Transcription of the genes for 5S ribosomal RNA and transfer RNA in isolated mouse myeloma cell nuclei

Marzluff, William F.; Murphy, Edwin C.; Huang, Ru Chih C.

doi:10.1021/bi00715a011

Cited by 98 publications

(77 citation statements)

References 18 publications

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“…We have calculated that sea urchin blastula embryos, to satisfy the requirement for synthesis of one of the most abundant RNAs in the cell, synthesize about one molecule of Ul RNA per gene per min (30). Given this high rate of expression and the fact that the RNA is small and should take less than a minute to transcribe, it seemed possible that at least a portion of the synthesis of this RNA in vitro resulted from de novo initiation, as has been found for the small RNA polymerase III transcripts (24,36). To test the possibility of initiation, we used P-S-and y-S-ribonucleotide triphosphates as substrates.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Morris¹,

Marzluff²

1985

Mol. Cell. Biol.

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Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin Ul RNA. This RNA is synthesized by RNA polymerase II and, in a hybridizationselection experiment, was selected by the cloned sea urchin Ul gene. The Ul RNA was initiated with ATP, but not GIP, in isolated nuclei with P-S-and y-S-ribonucleotide triphosphates as substrates. The Ul RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.

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Section: Methodsmentioning

confidence: 99%

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Morris¹,

Marzluff²

1985

Mol. Cell. Biol.

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“…They are excellent templates for the RNA polymerase of E. coli. Whereas the transcription in vitro of nuclei and chromatin generally involves the elongation of pre-existing chains [35,46,47,71] all synthesis in nucleoids depends upon the initiation of new RNA chains. Since transcription in vivo is likely to be controlled at initiation these features make nucleoids attractive for studies on the control of transcription.…”

Section: Discussionmentioning

confidence: 99%

“…Since we hope eventually to study RNA synthesis directed by templates constructed from nucleoids and selected proteins, we chose assay conditions known to be favourable for the initiation and elongation of RNA chains in isolated nuclei and chromatin [35,46,471. Fig.9 illustrates how variations in the composition of the assay mixture affect transcription of HeLa nucleoids and Xenopus DNA.…”

Section: Composition Of' the Assay Mediummentioning

confidence: 99%

Transcription of Superhelical DNA from Cell Nuclei

Colman

Cook

1977

European Journal of Biochemistry

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Structures resembling nuclei may be released by gently lysing human or frog cells in solutions containing non-ionic detergents i n d 1.95 M NaC1. These structures, or nucleoids, sediment in sucrose gradients containing intercalating agents in the manner characteristic of DNA that is intact, supercoiled and circular. They are depleted of nuclear protein and contain no endogenous RNA polymerase activity. We describe conditions for the transcription in vitro of these nucleoids by the RNA polymerase of Escherichia coli. We compared the kinetics of RNA synthesis directed by nucleoids and by DNA prepared using conventional procedures : nucleoids direct RNA synthesis at three to four times the rate of an equivalent weight of pure DNA and under appropriate conditions the DNA of each nucleoid can direct the synthesis of twice its own weight of RNA. Most of the newlysynthesized RNA remains within the nucleoid. Experiments with the inhibitor, rifampicin, reveal that all RNA synthesis depends upon the initiation of new RNA chains and that nucleoids and pure DNA possess similar numbers of initiation sites. RNA synthesis directed by nucleoids has unusual kinetics : maximal rates are attained only after a lag of about ten minutes. Almost no lag is seen with pure DNA. The lag is due to the slow rate of formation of complexes of polymerase with nucleoid DNA that can initiate. Removal of supercoils by irradiation with y-rays or by the addition of the unwinding ligand, ethidium bromide, decreases the numbers of polymerase molecules able to initiate synthesis rapidly and increases the lag before maximal synthetic rates are achieved. Loss of supercoiling does not alter the maximum synthetic rate. Supercoiling in eukaryotic DNA clearly influences the initiation of RNA synthesis. These results are discussed with reference to the effects of supercoiling on the transcription of prokaryotic templates.Structures resembling nuclei may be released by lysing cells gently in solutions containing non-ionic detergents and high concentrations of salt. These structures, which we call nucleoids, sediment in sucrose gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled, and circular. These characteristic changes in sedimentation rate are abolished by irradiating the nucleoids with y-rays, a procedure known to break DNA. We interpret these results as showing that nucleoids contain DNA that is subject to the same kind of topological constraint restricting rotation of one strand of the double helix of DNA about the other as that found in intact and circular DNA [1,2]. When the DNA of Escherichia coli is isolated using the procedures of Stonington and Pettijohn [3] similar constraints are found [4].Supercoils are lost spontaneously from circles of DNA when only one phosphodiester bond is broken so that supercoiling is a sensitive index of DNA Enzymes. RNA polymerase (EC 2.7.7.6); ribonuclease A (EC 3.1.4.22).integrity. Since very low doses of y-rays sufficient to introduce few breaks into DNA remove the...

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“…We have reported previously that myeloma nuclei are capable of synthesizing RNA at a high rate (9,10). The RNA product resembles nuclear RNA with respect to size range as determined by sucrose gradient centrifugation and contains the same specific RNA species as analyzed by gel electrophoresis.…”

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confidence: 81%

“…The cells used were 66-2 mouse myeloma cells which produce a K-chain. They were grown to a concentration of 3 to 5 X 105/ml (9,10).…”

Section: Methodsmentioning

confidence: 99%

Chromatin directed transcription of 5S and tRNA genes.

Marzluff¹,

Huang²

1975

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

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Chromatin prepared by gentle methods from mouse myeloma cells retained its ability to synthesize RNA using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). The transcription resembles that observed in vivo in several respects. The lowmolecular-weight RNA species, 5S RNA and the 4.5S precursor to 4S RNA, are transcribed accurately and transcription is reinitiated continually in vitro. Their synthesis was not inhibited by a-amanitin (1 ,ug/ml) as was found previously for these species in isolated nuclei.Several chromatin-transcription systems for specific genes were described recently (1-6). Using purified globin cDNA as the probe and Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), Axel, et al. (1) and Gilmour and Paul (2) found that globin sequences were transcribed from the chromatin of erythroid tissues but not from the chromatin of other systems. Steggles et al. (3) have extended these studies to show that homologous RNA polymerase also transcribes the globin sequences and these constitute a slightly larger percentage of the total product than when E. coli RNA polymerase is used. Astrin (4) and Shih et al. (5) showed that E. coli RNA polymerase transcribed SV40 viral sequences using chromatin from SV40 virus-transformed cells as template. The same strand of SV40 DNA was transcribed both in vivo and in vitro, whether DNA or chromatin was used as template (5). Most In facing such concerns, we have concentrated our efforts on developing an in vitro synthesis system which hopefully will yield the same transcriptional products as are found in living cells. We have reported previously that myeloma nuclei are capable of synthesizing RNA at a high rate (9, 10). The RNA product resembles nuclear RNA with respect to size range as determined by sucrose gradient centrifugation and contains the same specific RNA species as analyzed by gel electrophoresis. In particular, these nuclei reinitiate continually the synthesis of 5S RNA and of 4.5S precursor to tRNA. Other workers have reported faithful transcription of rRNA in isolated nuclei (11,12) and of viral-specific RNAs in nuclei from virus-infected cells (13,14).As a step further, we report here the isolation of myeloma chromatin with high endogenous RNA polymerase activity. The chromatin faithfully synthesizes specific RNA species, and 5S genes are transcribed continually in vitro with correct DNA strand selection. This chromatin, which contains only half of the nuclear protein and 10% of the nuclear RNA, promises to be useful for the study of the control of specific gene expression in eukaryotes. MATERIALS AND METHODSCells. The cells used were 66-2 mouse myeloma cells which produce a K-chain. They were grown to a concentration of 3 to 5 X 105/ml (9, 10).Preparation of Chromatin. Nuclei were prepared exactly as described previously (9). The nuclei were suspended in incubation medium (25% glycerol, 5 mM Mg...

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Transcription of the genes for 5S ribosomal RNA and transfer RNA in isolated mouse myeloma cell nuclei

Cited by 98 publications

References 18 publications

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Transcription of Superhelical DNA from Cell Nuclei

Chromatin directed transcription of 5S and tRNA genes.

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