mRNA was isolated from various Ac-containing maize lines and tissues (seedlings, endosperm, adult plant) and hybridized with Ac-derived probes. A 3.5 kb band segregates with Ac; other hybridizing bands are detected also in Ac-free material. Without exception, the 3.5 kb band is seen on Northern blots when the plant genome contains Ac. The expression level varies only slightly between different plant tissues.
cDNA libraries were prepared and screened with Ac. Three overlapping clones were isolated which are probably derived from the 3.5 kb transcript, and sequenced. They span together 3.1 kb on the Ac sequence, not including the 4 introns. A long untranslated leader precedes an ORF 807 amino acids in length. The first 2 ATG's to open this frame are not enclosed by Kozak's consensus sequence. To elucidate the ability of the translation apparatus to accept this first ATG as a start signal, the cDNA was transcribed in vitro and the products translated in vitro in two systems. The products were slightly larger than expected when the first ATG was used as the start. In addition, the N-terminal end of the protein was sequenced and by this means it was clearly demonstrated that the first ATG is used as a start site in vitro. The 5'-end of the transcript could not be isolated as cDNA. Instead, a 1 kb long uniformly labeled single-stranded DNA fragment was used as a probe in a S1-protection assay.
This probe overlaps terminally 158 bases with the 5'-end of the cDNA, its 3'-end extends beyond the Ac element. Upon S1 digestion a cluster of bands spanning a range of nearly 80 bases is detected. There appear to be two major start sites, 334 and 358 bases distant from the end of Ac. To exclude the possibility of the existence of another short exon close to the 5'-end of Ac, a 100 bases long M13 probe starting 54 bases upstream of the more 5' located major transcription start site was constructed. This probe does not hybridize on a Northern blot with the 3.5 kb band, whereas a similar probe reaching 120 bases farther downstream and thereby overlapping 65 bases with the more 5' located major start site clearly hybridizes with this 3.5 band. The detection limit under these hybridization conditions was shown to be better than 20 bases. First results from primer extension experiments seem to confirm the S1 results.
Reinhard Kunze, Ursula Stochaj, Ulrike Courage, Jurgen Laufs and Peter
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