Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa

Abstract

A cDNA complementary to the mRNA of the ADP/ATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cell-free translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony filter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRI fragment of total Neurospora DNA, and the start of the mRNA was determined by S1 nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP/ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rich promoter region. The mRNA has a 46-bp 5' end and a 219-bp 3' end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.