G. D. Clark-Walker

The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function.

Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasites life cycle. Here we report that silencing expression of the α subunit of the mitochondrial F1‐ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the γ subunit of F1 that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.

Rolling circle replication of DNA in yeast mitochondria.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2–7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single‐ or double‐stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2–7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of bakers yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

Evolution of Mitochondrial Genomes in Fungi

Publisher Summary This chapter describes sizes, gene topology, and structures contributing to size variation of mitochondrial DNA (mtDNAs) in fungi. It also presents mechanisms for generating length mutations and rearrangements together with a description of mtDNA codon variations. Two unusual features of mtDNA from oomycetes and hypochytridiomycetes groups include (1) the circular mtDNA in Achyla ambisexualis has inverted repeats, and (2) genome complexity—namely, single copy length, falls in a narrow range between 36.2 kb and 45.3 kb. Macro structural changes to fungal mtDNAs can involve length mutations and rearrangements. A micro structural change can lead to variation to the genetic code both between different groups of fungi and in relation to other organisms. Intron loss from mitochondrial genomes can be achieved experimentally and the mechanism is thought to proceed by an RNA intermediate and reverse transcription. Intron polymorphisms may produce subtle changes allowing organisms to exploit different niches. Loss of intergenic regions may occur through recombination–excision processes or, for small regions, by slipped-strand mispairing.

Location of transcriptional control signals and transfer RNA sequences in Torulopsis glabrata mitochondrial DNA

Determination of sequences from the nine regions separating the large genes in the 19‐kbp mitochondrial DNA from Torulopsis glabrata has led to the identification of 23 tRNA genes and to the recognition of two types of short repeated sequence implicated in mitochondrial genome expression. The two short repeated sequences are a nonanucleotide motif, 5′‐TATAAGTAA‐3′ and a dodecanucleotide motif, 5′‐TATAATATTCTT‐3′. By RNA sequence determination it has been found that primary transcripts of the small and large rRNAs commence at the 3′ penultimate adenine of the nonanucleotide sequence. This motif has also been found in the DNA sequence upstream from f‐methionine, phenylalanine, leucine, tyrosine and glycine tRNAs, cytochrome oxidase subunit 2 and ATPase subunit 9. The dodecanucleotide sequence is found at least once in each of the nine regions between the large genes. Determination of the 3′ ends of the small and large rRNAs has shown their location to be 8 and 23 nucleotides downstream from the dodecanucleotide sequence. This motif is thought to be involved in signalling processing of polycistronic transcripts. Such transcripts are invoked to account for the production of mRNAs for cytochrome b, cytochrome oxidase subunits 1 and 3, and the joint mRNA for ATPase subunits 8 and 6 genes that lack an adjacent upstream nonanucleotide transcription initiation signal sequence. Processing of polycistronic transcripts at tRNA sequences is also implicated in the formation of mature mRNAs. From the position of tRNA genes relative to the nonanucleotide motif it appears that clusters of these genes are co‐transcribed with downstream sequences for cytochrome oxidase subunits 1 and 3.

Contrasting mutation rates in mitochondrial and nuclear genes of yeasts versus mammals

SummaryBase substitutions have been compared in two mitochondrial and two nuclear genes from three yeasts and three mammals. In yeasts, the two mitochondrial genes, cytochrome oxidase subunit 2 (COX2) and apocytochrome b (CYB), have fewer changes on a percentage basis than the nuclear-encoded cytochrome c (CYC) gene. By contrast, in mammals, the same mitochondrial genes have more mutations than CYC on a percentage basis. Sequence comparisons of the nuclear small-subunit ribosomal RNA (nSSU) gene shows that there are more substitutions per unit length in the three yeasts than in the three mammals. This result suggests that although the yeasts are more distantly related than the mammals, their mitochondrial genes have accumulated fewer changes.

Does mitochondrial DNA length influence the frequency of spontaneous petite mutants in yeasts

SummaryResults from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.

A and B subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Abstract Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ−) and can survive complete loss of the organellar genome (ρo), the genetic factor(s) that permit(s) survival of ρ− and ρo mutants remain(s) unknown. In this report we show that a function associated with the F1-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ɛ subunit of F1, renders cells petite-negative. The F1 complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρo cells and those impaired in electron transport. The activity of F1 that suppresses ρo lethality is independent of the membrane Fo complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F1 subunits to suppress ρo lethality has been achieved by simultaneous expression of S. cerevisiae F1α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ− or ρo mutations in S. cerevisiae.

Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts

SummaryRestriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species.

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron

SummaryThe cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8 826 bp and contains five exons (termed E1–E5) totalling 1 602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1–1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5a, and 1.5b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5a and K1 cox1.4/Sc cox1.5b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.

Magnification of the rDNA cluster in Kluyveromyces lactis

SummaryBy employing pulsed field gel electrophoresis we find that slow growing strains of Kluyveromyces lactis have only 43%–55% of the wild-type level of ribosomal DNA (rDNA) repeats. When subjected to prolonged vegetative growth these strains can increase both the number of rDNA repeats and their growth rate.