Transcription in vitro of DNA in isolated bacterial nucleoids

Giorno, Ralph; Stamato, Thomas D.; Lydersen, Bjorn K.; Pettijohn, David E.

doi:10.1016/0022-2836(75)90344-7

Cited by 28 publications

(10 citation statements)

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“…At various times after being placed in a 370C bath, 0.4-ml samples were removed and incubated for 30 sec at 370C with 5 jACi of [3H]thymidine; then cold 5% trichloroacetic acid was added. Acid-precipitable [3H]DNA in each 0.4-ml sample was determined as described (21). molecules and, therefore, band separately at denser positions.…”

Section: Resultsmentioning

confidence: 99%

Supercoils in prokaryotic DNA restrained in vivo.

Pettijohn¹,

Pfenninger²

1980

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

128

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Cells of Escherichia coli containing the plasmid F were 7-irradiated with various doses to introduce determined numbers of single-strand breaks in the F DNA. The cells were then incubated to permit repair of the breaks while DNA gyrase was inhibited with coumermycin to limit restoration of any relaxed supercoils. Repaired, covalently continuous F DNA was isolated and its superhelical density was measured by two different methods. Both indicated that a major part (50-60%) of the negative superhelical turn's were maintained in the repaired molecules, suggesting that the supercoils are restrained in vivo.In eukaryotic chromatin and chromosomes, the first order of DNA packaging seems to be attained via the nucleosome structure (for reviews, see refs. 1 and 2). The double-helical DNA in this particle is coiled through its interaction with the core histones into about 13/4 toroidal superhelical turns per core nucleosome (3, 4). Prokaryotic DNA has superhelical densities similar to those found in eukaryotes (5, 6), but thereis no clear evidence for restraint of the supercoils in structures analogous to the nucleosome. Histone-like proteins are found in prokaryotes (7,8), and their mode of interaction with DNA in prokaryotic chromosomes may have some similarity with nucleosomic histones (8, 9). Also, beaded structures having the appearance of nucleosomes have been seen in an electron microscope study of DNA from partially lysed bacteria (10).However, pure DNA can spontaneously form beaded structures in certain solvents (11), and electron microscopic evidence alone cannot be considered sufficient to establish the existence of the prokaryotic equivalent of a nucleosome.DNA gyrase seems to play a vital role in forming DNA supercoils in prokaryotic chromosomes. This enzyme can catalyze an ATP-driven reaction that has the topological effect of decreasing the linking number between the two continuous strands of a DNA double helix, thereby introducing negative superhelical turns into the DNA (12). When the DNA gyrase is inhibited by coumermycin (or oxolinic acid) in intact Escherichia coli cells, X DNA molecules, which are converted in vivo to the covalently circular form, attain only a small fraction of their normal superhelical density (13,14). This result indicates that the DNA gyrase activity is necessary for the formation (or the maintenance or both) of at least part of the DNA supercoils in prokaryotic cells. Thus, it seems possible that, in viwo, supercoils in prokaryotic DNA may not be stabilized by a nucleosome-like structure and that the Major reason for both the formation and maintenance (15) of DNA supercoils is the activity of DNA gyrase. Other alternatives include the possibility that DNA supercoils catalyzed by the DNA gyrase are subsequently restrained in a nucleosome-like structure by interactions involving molecules other than DNA gyrase (9). These two alternatives differ in that in the former case the double helix (17). After irradiation, the 32p-labeled cells kept at 0C were divided into several p...

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Section: Resultsmentioning

confidence: 99%

Supercoils in prokaryotic DNA restrained in vivo.

Pettijohn¹,

Pfenninger²

1980

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

128

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“…In the phasecontrast microscope nucleoids resemble nuclei depleted of protein; nucleoli are clearly visible and membrane ghosts and cytoplasmic material adhere to the nucleoids (Fig. 1) 21. The original experiments concerned nucleoids prepared from HeLa and XTC cells in 1.0 M NaCl; we have since shown that HeLa nucleoids may be isolated freer of histones in 1.95 M NaCl, and that they can be pipetted without loss of supercoiling [8].…”

Section: Characterization Of Nucleoidsmentioning

confidence: 98%

“…The size of the RNA transcripts made in vitro was determined by a modification of the method of Giorno et al [21] which involves sedimenting the denatured transcripts in sucrose gradients containing formaldehyde. 50-yl aliquots containing radioactive RNA were removed from the assay mixture at specified times and added to 10 pl containing 1.13 % sodium dodecyl sulphate and 40mM EDTA to stop the reaction.…”

Section: Determination Of the Size Of Transcriptsmentioning

confidence: 99%

“…In this respect nucleoids resemble DNA [34] and bacterial nucleoids [21] rather than isolated nuclei in which RNA is synthesized most rapidly at 25 "C [33,35 -…”

Section: Effect Of Temperature On R N a Synthesismentioning

confidence: 99%

“…Rifampicin was added simultaneously with the triphosphates to prevent reinitiation. After 3 or 30 min, samples were removed and the size of the newly-synthesized and labelled RNA determined by centrifugation in sucrose gradients containing formaldehyde [21]. As the weightaverage and number-average molecular weights of the RNA synthesized on the two templates after identical periods are similar (Fig.…”

Section: Effect Of Ethidium On Transcription In Nucleoidsmentioning

confidence: 99%

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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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Characterization, Assay, and Use of Isolated Bacterial Nucleoids

Hirschbein

Guillén

1982

Methods of Biochemical Analysis

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Transcription in vitro of DNA in isolated bacterial nucleoids

Cited by 28 publications

References 28 publications

Supercoils in prokaryotic DNA restrained in vivo.

Supercoils in prokaryotic DNA restrained in vivo.

Transcription of Superhelical DNA from Cell Nuclei

Characterization, Assay, and Use of Isolated Bacterial Nucleoids

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