Abstract
The nuclear gene MIP1 is strictly required for mitochondrial DNA replication and mitochondrial DNA polymerase activity (Genga, A., Bianchi, L., and Foury, F. (1986) J. Biol. Chem. 261, 9328-9332). The MIP1 gene was cloned by genetic complementation of the mip1-1 allele after cell transformation with a yeast genomic library and was mapped to the right arm of chromosome XV about 20 centimorgans distal to the cpa1 gene by Southern blot hybridization and tetrad analysis. The mapping of the 5' ends of the MIP1 transcript and the nucleotide sequence analysis of a 4.7-kilobase DNA fragment complementing the mip1-1 allele allowed the determination of an open reading frame of 3762 nucleotides encoding a basic protein of 143.5 kDa. The following data show that the MIP1 gene encodes the catalytic subunit of the replicative mitochondrial DNA polymerase. 1) The mutant ts71 exhibits both a thermosensitive mitochondrial DNA replication in vivo and a thermosensitive mitochondrial DNA polymerase activity is observed, when compared to that of the wild type strain. 3) Chromosomal disruption of the MIP1 gene by an 80% deletion of the gene and its replacement by URA3 gene is not lethal to the cell but elicits total loss of mitochondrial DNA and mitochondrial DNA polymerase activity. 4) The MIP1 protein exhibits sequence similarities with both eukaryotic nuclear DNA polymerases and reverse transcriptases. There is no significant resemblance with prokaryotic DNA polymerases.
Highlights
The nuclear gene M I P l is strictly required for mitochondrial DNA replication and mitochondrial DNA polymerase activity
M I P l gene was cloned by genetic complementation of the m i p l - 1 allele after cetlrl ansformation with a yeast genomic library and was mapped to the right arm of chromosome XV about 20 centimorgans distal to the cpal gene by Southern blot hybridization and tetrad analysis
The following data show that the M I P l gene encodes the catalytic subunit of the replicative mitochondrial DNA polymerase
Summary
From the Laboratoire d’Enzymologie, Unite de Biochimie Physiologique, Uniuersite de Louuain, Louuain-la-Neuue, Belgium. Cloning of the MZPZ Gene-Yeast genomic libraries constructed in the shuttlevectors YEpl, YEp24, and YCp50 were used to transform the mipl-1 rho" mutant WF6-5D, Leu2+, or Ura3+ transformants were gridded on WO medium supplemented with the required amino acids, and in a second step, they were cross-replicated onto YD lawns of the strain F71-5A and incubated at 30 "C for 1 day. After centrifugation at 3,000 X g for 10 nlin to eliminate the particulate cell walls and unlysed cells, the mitochondria were obtained by differential centrifugation at 15,000 X g for 10 min, and they were washed in a medium containing 0.6 M mannitol, 0.1% serum albumin, 10 mM imidazole, p H 6.8 This step was immediately followed by mechanical extraction of the mitochondrial DNA polymerase in an ultra-turrax homogenizer in the presence of0.4 M NaCl [9]. The mipl-1 S U P double mutant ts is rho+ at25 "C and becomes rho" at 36 "C
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