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. Fractionation of the products of a digestion of the permuted linears with a single-hitting restriction endonuclease by gel electrophoresis yields a series of bands that can be used to determine relative molecular weights of the DNA fragments in the gel without the introduction of standards. It is possible to determine the relative molecular weight of a fragment to within +/-2.5%. These molecular weights immediately allow the determination of the HindIII and Hpa I maps of simian virus 40. The HindIII map of bacteriophage PM2 was determined by this method with one ambiguity that was resolved by using traditional techniques.

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. We have also developed an approximate method based on photographic theory, avoiding the use of digital integration which is required by the rigorous method.

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. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs. The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images

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 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.

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 mtDNA from freshly removed thyroids was isolated by buoyant density centrifugation in ethidium bromide/CsCl gradients after DNAase treatment of the mitochondrial pellet. Typically, more than 90% of the recovered mtDNA was found in the lower band, indicating a low rate of nicking during isolation. A sample of the total mtDNA (upper and lower bands) was examined by electron microscopy after preparation by the aqueous protein film technique. 3. The frequency of circular dimers generally ranged from 0.1 to 0.3%. However, in an mtDNA sample from cow thyroid, the frequency of circular dimers was 0.6% (0.9% if circular dimers occuring in catenanes are included(, differing significantly from the frequency of these forms in bull thyroid, 0.1%. A small but significant variability also occurred in the frequency of catenanes ranging from 2 to 8% in the different groups; this variation is within the limits usually observed in normal tissues. 4. These observations indicate that thyroids, like other normal tissues examined so far, have a low content of circular dimers. A high frequency of these forms seems to be the trademark of some genetically and physiologically abnormal cells such as certain established cell lines, virus-transformed cells and malignant or otherwise pathological tissues.

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. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs. Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments. A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165+/-10 nucleotide pairs.

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., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution.

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. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaCl. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed-circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was -0.098 +/- 0.001. The corresponding positive superhelix density has been calculated to be + 0.023. A nicking--swivelling--closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed-circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process.

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. The origin and the direction of replication have been designated in each of the cleavage maps. Polyacrylamide gel electrophoretic analyses demonstrated that additional fragments not detectable in the electron microscope and larger than 50 nucleotide pairs were not present.

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. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.

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 13.0 of RNA and of mitochondrial DNAs indicates that there are no more than 10 ribonucleotides in the major fraction of the mitochondrial DNAs.