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

摘要

Analysis of clones obtained by rapid amplification of the 5' end and by primer extension of the mRNA for carrot bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) showed transcripts of differing lengths that belonged to two sub-populations. The longer transcripts contained a translation start site 147 nucleotides upstream of, and in frame with, the one which was present in the shorter transcripts. The ORF that began at this ATG codes for a protein of 64 714 Da, which is much largerthan the mature DHFR-TS subunit. The N-terminus region of this polypeptide showed features typical of plant transit peptides. Immunogold labelling studies and immunorecognition of the plastid-containing sub-cellular fraction suggested a plastidial localisation of the bifunctional protein. Although plant cells were shown to contain folate pools in plastids, in mitochondria and in the cytosol, few enzymes of the folate pathway have been associated with any sub-cellular compartment. Thus, this is the first indication for the presence of an enzyme of the folate biosynthetic pathway in plastids. The longer transcripts revealed the presence of a TC microsatellite at the 5'-untranslated end. Nucleotide sequence data have been deposited in the EMBL/GenBank/DDBJ databases under accession number Z33383 (DRTSRACE).