IDENTIFICATION OF NITRATE TRANSPORTER GENES IN CHLAMYDOMONAS-REINHARDTII

The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate regulated gene cluster containing the nitrate reduct ase structural gene nit-1, have been related to nitrate transport. Mut ant strains defective in nitrate transport and having an active nitrat e reductase have been genetically constructed. Their nitrate non-utili zing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mut ants resulted in the expression of these nar transcripts and the recov ery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was als o achieved by co-transformation with plasmids containing nar-2 and nar -3 genes or nar-2 and nar-4, but not with single plasmids containing e ach individual gene. In addition, DNA sequences of a practically compl ete cDNA of nar-3 and a partial one of nar-4 have been generated and t he deduced amino acid sequences showed a very significant identity wit h that of the nitrate transporter gene (crnA) from Aspergillus nidulan s. These data strongly support the hypothesis that the nitrate transpo rt system in C. reinhardtii contains at least two protein components e ncoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a pr otein with a high identity to that of nar-3.