Multiple transcription start sites of the carrot dihydrofolate reductase thymidylate synthase gene, and sub-cellular localization of the bifunctional protein

The analysis of clones obtained by rapid amplification of the 5 endand by primer extension of the mRNA for carrot bifunctionaldihydrofolate reductase-thymidylate synthase showed transcripts ofdiffering lengths that belonged to two sub-populations. The longertranscripts were found to contain a translation start site 147 ntupstream of, and in frame with, the one which is present in the shortertranscripts. The ORF that begins at this ATG codes for a protein of64714 Da, which is much larger than mature DHFR-TS subunit. TheN-terminusregion of this polypeptide shows features typical of plant transitpeptides. Immunogold labelling studies and immunorecognition of theplastid-containing sub-cellular fraction suggested a plastidiallocalisation of the bifunctional protein. Although plant cells wereshown to contain folate pools in plastids, in mitochondria and in thecytosol, few enzymes of the folate pathway have been associated with anysub-cellular compartment. Thus, this is the first indication for thepresence of an enzyme of the folate biosynthetic pathway in plastids.The longer transcripts revealed the presence of a TC microsatellite atthe 5-untranslated end.