Isolation and nucleotide sequence of a gene encoding tRNA nucleotidyltransferase from Kluyveromyces lactis

A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae ccal-1 mutation, Sequencing of a 2665bp EcoRI -SpeI restriction fragment revealed an open reading frame potentially encod ing a protein of 489 amino acids with 57% sequence similarity to its S. cer evisiae homologue, Southern hybridization revealed a single copy of KlCCA1 in the K, lactis genome, KlCCA1 was able to complement both the mitochondri al and cytosolic defects in the cca1-1 mutant, suggesting that, as in S, ce revisiae, the K, lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol, An altered KlCCA1 gene encodin g a tRNA nucleotidyltransferase that lacked its first 35 amino acids was ab le to complement the nuclear/cytosolic but not the mitochondrial defect in the S, cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amin o acids are necessary for targeting to mitochondria but are not required fo r enzyme activity, Our results suggest that the mechanisms for production a nd distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltran sferase in K, lactis differ from those seen in S, cerevisiae. This DNA sequ ence has been assigned GenBank Accession No, AF207744, Copyright (C) 2000 J ohn Wiley & Sons, Ltd.