IDENTIFICATION AND EXPRESSION OF A CDNA FROM DAUCUS-CAROTA ENCODING ABIFUNCTIONAL ASPARTOKINASE-HOMOSERINE DEHYDROGENASE

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) c atalyze steps in the pathway for the synthesis of lysine, threonine, a nd methionine from aspartate. Homoserine dehydrogenase was purified fr om carrot (Daucus carota L.) cell cultures and portions of it were sub jected to amino acid sequencing. Oligonucleotides deduced from the ami no acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture m RNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Toget her the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature prot ein and a long chloroplast transit peptide. The deduced amino acid seq uence has high homology with the Escherichia coli proteins aspartokina se 1-homoserine dehydrogenase I and aspartokinase II-homoserine dehydr ogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.