Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli

Sharp, Phillip A.; Hsu, Ming; Ohtsubo, Eiichi; Davidson, Norman

doi:10.1016/0022-2836(72)90363-4

Cited by 341 publications

(241 citation statements)

References 39 publications

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“…Previous workers have shown that there is a narrow range of pH (about 12.0-12.5) within which denaturation of linear DNA but not CCC-DNA occurs and that this property can be used for purifying CCC-DNA (7)(8)(9)(10)20,21). We have utilized this approach for developing a rapid extraction method for plasmid DNAs.…”

Section: Principle Of the Alkaline Extraction Methodsmentioning

confidence: 99%

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Birnboim

Doly

1979

Nucleic Acids Research

13,802

4,673

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A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

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Section: Principle Of the Alkaline Extraction Methodsmentioning

confidence: 99%

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Birnboim

Doly

1979

Nucleic Acids Research

13,802

4,673

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“…Most of these molecules appear to be entirely duplex with a length that is a multiple of the unit length. Of 122 oligomers traced and measured, there are 86 dimers, 24 trimers, 6 tetramers, 4 pentamers, and 2 hexamers. These molecules had very few observable singlestrand branches.…”

Section: Specificity Of the Nicksmentioning

confidence: 99%

“…guanidine'hydrochloride, 3.86 M hydroxylamine (pH 4.23), and in 98% formamide. Most of the molecules in the nicked circular DNA fraction contained one nick.…”

mentioning

confidence: 99%

Structure of a nicked DNA-protein complex isolated from simian virus 40: covalent attachment of the protein to DNA and nick specificity.

Kasamatsu¹,

Wu²

1976

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

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formamide. Based on the stability in the presence of these reagents, the protein-DNA complex is regarded as covalently bonded. We also report that a single nick occurs in the circular DNA, but on either of the two complementary strands. The position of the nick in one strand is staggered with respect to the position in the other strand. The above conclusion is based on the appearance in the electron microscope of the DNA forms after self-annealing and partial denaturation of the self-annealed molecules.MATERIALS AND METHODS SV40 was purified from infected TC-7 cells as described (1), except for the use of a CsCl step gradient purification before the final banding of virus. Band velocity sedimentation was performed by layering the sample, after 2-fold dilution by TD buffer (0.14 M NaCl-5 mM KCI-0.7 mM Na2HPO4-2.5 mM Tris.HCI, pH 7.5) of virus in CsCl (p = 1.35 g/cm3), onto a 4-ml step gradient of CsCl (1.0 ml each of CsCl 1.25 g/cm3, 1.34 Abbreviations: SV40, simian virus 40; EM, electron microscopy; ss-DNA, single-stranded DNA; ds-DNA, double-stranded DNA; nicked DNA, ds-DNA that contains at least one single-strand scission; TD buffer, 0.14 M NaCI-5 mM KCI-O.7 mM Na2HPO4-25 mM Tris-HCI, pH 7.5; NaDodSO4, sodium dodecyl sulfate; BAC, benzyldimethylalkylammonium chloride. g/cm3, 1.4 g/cm3, and 1.6 g/cm3 all in TD buffer) and centrifugation for 2 hr at 33,000 rpm at 20°in an SW50. 1 rotor. Viruses were stored in TD buffer at 40 or at -20°.Viruses (10 mg) were deproteinized in 5% NaDodSO4-5 mM EDTA-5 mM dithiothreitol-TD buffer as described (1) In order to visualize proteins attached to single-stranded DNA (ss-DNA) by EM, we modified the benzyldimethylalkylammonium chloride (BAC) spreading method from that described by Vollenweider et al. (5). Immediately before spreading, 0.5 id of 2 M triethanolamine (pH 8.7), 0.5 ,Ad of DNA solution (50 ,g/ml), and 1 gl of 0.25% BAC stock solution prepared from Zephiran (Winthrop Inc.) in formamide, were added in that order to 98 ul of formamide. Twenty-five microliters of this solution were spread onto a hypophase of redistilled water. The DNA film was picked up on carbon-coated grids, dehydrated in ethanol, stained with uranyl acetate, and shadowed with Pt. RESULTS

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“…The enzymes, HindIII (from Hemophilus influenzae Rd) and EcoRI (from E. coli RY13), were prepared by R. J. Roberts scribed by Sharp et al (13). Micrographs were taken at a magnification of 10,000 with an RCA model EMU4.…”

mentioning

confidence: 99%

Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Bukhari¹,

Taylor²

1975

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

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Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such-insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.The temperate bacteriophage Mu is characterized by the highly promiscuous integration of its DNA into the genome of its host bacterium Escherichia coli (1-3). The prophage Mu DNA can be excised precisely from the different integration sites, and as a result wild-type function of the gene into which Mu was inserted can be restored (4). This reversal of Mu integration can be seen if the prophages contain the X mutations, which block the lethal and essential phage functions.Mu X mutants revert to wild type at a frequency of about 10-8. However, no X amber mutants have been found. Thus, we proposed that the X mutations are insertions (4). We have investigated the nature of the X mutations by agarose-gel electrophoresis of Mu DNA fragments produced by specific endonucleases, and by electron microscopic examination of Mu DNA heteroduplexes. These studies have shown that the X mutations are insertions near the immunity (c) end of Mu DNA. In one X mutant, the insertion is demonstrated to be of about 720 base pairs, located 4 kilobases (kb) away from the c end.The isolation of insertions in Mu DNA has allowed us to analyze further some aspects of Mu DNA structure. Mu DNA, a double-stranded linear duplex of about 37 kb (5), has some interesting features. When it is denatured and reannealed, two types of homoduplex molecules are obtained (Fig. 1). All the molecules show single-stranded tails or split ends, measuring about 4% of the molecular length. The split ends, which apparently represent sequences picked up randomly from the, E. coli chromosome, are always found at the S gene end, called here the SE end, of Mu DNA (6-8). Some molecules show another nonrenaturable region, termed the G bubble, near the SE end (9). Recently, we observed that the c end of Mu DNA is also not fixed, and probably varies in length by about 100 base pairs (10, 11).Abbreviation: kb, kilobase. t To whom correspondence should be directed.One plausible model for the origin of the heterogeneity of the SE end is that Mu DNA is packaged into phage heads from maturation precursors which contain both Mu DNA and host sequences. This hypothesis implies that...

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Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli

Cited by 341 publications

References 39 publications

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Structure of a nicked DNA-protein complex isolated from simian virus 40: covalent attachment of the protein to DNA and nick specificity.

Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

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