translation of plasmid DNA (12).…”

supporting

confidence: 65%

supporting

confidence: 64%

“…Eckstein and Gindl (1) showed that the ATP analogue adenosine 5'-O-(l-thiotriphosphate) (ATP[a-S]) was utilized as a substrate by Escherichia coli RNA polymerase. Adenosine 5'-O-(3-thiotriphosphate) (ATP[y-S]) (2) is effectively utilized for RNA chain initiation as catalyzed by both prokaryotic (3,4) and eukaryotic RNA polymerases (5)(6)(7)(8). A great advantage of phosphorothioate incorporation is that transcripts initiated with ATP[y-S] can be recovered selectively by affinity chromatography on organomercurial-Sepharose columns.…”

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confidence: 99%

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Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

Sun¹,

Allfrey²

1982

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

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African green monkey CV-1 cells infected with simian virus 40 (SV40) were incubated in the presence of sodium [MS]thiophosphate. The viral DNA was prepared and the newly synthesized SV40 DNA molecules containing phosphorothioate groups were selectively recovered by affinity chromatography on organomercurial-Sepharose columns. The thio-derivatized viral DNA occurred in both superhelical (form I) and relaxed circular (form II) configurations, and it gave the expected cleavage products upon digestion with the restriction endonucleases Kpn I and HindIl. Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recover selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [ The method has been employed to study the initiation of 5S RNA in myeloma nuclei (S), ofpreribosomal RNAs in Physarum polycephalum (6) and Xenopus laevis oocyte nuclei (7), and of pre-tRNAs in yeast nuclei (8).Thio-derivatized deoxynucleoside triphosphates substituted in the a position have been employed as precursors for the in vitro synthesis ofviral DNAs (9, 10), and they permit an analysis of the stereochemistry of the DNA polymerase reaction (11). Some interesting recent applications include the synthesis of a 'S-labeled cDNA of rabbit globin mRNA (12) and the use of dATP [a-35S] for the nick-translation of plasmid DNA (12).While it is clear that the phosphorothioate analogues of nucleoside and deoxynucleoside triphosphates have many important applications, such compounds are not effective substrates for the in vivo synthesis of polynucleotides, due to restricted permeation through the cell membrane. What is needed is a thio-derivatized precursor that can freely enter the nucleotide pools of living cells. We have recently shown that [3S]thiophosphate fulfils this requirement and effectively labels ATP, GTP, and other nucleotides of HeLa cells (13). The subsequent transfer of thiophosphate to nuclear proteins permits their purification by Hg-affinity chromatography and facilitates the analysis of different sites of modification within the polypeptide chain.Given the evidence that inorganic thiophosphate readily enters the ribonucleotide pools of cultured cells, we decided to test whether deoxyribonucleotide pools were also labeled to a degree permitting the isolation of thio-derivatized DNA. The experiments to be described indicate that [3S]thiophosphate can be used to label the DNA of simian virus 40 (SV40) in infected CV-1 cells and that the substitution permits purification of new SV40 molecules by Hg-affinity chromatography. MATERIALS AND METHODSThe African green monkey cell line CV-1 and SV40 were the generous gifts of M. T. Hsu of the Rockefeller University. The growth and maintenance of the cell line and propagation of SV40 were carrie...

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supporting

confidence: 65%

supporting

confidence: 64%

mentioning

confidence: 99%

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Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

Sun¹,

Allfrey²

1982

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

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“…Filter hybridization analyses were used initially to demonstrate initiation of pre-rRNA chains with nucleoside thiotriphosphates in nuclei isolated from Physarum polycephalum (15) and from Xenopus laevis oocytes (16). In the studies described here, use of the S1 nuclease mapping technique along with the thionucleotides allowed us to determine the precise site of transcription initiation in the isolated nuclei; the data unambiguously demonstrate that authentic initiation of pre-rRNA occurs in nuclei from rat and mouse cell culture lines.…”

Section: Discussionmentioning

confidence: 99%

Hormonal regulation of transcription of rDNA: use of nucleoside thiotriphosphates to measure initiation in isolated nuclei

1984

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Nuclei isolated from cultured mouse and rat cell lines initiated pre-rRNA chains at the correct site and with the correct nucleotide specificity (A for mouse, G for rat). Nucleic acid filter hybridization and S1 nuclease mapping were used to analyze the RNA products initiated with nucleoside (P-S)triphosphates. Initiation of pre-rRNA was completely resistant to a-amanitin but was inhibited by either actinomycin D or heparin. Experiments with P1798 mouse lymphoma cells indicated that the antiproliferative effects of glucocorticoids on lymphoid cells includes a reduction in the ability of nuclei to initiate pre-rRNA chains.

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“…Comparison of this profile with the transcription map shown in Fig. 2D leads to the conclusion that the DNA sequences corresponding to the pre-rRNA, which extend from about 8 to 29% on the map (28), are about six times more accessible to the intercalating drug than the central spacer region and about four times more accessible than the terminal spacer. This conclusion is qualitatively similar to that drawn for Tetrahymena rDNA by Cech and Karrer (2).…”

Section: Resultsmentioning

confidence: 86%

Accessibility of ribosomal genes to trimethyl psoralen in nuclei of Physarum polycephalum.

Judelson¹,

Vogt²

1982

Mol. Cell. Biol.

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We have probed the accessibility of the genes for rRNA in Physarum polycephalum by using the photoreactive DNA cross-linking agent 4,5',8-trimethyl psoralen. Nuclei isolated from actively growing Physarum were treated with trimethyl psoralen and irradiated with 360-nm light in order to form cross-links. The palindromic, extrachromosomal rDNA then was isolated, and the positions of cross-links were determined by electron microscopy of the DNA under totally denaturing conditions. The results indicate that the frequency of cross-linking, after correction for base sequence bias of the reaction, is up to sixfold higher in the transcribed regions than in the central or the terminal spacer regions. There is no detectable heterogeneity among the different rDNA molecules or between the halves of a single molecule. Cross-linked molecules invariably occur in a linear as opposed to a cruciform structure. The preferential cross-linking of the transcribed region is nearly eliminated in spherules, a dormant transcriptionally inactive form in the Physarum life cycle.The genes coding for ribosomal RNA (rDNA) in the acellular slime mold Physarum polycephalum are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases (kb) (30). Each of the several hundred molecules per nucleus has the structure of a palindrome, with a transcription unit near each end separated by a large central spacer region. The facts that rDNA is repetitious and that it is unlinked to chromosomal DNA and differs from it in GC content facilitate the purification and therefore the study of these genes.The structure of the repetitive unit common to all eucaryotic chromatin, the octamer of four different histones called the nucleosome, has been elucidated over the past several years (8, 16). Much less is known about the role of nonhistone proteins in chromatin, however, or about the features that distinguish those portions of the chromatin that are actively transcribed from those that are not. Both types of regions appear to contain nucleosomes, as defined by staphylococcal nuclease digestions (for example, see reference 32). At the same ionic strength, transcribed chromatin appears by electron microscopy to be more extended than nontranscribed chromatin (22,23,29). At least for certain vertebrate and invertebrate genes, an operational characteristic of transcribed chromatin is that it is preferentially attacked by DNase 1 (9,10,18,21,31,35 proteins HMG-14 and HMG-17 appear to play an essential role in this DNase I sensitivity (32,33). However, it is not certain that DNase I sensitivity is either a necessary or a sufficient condition for active transcription of chromatin. Experiments with yeast (17) as well as our own experiments with Physarum (Swofford and Vogt, unpublished results) failed to demonstrate differential DNase I sensitivity. Also, some chromatin has been found to be DNase I sensitive without concurrent transcription (18,20,26,31).Recently Hearst and collaborators (12,34) and Cech and collaborators (2,4,5) have shown that the...

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Initiation of transcription of ribosomal deoxyribonucleic acid sequences in isolated nuclei of Physarum polycephalum: studies using nucleoside 5'-[.gamma.-S]triphosphates and labeled precursors

Cited by 31 publications

References 38 publications

Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

Hormonal regulation of transcription of rDNA: use of nucleoside thiotriphosphates to measure initiation in isolated nuclei

Accessibility of ribosomal genes to trimethyl psoralen in nuclei of Physarum polycephalum.

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