A cell suspension of the Black Mexican Sweet line of maize contains two circular DNA molecules of 1.94 kb and 1.4 kb in addition to a 2.3 linear DNA and the principal mtDNA genome. A previous report showed that under certain conditions the 1.94 kb molecule could replicate preferentially with respect to the other mitochondrial DNAs (Smith, Pring, and Chourey, MNL 57:47-48, 1983). This characteristic of the 1.94 kb DNA led to an investigation of its distribution and transcription.
MtDNA was prepared from several lines, electrophoresed, transferred to nitrocellulose, and hybridized with nick-translated probes. The probes were either a cloned BamH1 fragment of the 1.94 kb DNA or a full-length clone of the 1.4 kb molecule. No homology was found between either of the cloned molecules, the other known linear small mtDNAs (S1, S2, 2.3 kb, 2.1 kb) or the restricted mtDNA of any cytoplasm tested. Many cytoplasms tested for presence of the DNAs carried the 1.94 kb DNA but not the 1.4 kb DNA; no cytoplasm was found to contain the 1.4 kb DNA without the 1.94 kb DNA. The 1.94 kb DNA was present in Black Mexican (cell suspension and plant), Wf9(N), Wf9(T), Wf9(C), Wf9(S), a fertile cytoplasmic revertant of S cytoplasm, S cytoplasm in two additional backgrounds, and in cytoplasms RB, BB, PR, and ES, members of the C group of male-sterile cytoplasms. The 1.4 kb DNA was present in Black Mexican (cell suspension and plant), 38-11(S), A619(C), Wf9(C), and in BB, PR, and ES cytoplasms. The 1.94 kb and 1.4 kb DNAs were undetectable in several cytoplasms tested, including VG, fertile cytoplasmic revertants of VG, ME, CA, LBN, B, and R. The 1.4 kb DNA was absent in Wf9(N), Wf9(S), Wf9(T), and RB cytoplasm. Although mtDNA prepared from 38-11(ME) showed no homology to either the 1.94 to 1.4 kb DNAs, nuclear DNA prepared from this line and restricted with either BamH1 or EcoRI, electrophoresed, and transferred to nitrocellulose, showed homology to the cloned probes. The 1.94 kb molecule hybridized to BamH1 fragments of 6.7 and 9.5 kb, while the 1.4 kb molecule hybridized to a 6.8 kb EcoRI fragment.
To detect transcription of the 1.94 and 1.4 kb DNAs, total mtRNA was extracted from Black Mexican Sweet line cell suspension, glyoxylated, electrophoresed in 10 mM phosphate 1.6% agarose gels, transferred to nitrocellulose, and probed with the DNAs. Two transcripts were detected for each probe. The 1.94 kb DNA hybridized to transcripts of approximately 1,005 and 600 nucleotides. The 1.4 kb DNA hybridized to transcripts of approximately 890 and 460 nucleotides. Both probes also hybridized to contaminating DNAs in the RNA preparations which corresponded to their own molecular size. This hybridization may have obscured the detection of full-length transcripts.
The transcription of these small circular DNAs within the mitochondrion could indicate that these molecules may play a role in mitochondrial function. However, the existence of cytoplasms which apparently lack one or both of these molecules indicates that they are not indispensable to the mitochondrion. The nuclear homology to these molecules may be an indication of transposition of the DNAs between the mitochondrion and the nucleus, and a possible mechanism to compensate for the absence of these DNAs in the former.
A. G. Smith, D. R. Pring and P. S. Chourey
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