MOLECULAR ANALYSIS OF THE ASPARTATE KINASE-HOMOSERINE DEHYDROGENASE GENE FROM ARABIDOPSIS-THALIANA

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-asparta te 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA lib raries using the carrot ak-hsdh gene as the hybridizing probe. Two gen omic libraries from different A. thaliana races were screened independ ently with the ak probe and the hsdh probe. Nucleotide sequences of th e A, thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)(+)-mRNA extra cted from cell suspension cultures. Sequence comparison between the Ar abidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak- hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A, th aliana in single copy and functional as evidenced by hybridization ana lyses. The apoprotein-coding region is interrupted by 15 introns rangi ng from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified . A signal sequence is proposed starting from a functional ATG initiat ion codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5' non-coding leader sequence and the othe r in the putative chloroplast targeting sequence. 5' sequence analysis revealed the presence of several possible promoter elements as well a s conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN 4-like recognition element might be relevant for such a gene coding fo r an enzyme limiting the carbon-flux entry to the biosynthesis of seve ral essential amino acids. 3' sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site. T his work is the first report of the molecular cloning of a plant ak-hs dh genomic sequence. It describes a promoter element that may bring ne w insights to the regulation of the biosynthesis of the aspartate fami ly of amino acids.