Recently we reported finding unique, plasmid-like DNAs associated with mitochondria from the S-type of cytoplasmic male-sterile maize (PNAS 74:2904-2908). These unique DNAs have never been observed in mitochondrial DNA preparations from normal, T, C and EP cytoplasms. Conversely, they have been found in every S cytoplasm studied regardless of source or nuclear background. These associations have suggested a causal relationship between the unique DNAs and the S type of male sterility. Importantly, the unique DNAs are only isolated from mitochondrial preparations and not from chloroplasts or nuclei. Very recently, strict maternal transmission of the unique DNAs has been demonstrated.
The unique DNAs associated with the S cytoplasm (S-S and S-F) were isolated from mitochondria and separated by gel electrophoresis. Examination by electron microscopy revealed that the S-S and S-F DNAs were linear molecules of 6212 and 5227 base pairs, respectively. For comparison, the phage, fX174, contains a DNA molecule of 5375 base pairs which codes for nine genes.
When the S-S or S-F DNAs were denatured in 95% formamide and renatured in 50% formamide at room temperature for five minutes, lariat-like configurations were observed by electron microscopy of formamide spreads (Fig. 1). The lariat-like structure consisted of a short double-stranded (DS) stem and a large singlestranded (SS) loop. The double-stranded stem contained 195 and 168 base pairs for the S-S and S-F DNA, respectively. This constitutes 3.1 and 3.2 percent of the S-S and S-F molecules, respectively.
The occurrence of stem-loop structures is interpreted as being due to terminal inverted repeats on the S-S and S-F molecules (Fig. 2). Intrastrand stem-loop structures form with first-order kinetics when the terminal inverted repeats pair. Figure 2 gives a diagrammatic presentation of the events leading to the formation of stem-loop structures.
The importance of terminal inverted repeats on the unique DNAs from the S cytoplasm is not clear. Inverted repeats are often prominently involved with insertional events in lower organisms. It is tempting to hypothesize that the unique DNAs described here are in some way related to the unstable nature of the S cytoplasm.
C. S. Levings III, W. W. L. Hu, D. H. Timothy and D. R. Pring*
*ARS, USDA and Department of Plant Pathology, University of Florida
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