Sedimentation Rate as a Measure of Molecular Weight of DNA

Burgi, Elizabeth; Hershey, Alfred Day

doi:10.1016/s0006-3495(63)86823-x

Cited by 716 publications

(151 citation statements)

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“…4 somal RNA were used as standards for sucrose-gradient centrifugation. Based upon these standards, the major part of Stentor DNA was estimated to be between 25 and 40S, according to the relation of Burgi & Hershey (1963), and corresponds to a molecular weight of 5 x 10 6 to 4 x 10 7 daltons. Direct measurements on electron micrographs shows molecules of variable length.…”

Section: Dna Characterization: Resultsmentioning

confidence: 99%

“…At the end of the run, fractions were collected from the bottom of the tube and their optical density read in a spectrophotometer at 260 nm. Determination of the molecular weight of DNA was carried out using the relationship developed by Burgi & Hershey (1963). For 3 H counting the DNA was precipated with an equal volume of 10% TCA, collected on Whatman GF/F glass filters, washed with cold 5 % TCA, cold 95 % ethanol and dried.…”

Section: (Vi) Analytical Cscl Gradient Ultracentrifugationmentioning

confidence: 99%

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Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

Pelvat

Haller

1976

Genet. Res.

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SUMMARYThe macronuclei of Stentor coeruleus were isolated on a discontinuous sucrose gradient and their DNA was purified by conventional methods. The GC content was 32 mole %. The DNA banded as a single peak on analytical ultracentrifugation at 1-691 g/cm 3 . The molecular weight of the DNA was 5 x 10 6 to 4 x 10 7 daltons. Genome size determined by DNA-DNA reassociation kinetics was 6 x 10 10 daltons. The macronuclear genome was mostly simple, about 85 % being made of non-repetitive sequences.

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Section: Dna Characterization: Resultsmentioning

confidence: 99%

Section: (Vi) Analytical Cscl Gradient Ultracentrifugationmentioning

confidence: 99%

Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

Pelvat

Haller

1976

Genet. Res.

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“…After 3 h of centrifugation and collection of the gradicnt, 0.1 ml of each fraction was placed on glass filter, dried and counted. The sYu, values werc estimated from the position of the peak fractions, according to Burgi and Hershey [21], using as standard SPPl DNA having an s;, value of 36s in alkali. The M , were calculated by the Studier equation [22] SPPl [3H]DNA were incubated with fraction VI in conditions that cause 2.1 %, of acid solubilization.…”

Section: Characterization Of Activity On D N Amentioning

confidence: 99%

An Intracellular Endonuclease of Bacillus subtilis Specific for Single‐Stranded DNA

Ciarrocchi¹,

Fortunato²,

Cobianchi³

et al. 1976

European Journal of Biochemistry

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We have fractionated from extracts of Bacillus subtilis the DNase activity specific for singlestranded DNA; the activity separates in two main fractions on Sephadex G-200, a larger one ( M , > 400000) and a smaller one ( M , z 30000). We have purified the smaller, more abundant fraction nearly 3000-fold. The purified enzyme has a pH optimum close to 8, is activated by Ca", and is inhibited by EDTA ; the enzyme hydrolyses single-stranded DNA at a rate approximately 40 times greater than double-stranded DNA. The mode of action is endonucleolytic on both substrates, but the possibility that the two activities may reside on different molecules is not ruled out. The products have 5'-P and 3'-OH ends.The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single-stranded DNA (except one) and have all an exonucleolytic mode of action.A description of the properties of the deoxyribonucleases of Bacillus subtilis is essential to understand the pathways of genetic recombination in this organism. Chestukhin et al. [l], Doly and Anagnostopoulos [2] and Ohi and Sueoka [3] have described an ATPdependent DNase from this organism; two mutants, belonging to two different genes have been described which are reduced in the level of that enzyme [1,2] and are deficient in recombination and in several repair properties, in analogy with the comparable mutants of Diplococcuspneumoniae [4] and Escherichia coli [5]. No other enzyme of Bacillus subtilis has been associated with the recombination process.In our laboratory we have isolated a number of mutants of Bacillus subtilis deficient in recombination and/or in several DNA repair properties [6-81. A characterization of the levels of some enzymes in these mutants led to the identification of a polA mutant [6,9] and to the observation of a partial defect in an intracellular DNase acting on singlestranded DNA in another mutant named rec-30 [XI.We have thus begun a study of the intracellular nucleases hydrolyzing single-stranded DNA. We report here the initial results of our work, describing one of the enzymes which catalyze this reaction. Enzymes. Pancreatic DNase (EC 3

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“…2). On the assumption that the empirical Burgi-Hershey relationship (18) can be extrapolated to these higher molecular weights and these conditions of sedimentation, a molecular weight of 1.5 X 109 was calculated for the DNA in the main sedimentation band of Fig. 2.…”

Section: Dna Packagingmentioning

confidence: 99%

The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex

Stonington

Pettijohn

1971

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

267

135

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The DNA of Escherichia coli has been isolated in a compact structure containing small amounts of protein and RNA and having a sedimentation coefficient of approximately 3200 S. The molecular weight of the DNA in the complex is very large (probably higher than 109); the protein is predominantly core RNA polymerase; the RNA is chiefly nascent messenger and ribosomal chains. Solutions containing the complex have low vicosities; this plus its sedimentation rate suggest that the DNA is in a tightly folded conformation. The DNA unfolds after exposure to RNase or heat; this indicates that an RNA component of the complex is involved in stabilizing the structure..The DNA in bacterial cells as seen under the electron microscope is confined in regions of the cell called "nuclear bodies," but no nuclear membrane is observed (see, for example, refs. 1 and 2). Yet these chromosome-like structures have defined boundaries, which reorganize during the nuclear segregation that accompanies cell division (3). This implies that DNA folding within the nuclear body is maintained-at least partly--by internal interactions between the elements or regions of the nuclear body. The tertiary conformation of DNA within this body is unknown; however, studies of the rates at which different operons can be transcribed and regulated suggest that a considerable portion (perhaps all) of the genome is available for free interaction with other macromolecules (for examples see refs. 4-7). The packaging of the DNA must also be compatible with the requirements of semiconservative DNA replication and the eventual segregation of daughter double-helices (8, 9). These considerations imply that the-, tertiary structure of DNA within the cell is organized; however, this structure and the interactions that stabilize it are still to be determined.We describe here the isolation from E. coli of a DNA complex in which the DNA genome remains folded into a structure which is compact and nearly homogeneous in sedimentation. The complex is approximately 80% DNA by weight; it includes a small amount of protein and also contains nascent RNA. At least some of the RNA of the complex is essential for stabilizing the packaged structure, since the DNA unfolds as a result of RNase treatment. MATERIALS AND METHODSThe E. coli strain D-10 (RNase I-, ref. 10) was used in these studies. The bacteria were grown into the log phase in M9 media supplemented with 0.2% casamino acids.Viscosities of solutions of the DNA complex were measured at 250C in a falling-ball viscometer.Proteins were obtained from a DNA complex that had been purified by a scaled-up modification of the procedure described in the legend to Fig. 1 When E. coli cells are gently opened in solutions of high ionic strength, DNA is released from the cells but the solution does not become viscous. A DNA complex, having a nearly homogeneous sedimentation profile, can be isolated from the bulk of the ultraviolet-absorbing material of the lysate ( Fig. 1; see also ref. 12). This complex contains: nearly all of the DN...

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Sedimentation Rate as a Measure of Molecular Weight of DNA

Cited by 716 publications

References 6 publications

Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

An Intracellular Endonuclease of Bacillus subtilis Specific for Single‐Stranded DNA

The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex

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