A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.

https://www.pnas.org/content/pnas/76/10/5269.full.pdf

Mathematical model for studying genetic variation in terms of restriction endonucleases

Mitochondrial DNA was purified from four species of higher primates (Guinea baboon, rhesus macaque, guenon, and human) and digested with 11 restriction endonucleases. A cleavage map was constructed for the mitochondrial DNA of each species. Comparison of the maps, aligned with respect to the origin and direction of DNA replication, revealed that the species differ from one another at most of the cleavage sites. The degree of divergence in nucleotide sequence at these sites was calculated from the fraction of cleavage sites shared by each pair of species. By plotting the degree of divergence in mitochondrial DNA against time of divergence, the rate of base substitution could be calculated from the initial slope of the curve. The value obtained, 0.02 substitutions per base pair per million years, was compared with the value for single-copy nuclear DNA. The rate of evolution of the mitochondrial genome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10. This high rate may be due, in part, to an elevated rate of mutation in mitochondrial DNA. Because of the high rate of evolution, mitochondrial DNA is likely to be an extremely useful molecule to employ for high-resolution analysis of the evolutionary process.

https://www.pnas.org/content/pnas/76/4/1967.full.pdf

Rapid evolution of animal mitochondrial DNA.

The 23S twisted circular form of ColE1 DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE1 DNA even in the presence of the ColE1-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE1-protein complex after treatment with these various agents is largely owing to an induced transition of ColE1 DNA from the supercoiled to the open circular state.

https://www.pnas.org/content/pnas/62/4/1159.full.pdf

SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM

A preparative method for isolating high-molecular-weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene-diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number-average molecular weight of the native DNA is 190 × 106, whereas it is 90 × 106 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks. The native DNA molecules range in molecular weight from 40 × 106 to more than 500 × 106.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02881.x

Isolation of high-molecular-weight DNA from mammalian cells.

In vitro insertional mutagenesis with a selectable DNA fragment

Early and late helix-coil transitions in closed circular DNA. The number of superhelical turns in polyoma DNA.

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.

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

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.

The Presence of Ribonucleotides in Mature Closed-Circular Mitochondrial DNA

Previously, DNA polymorphisms in the HLA gene cluster have been analyzed using radioactive probes in Southern blot experiments; the restriction fragment length polymorphisms (RFLPs) revealed by this analysis are capable of subdividing HLA serological types. Here, we report the use of DNA probes labeled with biotinylated psoralen to provide nonisotopic detection of HLA class II RFLP patterns. These biotinylated probes contain cDNA sequences encoding the alpha and beta chains of DP, DQ, and DR HLA class II genes as inserts in M13 vectors. The recombinant M13 molecules are partially double-stranded with single-stranded HLA cDNA regions and contain biotinylated psoralen covalently linked to duplex DNA by UV irradiation. Following hybridization, the presence of biotinylated probe bound to target DNA is detected using a streptavidin-horseradish peroxidase conjugate, which converts the colorless substrate 3,3',5,5'-tetramethylbenzidine to a blue precipitate in less than 1 hr. The probe and detection system described here can detect single-copy genes in less than 0.5 microgram of total human DNA on Southern blots and generates the same specific RFLP patterns as do probes labeled with 32P by nick-translation. These biotinylated HLA class II probes have been applied to tissue typing for bone marrow transplantation and the study of insulin-dependent diabetes susceptibility, revealing in each case relevant polymorphisms not detected by serologic typing.

Use of nonisotopic M13 probes for genetic analysis: application to HLA class II loci.

Mitochondrial DNA from an Old World mouse, Mus musculus, and from an Old World rat, Rattus norvegicus, contain 19 and 22 distinct sites, respectively, for the 8 restriction endonucleases, BamHI, EcoRI, HaeII, HhaI, HincII, HindIII, HpaI and PstI. The relative positions of the sites have been mapped by the study of partial and double enzyme digests. Some sites may been conserverd between the mouse and rat mitochondrial genomes.

Restriction endonuclease cleavage maps of rat and mouse mitochondrial DNAs

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