Alternative transcription initiation sites generate two LCA1 Ca2+-ATPase mRNA transcripts in tomato roots

The tomato LCA1 gene encodes a Ca2+-ATPase and gives rise to two major mRNA transcripts and two distinct protein products of different size in tomato roots. The basis of the transcript size difference was investigated to asse ss whether the mRNA transcripts encoded distinct protein products. Primer e xtension and S1 nuclease analysis identified two transcription initiation s ites at -72 and -1392 from the start of translation. RNA gel blot analysis of poly(A)(+) RNA isolated from phosphate-starved tomato roots using probes designed to domains of the 5'-untranslated region (UTR) or the full-length LCA1 cDNA identified mRNAs of 4.7 and 3.6 kb, corresponding to mRNA origin ating from transcription initiation sites -1392 and -72, respectively. Scre ening of a cDNA library derived from phosphate-starved tomato roots yielded three cDNA clones, LCA1A, LCA1B and LCA1C (3.6, 4.5 and 5.1 kb respectivel y). These cDNAs contain full-length LCA1 mRNA sequence derived from each tr anscription initiation site, with LCA1C additionally containing an intron o f 0.6 kb. Sequence analysis indicated 100% identity between the three size classes of cDNA clones except for the differential 5'-UTR and the unspliced intron. Overall, the results indicate that the two major LCA1 mRNA transcr ipts are derived by differential transcription initiation and that two of t he mRNAs may encode identical protein products, while a third mRNA may corr espond to a non-functional truncated protein.