Exercise 13: Mitochondrial DNA (mtDNA) Analysis

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

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Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072733 ◽

1973 ◽

Vol 31 ◽

pp. 458-459

Author(s):

K. S. McCarty ◽

R. F. Weave ◽

L. Kemper ◽

F. S. Vogel

Keyword(s):

Mitochondrial Dna ◽

Ethidium Bromide ◽

Microscopic Examination ◽

Agaricus Bisporus ◽

Osmotic Shock ◽

Electron Microscopic Examination ◽

Electron Microscopic ◽

Isolated Mitochondria ◽

Dna Strands ◽

Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

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