Temporal synthesis of mitochondrial DNA in maize cell suspension culture

The maize mitochondrial genome is composed of small circular and linear DNAs, in addition to a large chromosomal DNA. Kemble and Bedbrook (Nature 284:565, 1980) described a 2.3 kb linear molecule and a 1.94 kb circular molecule in N cytoplasm maize. The circular molecule is detected as linear, open-circular, and covalently closed circular molecules in agarose gels. Cell suspension cultures of the Black Mexican Sweet line (N cytoplasm) contained the same family of low molecular weight DNAs as is characteristic of the intact plant (Chourey and Kemble, 5th International Cong. of Plant Tissue and Cell Culture, Tokyo, Japan, in press, 1982). The Black Mexican Sweet line also contains an additional small DNA detected as a covalently closed circle, open circle, and a linear molecule of approximately 1.4 kb. The relative levels of the 2.3 kb and 1.94 kb molecules are higher than the 1.4 kb molecules. In the present report, we describe replicative properties of the low molecular weight DNAs using Black Mexican Sweet cell suspension cultures which readily incorporated 32P-orthophosphate into mtDNA. The relative level of DNA synthesis in each class of DNA was monitored through UV fluorescence of ethidium bromide stained agarose gels and autoradiography intensities on X-ray film of the corresponding gels.

15 uCi 32P per ml of culture medium was added to cultures in either logarithmic phase (7 days post subculture) or stationary phase (15 days post subculture), and allowed to incubate for 24 hours. The cells were harvested and the mitochondrial DNA was extracted and electrophoresed. The DNA extracted from logarithmic phase cultures showed 32P incorporation into all classes of mitochondrial DNA. The 2.3 kb and 1.94 kb molecules had equal intensities in both ethidium bromide staining and autoradiography. The three forms of the 1.4 kb molecule were present, but in much lower intensity. However, DNA extracted from stationary phase cultures had the same relative intensities of DNA when stained with ethidium bromide, but no 32P was incorporated into the DNA. At this sensitivity, no mitochondrial DNA synthesis could be detected, although the majority of cells were still viable, as evidenced by their fluorescence in the presence of fluorescein diacetate stain: Furthermore, if the stationary phase cells were removed from the exhausted medium and placed into new medium with 32P for 24 hours, there was renewed incorporation of label into mitochondrial DNA. However, the stoichiometry of the low molecular weight DNAs had shifted so that the 1.94 kb molecule was in much higher concentration and had incorporated more 32P, relative to the 2.3 kb and 1.4 kb molecules. When stationary cells are incubated for prolonged periods in fresh medium prior to the 24 hours labeling with 32P, stoichiometry and 32P incorporation characteristic of cells in logarithmic growth was observed.

These data indicate that mitochondrial DNA is being rapidly synthesized in cells from logarithmic growth phase cultures. The progression into stationary phase was correlated with a suspension of mitochondrial DNA synthesis, as seen by a failure of cultures to incorporate 32P into mitochondrial DNA over a 24-hour period. The addition of new media to the stationary cells resulted in a preferential synthesis of the 1.94 kb molecule, relative to the other classes of low molecular weight DNA.

A. G. Smith, D. R. Pring and P. S. Chourey

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