Transcriptional analysis of the mitochondrial gene URF13-T in T cytoplasm

The unique mitochondrial gene, URF13-T, in Texas male-sterile cytoplasm maize, has a complex transcriptional pattern. This is related to its association with a repeated region which is 5' to both URF13-T and atp6, and to processing of the transcripts (Dewey et al., Cell 44:439, 1986). By using small DNA probes (31-630 bp), we have Northern-walked from a region -1215 through the coding region of URF13-T and a co-transcribed gene, ORF25. The first five probes also represent a region of -1590 to -444 5' to atp6. Other atp6-specific probes were used to cover the remaining 5' region and the coding region for this gene.

The majority of the transcripts for both atp6 and URF13-T appear to initiate within the repeated region and undergo RNA processing events. No differences were detected in the transcript pattern between sterile and restored lines using atp6-specific probes, however, differences between N and T cytoplasms were seen. A unique transcript of 1.55 kb occurred in T cytoplasms from five different nuclear backgrounds. This transcript may be related to differences in DNA sequence seen between -580 and -562 of atp6. There are two small insertions in T, of 4 and 5 base pairs, at -580 to -577 and -566 to -562, respectively, relative to the sequence in N cytoplasms. One insertion disrupts an AluI restriction site, allowing for an easy assay of many genotypes. The insert(s) is present in all T cytoplasms surveyed and absent in all N, C, and S cytoplasms we have examined.

The effect of dominant nuclear restorer genes on URF13-T has been described by Dewey et al. as differential processing of transcripts, seen with a probe representing the region + 21 to + 41. We find that a differential effect of restorers is detected on transcripts using a probe within the repeated region, representing positions -202 to -81 as well as in the region + 2 to + 200. The region from -80 to + 4 is characteristic of an intron region, as a discrete transcript (1.6 kb) is missing from both T and T-restored mitochondria that is seen with flanking probes in T-restored mitochondria. A 1.5 kb transcript is seen in both T and T-restored mitochondria with probes covering the region + 200 through the coding region. This transcript appears to be the result of an RNA processing event, whereas the 1.6 kb transcript could be the result of RNA splicing, which is unique to T-restored mitochondria. The current model to explain the effect of the nuclear restorer genes on the expression of URF13-T is that splicing and processing of larger transcripts (2.0 and 1.8 kb) decrease their relative copy number, lowering the effective number of transcripts that could encode the entire open reading frame of URF13-T. Antisera to this gene have detected a protein of Mr 13,000 which is unique to T cytoplasms (Wise et al., MNL, this volume). Synthesis of this protein is reduced in T-restored mitochondria (Forde and Leaver, Proc. Natl. Acad. Sci. 77: 418, 1980).

John C. Kennell, Roger P. Wise and Daryl R. Pring
 
 


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