HETEROSPECIFIC CLONING OF ARABIDOPSIS-THALIANA CDNAS BY DIRECT COMPLEMENTATION OF PYRIMIDINE AUXOTROPHIC MUTANTS OF SACCHAROMYCES-CEREVISIAE .1. CLONING AND SEQUENCE-ANALYSIS OF 2 CDNAS CATALYZING THE 2ND, 5THAND 6TH STEPS OF THE DE-NOVO PYRIMIDINE BIOSYNTHESIS PATHWAY

An Arabidopsis thaliana cDNA library was used to complement Saccharomy ces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimid ine biosynthetic pathway could be complemented. We report here the clo ning, sequencing and computer analysis of two cDNAs encoding the aspar tate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosylt ransferase-orotidine-5'-phosphate decarboxylase (OPRTase-OMPdecase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last t wo steps of the pyrimidine biosynthetic pathway, as previously suggest ed by biochemical studies. The ATCase encoding cDNA sequence (P YRB ge ne) shows an open reading frame (ORF) of 1173 bp coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 bp coding for 476 amino acids. Computer analysis of the deduce d amino acid sequences of both cDNAs shows the expected high similarit y with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OP RTase and OMPdecase families. This heterospecific cloning approach inc reases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and un derlines its usefulness as a model for evolutionary studies.