MOLECULAR-CLONING AND EXPRESSION OF A CDNA SEQUENCE ENCODING HISTIDINOL PHOSPHATE AMINOTRANSFERASE FROM NICOTIANA-TABACUM

A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminot ransferase (HPA) was isolated by functional complementation of an Esch erichia coli histidine auxotroph (UTH780). The enzymatic assay has con firmed that the isolated cDNA encodes a functional HPA protein. Amino acid sequence alignment of the HPA protein from N. tabacum, Saccharomy ces cerevisiae and E. coli revealed that, despite the low degree of id entity, some residues were found to be highly conserved. The predicted protein contains a transit peptide sequence at the aminoterminal end, suggesting a chloroplastic localization of the HPA enzyme. Western bl ot analysis demonstrated that the deduced HPA protein and the mature H PA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively. Gene copy number estimation by Southern analysis indicat es the presence of at least two genes per haploid genome coding for th is protein in Nicotiana sp. From northern analysis results, the gene s eems to be highly expressed in green tissues and the detected transcri pt showed a single band of expected molecular size.