The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

For sequencing, mitochondrial DNA from Hansenula wingei yeast was digested with various restriction enzymes and the resultant DNA fragments were cloned into a pEMBL phasmid vector. Our clone bank consists of 39 overlapping clones which cover the entire 27,694 bp region of the H. wingei mitochondrial genome.

No abstract

To update sequenced data, we determined the 5' and 3' termini of yeast Hansenula wingei (Pichia canadensis) mitochondrial (mt) large subunit ribosomal RNA (LSU) which is encoded in the mt genome. The 5' end position was mapped downstream from a putative transcription starting site which is homologous to a Saccharomyces cerevisiae mitochondrial promoter sequence. This suggests that the primary transcript of LSU is processed from 5' end and then mature transcript is formed. This processing is different from that of S. cerevisiae mt LSU in which processing on its 5' end does not occur. Based on the sequence data of H. wingei mt LSU, we constructed its secondary structure, and compared it with those of the other fungal organisms. Conserved regions of H. wingei LSU were identified and used for subsequent phylogenetic analysis. In genome structure and gene content, H. wingei mt genome has several characteristics similar to those in filamentous fungi, but the phylogenetic analysis indicates closer kinship to yeast S. cerevisiae. This agrees with previous non-sequencing phylogenies and suggests that extraordinary rearrangements have occurred in yeast mt genomes during divergent evolution.