Clemson University

B37N mitochondria lack a mature transcript for URF-25

--J. Wang, J. Barth and A. Abbott

The study of mitochondrial gene expression in higher plants is hampered by the inability to easily manipulate the system; therefore, we have chosen to study maize lines which have naturally occurring variation in gene expression. Comparative molecular analyses of these expression variants with normal systems will define components essential to the process of mitochondrial gene transcription, processing and translation. Using Northern hybridization analysis, mitochondrial RNA's from diverse cytoplasmic backgrounds have been screened for variations in expression of a number of cloned genes or unassigned reading frames. Most sources of N cytoplasm have identical transcript patterns for the probes examined (N. H. Walker et al., Theor. Appl. Genet. 74:531, 1987). One line, however, B37N, shows an altered transcript pattern for the URF-25 reading frame originally described by R. E. Dewey et al. (Cell 44:439, 1986). In this line the mature URF-25 transcript is not present at the 2200 nucleotide size seen in B73N cytoplasm and all other lines examined. The major transcript for this URF in B37N is found at the probable precursor size of approximately 3800 nucleotides. Using flanking and reading frame specific probes in Northern hybridization analysis the putative precursor transcript has been characterized in B37N and B73N mitochondrial RNA's. These transcripts appear to be identical. One plausible explanation for the lack of a 2200 base transcript in B37N is the loss of a processing sequence on either the 5' or 3' end of the transcript. To examine this possibility clones containing URF-25 and flanking sequences from B37N and B73N mitochondrial DNA have been isolated and comparative restriction fragment analysis has been carried out. There does not appear to be any gross difference in the structural organization of this transcription unit in either cytoplasm. Using cloned probes, the 5' end of the mature transcript has been mapped and is being sequenced in both B37N and B73N to search for minor sequence changes. Similar experiments will be done on the 3' end. We are also testing the possibility that nuclear background may influence the expression of this URF. These experiments will delimit salient features involved in mature transcript size determination in higher plant mitochondria.

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