Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis.

The cleavage of DNA by restriction endonucleases can be limited by the addition of ethidium bromide. When closed circular DNA is used as a substrate, DNA with one-site cleavages of one or both strands can be made by adding appropriate amounts of dye. The singly cleaved DNA is a complete set of full-length permuted linear molecules.

The quantitation of fluorescence by photography.

A method based on theory has been developed for the photographic quantitation of fluorescent substances. DNA stained with ethidium in agarose gels is used as an example of an application of this method. In the course of developing this method we have demonstrated that the empirical methods employed by others authors can give rise to large systematic errors.

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity.

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21.

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.

Complex mitochondrial DNA in animal thyroids. A comparative study.

1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2.

The structures and fidelity of replication of mouse mitochondrial DNA-pSC 101 EcoRI recombinant plasmids grown in E. coli K12.

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (La9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12.

Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.

Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColE1, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by deltatau = +/-1, +/-2, etc.

Isolation and partial characterisation of the relaxation protein from nuclei of cultured mouse and human cells.

A protein, called relaxation protein because of its ability to remove superhelical turns in closed-circular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closed-circular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA.

Restriction endonuclease cleavage maps of animal mitochondrial DNAs.

The restriction endonuclease, HindIII, gives rise to three fragments in each of the three mitochondrial DNAs isolated from the established mammalian cell lines LA9 (mouse), HeLa (human), and BSC-1 (African green monkey). The restriction endonuclease, EcoRI, gives rise to three fragments in mitochondrial DNA from HeLa and to two in DNAs from LA9 and BSC-1. The sizes and the orders of the fragments in the respective genomes have been determined with data obtained from the electron microscope.

Mitochondrial DNA replication in sea urchin oocytes.

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [(3)H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells.

The presence of ribonucleotides in mature closed-circular mitochondrial DNA.

Mitochondrial DNA from mouse and HeLa cells is nicked by alkali and chick-embryo ribonuclease H. It has been concluded that ribonucleotides are present in the closed-circular duplex. A comparative study of the scission rates at pH 13.

Replication of mitochondrial DNA. Circular replicative intermediates in mouse L cells.

The frequency, composition, and structure of circular replicating forms of mitochondrial DNA, including two new forms described here, suggest a scheme for the mode of replication of this DNA.

Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid.

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved.