I. Occurrence and Structure of Complex Mitochondrial DNA in Human Leukemic Leukocytes and Normal Mammalian Tissues. II. Use of Alkali Metal Salts of Trichloroacetic Acid as Buoyant Denaturing Solvents for DNA

Citation

Clayton, David Alvin (1970) I. Occurrence and Structure of Complex Mitochondrial DNA in Human Leukemic Leukocytes and Normal Mammalian Tissues. II. Use of Alkali Metal Salts of Trichloroacetic Acid as Buoyant Denaturing Solvents for DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/AS6W-K850. https://resolver.caltech.edu/CaltechETD:etd-08092006-083749

Abstract

This thesis is divided into two parts. Part I is concerned with the occurrence and structure of complex forms of mitochondrial DNA isolated from human leukemic leukocytes and normal mammalian tissues. Complex mitochondrial DNA (M DNA) occurs in two forms. The covalently closed double-sized circle (10 [mu]) is called the circular dimer. Oligomers composed of interlocked 5 [mu] monomer submolecules are called catenanes. The catenane form is ubiquitous in nature. It has been observed in M DNA preparations from every mammalian source examined to date. These sources include various organs from rabbits, guinea pigs, and mice. The frequency of catenated dimers in normal and leukemic tissues varies from 5 to 11 percent, and the frequency of catenated higher oligomers from 0.1 to 8.0 percent.

In contrast, the circular dimer has not been observed in normal tissues. M DNA's from the peripheral blood of 14 patients with myelogenous leukemia contained a circular dimer form. No such structure could be found in M DNA's from three patients with nonmalignant proliferations of myeloid cells. The frequency of the circular dimer form is reduced upon treatment with antileukemic drugs. This result suggests that a significant relation exists between the formation and presence of the circular dimer M DNA form and myelogenous leukemia in man. Additional data presented in this section demonstrate that a sixteen-hour labeling of leukocyte M DNA results in a uniform labeling pattern of the circular dimer and monomer form.

DNA hybrids between the circular dimer and monomer were analyzed by electron microscopy and analytical centrifugation. The results indicate that the circular dimer and monomer are at least 90 percent homologous and that heterologous regions, insertions, or deletions exceeding 50 to 100 nucleotides in length do not occur. The electron microscope studies also show that monomer genomes in the dimer are connected in a head-to-tail structure, rather than in a head-to-head structure. In addition, the results show that leukemic leukocyte M DNA is substantially homogeneous in base sequence.

Part II contains a preliminary study of a denaturing buoyant system for DNA. It is shown that rubidium trichloroacetate is a potentially useful buoyant solvent for DNA. It is likely that most DNA's can be banded at neutral pH and room temperature in both the native and denatured states without introducing single-strand scissions. It is also concluded that Li, Na, and K trichloroacetate are potentially useful denaturing solvents for sedimentation velocity studies of DNA.

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