THE HUMAN DUTPASE GENE ENCODES BOTH NUCLEAR AND MITOCHONDRIAL ISOFORMS - DIFFERENTIAL EXPRESSION OF THE ISOFORMS AND CHARACTERIZATION OF A CDNA-ENCODING THE MITOCHONDRIAL SPECIES

We have previously identified distinct nuclear and mitochondrial isofo rms of dUTPase in human cells, reporting the cDNA sequence of the nucl ear isoform (DUT-N), We now report a cDNA corresponding to the mitocho ndrial isoform (DUT-M), The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted M-r of 26,704, The amino-terminal region of the protein contains an arginine-rich, 69-re sidue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA res ults in the production of a precursor protein with an apparent molecul ar mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a d UTPase specific monoclonal antibody, Mitochondrial import and processi ng studies demonstrate that the DUT-M precursor is processed into a 23 -kDa protein and imported into mitochondria in vitro, Isoelectric focu sing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values, Studies aimed at understandin g the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture sy stem, Northern blot analysis, employing an isoform specific probe, dem onstrates that DUT-N and DUT-M are encoded by two distinct mRNA specie s of 1.1 and 1.4 kilobases, respectively, Western and Northern blot an alysis reveal that DUT-M protein and mRNA are expressed in a constitut ive fashion, independent of cell cycle phase or proliferation status, In contrast, DUT-N protein and mRNA levels are tightly regulated to co incide with nuclear DNA replication status, Because DUT-N and DUT-M ha ve identical amino acid and cDNA sequences in their overlapping region s, we set out to determine if they were encoded by the same gene, The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing, These a nalyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alte rnative 5' exons, This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nucl ear and mitochondrial specific protein isoforms.