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Human Molecular Genetics Advance Access originally published online on August 29, 2006
Human Molecular Genetics 2006 15(19):2846-2855; doi:10.1093/hmg/ddl219
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans

Enrico Baruffini1,2,{dagger}, Tiziana Lodi1,{dagger}, Cristina Dallabona1, Andrea Puglisi3, Massimo Zeviani4,* and Iliana Ferrero1

1 Department of Genetics, Biology of Microrganisms, Anthropology, Evolution, University of Parma, 43100 Parma, Italy, 2 Institut des sciences de la vie, Faculté d'Ingénierie biologique, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium, 3 Department of Molecular Biology and NCCR Program ‘Frontiers in Genetics’, University of Geneva, Sciences III, CH-1211, Geneva 4, Switzerland and 4 Pierfranco and Luisa Mariani Center for Mitochondrial Disease, Division of Molecular Neurogenetics, National Neurological Institute ‘C. Besta’, 20126 Milano, Italy

* To whom correspondence should be addressed at: Division of Molecular Neurogenetics, National Neurological Institute, ‘C. Besta’ Via Temolo 4, 20126 Milano, Italy, Tel: +390 22394633; Fax: +390 22394619; Email: zeviani{at}istituto-besta.it

Received June 20, 2006; Accepted August 2, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase {gamma} (pol {gamma}). Mutations in pol {gamma} are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers–Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol {gamma} mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged ({rho}–) or no ({rho}0) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol {gamma} mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol {gamma} mutations in yeast and, possibly, in humans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different mutations in POLG, the gene encording the catalytic subunit of the mitochondrial DNA polymerase (polymerase gamma, pol {gamma}) (1) are associated with a spectrum of human mitochondrial disorders, including autosomal dominant (ad) and autosomal recessive (ar) progressive external ophthalmoplegia (PEO), juvenile spino-cerebellar ataxia and epilepsy (2) and infantile hepatopatic poliodystrophy, or Alpers–Huttenlocher syndrome (AHS) (3). Adult-onset adPEO and arPEO syndromes are characterized by the accumulation of multiple mtDNA deletions in affected tissues (4), whereas the most severe syndrome, AHS, shows marked reduction in the mtDNA copy number in liver (5) and, possibly, brain. Beside mutations in POLG, adPEO can also be caused by heterozygous mutations in ANT1, encoding the ADP/ATP translocator (6), and TWINKLE, encoding a mitochondrial helicase (7). ANT1 and POLG genes, but not TWINKLE, are highly conserved from humans to the facultative budding yeast Saccharomyces cerevisiae: the yeast AAC2 gene corresponds to ANT1 (8), whereas MIP1 corresponds to POLG (9). This makes it possible to introduce and study in AAC2 and MIP1 the mutations that are equivalent to those affecting mtDNA stability in humans (10–12).

Human pol {gamma} is composed of a 140 kDa catalytic subunit A and a 55 kDa accessory subunit B, which increases the processivity of mtDNA synthesis. Pol {gamma}A comprises a polymerase domain and an exonuclease proofreading domain, separated by a linker region of 482 amino acids. Over 60 PEO-associated mutations have been found in POLG (13,14). Most of the dominant POLG mutations are in the polymerase domain, whereas most of the recessive mutations are in the exonuclease or in the spacer domains. Only one mutation has been found in pol {gamma}B (14).

In the present work, we have introduced in MIP1 two mutations that are equivalent to two pathogenic mutations in pol {gamma}A, the Y955C and the G268A. The first mutation is associated with adPEO (1), and the second is associated with arPEO (15). We have demonstrated that the equivalent yeast mutations behave as dominant and recessive traits, respectively, and, by inducing severe damage to, or the loss of mtDNA, determined an increase of either {rho}– or {rho}0 petite colonies. We have also shown that the yeast phenotype can be suppressed by increasing the mitochondrial dNTP pool, through the overexpression of RNR1, encoding the ribonucleotide reductase (RNR1) (16,17), or the deletion of its inhibitor, SML1 (18,19). Finally, we have studied the effect of a reactive oxygen species (ROS) scavenger on our mutants. Our results indicate that ROS damage contributes to mtDNA instability in yeast.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of MIP1 alleles equivalent to PEO-associated hPOLG mutants on oxidative growth phenotype
Phenotypic analysis showed that the haploid mip1Y757C mutant strain ({Delta}mip1//mip1Y757C) was unable to grow on 2% ethanol, indicating the absence of respiration. As exemplified by the experiment shown in Figure 1, the spectrum profile of mitochondria isolated from this strain consistently lacked the peaks specific to cytochromes b and aa3, which are part of the mtDNA-dependent complexes III and IV of the respiratory chain, whereas the peak specific to cytochrome c, a nucleus-encoded protein, was normal. Contrariwise, the haploid mip1G224A mutant strain ({Delta}mip1//mip1G224A) was able to grow on ethanol, displayed a normal cytochrome profile (Fig. 1) and retained a normal respiratory activity (data not shown). The MIP1/{Delta}mip1 heterozygous diploid strain, carrying either the mip1Y757C or the mip1G224A mutant alleles (MIP1/{Delta}mip1//mip1Y757C and MIP1/{Delta}mip1//mip1G224A) both failed to show a significant growth defect; the cytochrome profile and respiration were normal in both strains.


Figure 2191
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  		Figure 1. Oxidized versus reduced cytochrome spectra of the {Delta}mip1 strain (DWM-5A), transformed with wt MIP1, pFL39 plasmid (no insert), mip1G224A and mip1Y757C mutant alleles cloned in pFL39. The peaks at 550, 560 and 602 nm (vertical bars) correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline of each spectrum is an index of cytochrome content.

 
Determination of petite frequency and evaluation of the nature of the petite mutations
In humans, both G268A and Y955C mutations induce the accumulation of multiple deletions of mtDNA. In S. cerevisiae, deletions of mtDNA are known as petite mutations. Conditions that increase mtDNA mutability also increase the frequency of petites (20). In order to evaluate whether an effect similar to that observed in humans was produced by the equivalent mutations in yeast, we measured the frequency of petite mutants in haploid {Delta}mip1 strains carrying either the mip1G224A or the mip1Y757C mutant allele. The results reported in Figure 2 indicate that, in the mip1Y757C haploid strain, the petite frequency was 100%, as previously described (12). In the mip1G224A haploid strain, the petite frequency was ~2.5-fold the frequency determined in {Delta}mip1 haploid strains transformed with the MIP1 wild-type allele.


Figure 2192
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  		Figure 2. Determination of petite frequency in (A) haploid {Delta}mip1 strain transformed with wt MIP1, pFL39 plasmid without insert, mip1G224A and mip1Y757C mutant alleles cloned in pFL39; (B) heterozygous diploid MIP1/{Delta}mip1 transformed with wt MIP1, pFL38 plasmid without insert, mip1G224A and mip1Y757C mutant alleles cloned in pFL38. More than 4000 colonies/strain were scored. All values are means of three independent experiments. In no case, the variation was higher than 15%. RD, respiratory deficient cells; RS, respiratory sufficient cells.

 
In the MIP1/{Delta}mip1 diploid strain carrying the mip1G224A mutant alleles, the petite frequency was equivalent to that of the background level observed in the same diploid strain carrying the MIP1 allele. This result indicates that mip1G224A behaves as a recessive allele in yeast, like the corresponding G268A mutation does in humans. In the same strain carrying the mip1Y757C mutant allele, the petite frequency was ~21-fold, indicating that the mutation is dominant in yeast, as it is in humans.

Petite mutants can be either {rho}– (that is, cells in which the mtDNA is partially deleted) or {rho}0 (that is, cells in which the mtDNA is completely lost). When {rho}– mutants are crossed with a mit– mutant carrying a point mutation, {rho}+ cells are produced by recombination, provided that the mit– point mutation maps in a region conserved in the {rho}– mtDNA. Contrariwise, {rho}0 mutants, which are completely devoid of mtDNA, are unable to produce {rho}+ cells. In order to evaluate the nature of the petite colonies produced by our mutant strains, we crossed 200 petite clones from the {Delta}mip1//MIP1 strain and 200 petite clones from {Delta}mip1//mip1G224A with four mit– mutants. Each mit– mutant harbours a different deleterious point mutation, namely, in the first and sixth exons of the cob gene, in the cox2 gene and in the cox3 gene (see Methods). The diploid clones resulting from the crosses were tested for their ability to grow on glycerol. Approximately 56% of the {Delta}mip1//MIP1 and 54% of the {Delta}mip1//mip1G224A petite clones were able to complement at least one of the mit– mutations, indicating that these clones were {rho}– (Table 1). None of the clones derived from the {Delta}mip1//mipY757C strain was able to complement any of the mit– mutations, indicating that they were {rho}0. This was consistently confirmed by Southern-blot analysis, as exemplified in Figure 3 (see also Methods).


Figure 2193
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  		Figure 3. Southern-blot analysis of mtDNA isolated from petite clones unable to complement any of the mit– mutants tested (lanes 1–10). mtDNA was prepared and analysed as described in Materials and Methods from cells grown on YNB medium supplemented with 2% glucose. mtDNA (1 µg) was digested with EcoRV and hybridized with an mtDNA-specific probe. Lane 7 corresponds to an mtDNA-rearranged {rho}– clone; lanes 1–6 and 8–10 correspond to mtDNA-less {rho}0 clones. The mtDNA hybridization pattern of a respiratory sufficient {rho}+ strain is shown as a normal control; examples of abnormal hybridization patterns were obtained from a respiratory-deficient mtDNA-less {rho}0 strain, and from two respiratory-deficient, mtDNA-rearranged {rho}– strains.

 


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  		Table 1. Percentage of petite ({rho}0+{rho}–) and percentage of {rho}0 mutants produced in different genetic backgrounds

 
The complementation test cannot be used to evaluate the nature of the petite mutants in wt/mutant diploid strains. To mimic the diploid condition, we have constructed heteroallelic strains by introducing the wt MIP1 or the mutated mip1Y757C alleles in the haploid wt strain W303-1B. We first established that the heteroallelic MIP1//mip1Y757C and the homoallelic MIP1//MIP1 strains produced petite colonies with a frequency similar to that observed in diploid strains carrying the corresponding mip1Y757C and MIP1 alleles (data not shown). The transformants were then crossed with the mit– testers. Approximately 74% of 200 MIP1//MIP1 homoallelic petite clones was able to complement each of the mit– mutations but only 49% of 500 heteroallelic MIP1//mip1Y757C petite clones was able to do so, again indicating that the Y757C mutation induces the loss of mtDNA (Table 1).

Determination of mtDNA point mutations
We next analysed whether our mip1 mutations increased the mtDNA point mutagenesis, as demonstrated by an increase in the frequency of erythromycin-resistant (EryR) mutants. Resistance to the drug erythromycin in yeast is acquired through specific point mutations in the mtDNA-encoded rRNA genes and therefore is a convenient, direct measurement of mtDNA point mutagenesis in vivo (21). Using this assay, we have measured mtDNA mutation frequencies in the haploid strain {Delta}mip1//mip1G224A and in the diploid strains MIP1/{Delta}mip1//mip1G224A and MIP1/{Delta}mip1//mip1Y757C. Both mip1G224A in haploid, and mip1Y757C in diploid conditions showed a 10-fold and a 20-fold increase, respectively, in mtDNA point mutagenesis, compared with that observed in the wild-type strain. However, the MIP1/mip1G224A heterozygous diploid strain failed to show any increase of EryR mutant frequency (Table 2), again indicating that this mutation behaves as a recessive trait also for this phenotype.


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  		Table 2. Frequency of EryR mutants

 
Effect of increasing dNTP pool on mitochondrial mutability
We next tested whether an increase in the level of the dNTP pool could rescue the mitochondrial mutability observed in the presence of the two pathological mip1 mutations. The increase of the dNTP pool was obtained either by overexpressing the RNR1 gene, which encodes the large subunit of the ribonucleotide reductase (22) or by deleting the SML1 gene, which encodes an inhibitor of the latter activity (18).

We transformed haploid and diploid strains harbouring the mip1G224A or the mip1Y757C alleles with a multicopy plasmid carrying the RNR1 gene (plasmid pWJ841). We also constructed a haploid {Delta}sml1, {Delta}mip1 strain and a diploid {Delta}sml1/{Delta}sml1 MIP1/{Delta}mip1 strain, which were transformed with either one of three different plasmids: the first plasmid carried the wt MIP1 allele, the second the mip1G224A allele and the third the mip1Y757C allele. The results reported in Figure 4 indicate that both conditions caused dramatic decreases of the petite frequencies in the mutant strains. The petite mutability was reduced approximately to the same extent, suggesting that the protective effect on mtDNA is likely due to the increase of the dNTP pool and not to an unrelated effect of the RNR1 overexpression or of the SML1 deletion.


Figure 2194
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  		Figure 4. Determination of petite frequency in the presence of RNR1 overexpression and SML1 deletion. Relevant genotypes of different strains are indicated on top of the bars. Bars indicate transformants with MIP1 or different mip1 mutant alleles, as indicated. (A) Haploid strains transformed with wt MIP1 and mip1G224A mutant allele cloned in pFL39; (B) diploid transformed with wt MIP1, pFL38 plasmid without insert, mip1G224A and mip1Y757C mutant alleles cloned in pFL38. More than 4000 colonies/strains were scored. All values are means of three independent experiments. In no case the variation was higher than 15%.

 
Decreased petite mutability was associated with decreased {rho}0 frequency (Table 1). In the presence of RNR1 overexpression, the proportion of haploid {Delta}mip1//MIP1 {rho}0 clones was reduced from 46 to 33% and in the presence of the SML1 deletion was reduced to 28%. Similar results were obtained in the haploid {Delta}mip1//mip1G224A strain. The heteroallelic strain, MIP1//mip1Y757C showed a reduction of {rho}0 clones from 51 to 35% in the presence of overexpressed RNR1 and to 29% in the presence of {Delta}sml1, whereas no reduction was observed in the MIP1//MIP1 homoallelic strain. The 4–5-fold decrease of petite mutability and the 7–10-fold decrease of {rho}0 frequency suggest that the increase of the dNTP pool contributed to maintain mtDNA integrity and stability.

Since the effects obtained by RNR1 overexpression were similar to those obtained with SML1 deletion, we evaluated the effect on mtDNA point mutability only in the {Delta}sml1 background. The number of EryR mutants was unchanged in the presence of the mip1G224A mutation, but decreased by ~2-fold in the presence of the mip1Y757C mutation (Table 2).

Role of ROS in mtDNA mutability
Increased production of ROS is associated with an increase in the accumulation of mtDNA rearrangements (23,24). To address this issue in our system, we analysed the effect of the ROS scavenger dihydrolipoic acid on the petite mutability of the heterozygous MIP1/{Delta}mip1 diploid strain carrying the mip1Y757C allele, as well as of the {Delta}mip1 haploid strain carrying the mip1G224A allele. Exposure to dihydrolipoic acid decreased significantly the petite mutability in both cases by a factor of six to eight. A clear, albeit less prominent, effect was also observed in the mip1 and MIP1/mip1 strains carrying the wild-type MIP1 allele (Fig. 5).


Figure 2195
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  		Figure 5. Determination of petite frequency in the presence/absence of 30 µM dihydrolipoic acid (A) haploid {Delta}mip1 strain transformed with wt MIP1 and mip1G224A mutant allele cloned in pFL39; (B) heterozygous diploid MIP1/{Delta}mip1 transformed with wt MIP1, pFL38 plasmid without insert, mip1G224A and mip1Y757C mutant alleles cloned in pFL38. More than 4000 colonies/strains were scored. All values are means of three independent experiments. In no case the variation was higher than 15%. –L, cells untreated with dihydrolipoic acid; +L, cells treated with dihydrolipoic acid.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The human Y955C pol {gamma} mutation is located in the polymerase domain. Its fidelity is 42-fold decreased in an exonuclease-deficient background and 2-fold decreased in an exonuclease-proficient background (25); its polymerase activity is 3000-fold decreased (26). The yeast mutation mip1Y757C, corresponding to Y955C, behaved like the mip1 null mutation when introduced into a {Delta}mip1 haploid strain. It induced a dramatic loss of mitochondrial DNA, as shown by the production of 100% petite mutants that were all {rho}0, that is, mtDNA-less cells. In a MIP1/mip1 heterozygous diploid strain, the mip1Y757C mutation caused a 20-fold increase in petite mutability compared with the MIP1 wt allele and a 4-fold increase compared to the mip1 null allele. The mip1Y757C mutation caused also an increase of point mutability of mtDNA, leading to a 10-fold increase of EryR mutations compared with the MIP1 wt allele and a 3-fold increase compared with the {Delta}mip1 null allele. These results indicate that the mip1Y757C behaves as a dominant allele, since its presence is more deleterious to mtDNA than the absence of the MIP1 gene itself. This behaviour mimics exactly what is observed in humans.

The human G268A is a missense mutation in the proofreading exonuclease domain of pol {gamma}A. In homozygous conditions, the human G268A mutation causes the accumulation of both mtDNA deletions and point mutations (27). We have observed reduced fidelity of the homologous mutation also in yeast, where the equivalent mip1G224A allele was associated with a 2.5-fold increase in the frequency of petite clones and a 10-fold increase in the point mutability of mtDNA. Therefore, the phenotype is recessive in yeast as it is in humans.

The recessive phenotype is formally indicative of a complementation exerted by the wild-type allele and is possibly due to the ability of the wild-type MIP1 polymerase to proofread errors introduced by the mutant polymerase.

Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans (28) and yeast (29).

The level of dNTP pools is under an evolutionary conserved surveillance system that maintains genomic stability. One important checkpoint of this control system is the RNR1. In yeast, mutations in RNR1 increase petite mutations (30). In both yeast and humans, the transcription of RNR1 is induced after DNA damage. In addition, the yeast mtDNA-damage-checkpoint proteins also regulate the RNR1 inhibitor Sml1p. After DNA damage or at S phase, the MEC1 and RAD53 gene products control the phosphorylation and the subsequent degradation of the Sml1 protein (31). This control contributes to increase the synthesis of dNTPs, necessary for DNA replication and repair. Overexpression of RNR1 (32) or deletion of SML1 (18), both are able to rescue the petite-inducing phenotype of a specific point mutation in yeast pol {gamma} (mip1-1).

Here we show that either RNR1 overexpression or SML1 deletion, two conditions that increase the dNTP pools of the cell, including the mitochondrial pool (19), induced a 4–8-fold reduction in the production of petite colonies of our recombinant mutant strains. However, neither RNR1 overexpression nor SML1 deletion decreased the frequency of EryR clones, which is an index of the propensity of mtDNA to accumulate point mutations. This result is similar to previous data (33) and indicates that induction of large-scale rearrangements, rather than increased point mutagenesis, is responsible for the petite mutability induced by the two mip mutations considered in our work. The human Y955C mutation displays a 45-fold reduction in affinity for the incoming nucleotide (25). Reduced affinity could determine a stalling on homopolymeric runs due to frequent reiteration of a single nucleotide. This could result in local depletion of that nucleotide and make it difficult for a defective polymerase to incorporate the subsequent nucleotide (34). By preventing pol {gamma} stalling, an increase of the dNTP pool could decrease the generation of mtDNA rearrangements, and thereby reduce the number of petite clones. However, the increase of the dNTP pool could also induce the rescue of petites by improving the efficiency of mtDNA repair, especially in the presence of the D268A mutation, which is an error-prone mutation, being contained in the proofreading domain of pol {gamma}A. The existence of mtDNA repair is supported by the recent demonstration that mitochondria share several repair systems with the nucleus, in both yeast (35) and humans (36).

MtDNA is attached to the mitochondrial inner membrane in close proximity to the respiratory chain. Mitochondrial respiration is a major source of ROS, which makes mtDNA vulnerable to oxidative damage (23). The latter effect can lead to the production of more ROS by impairing the electron transport chain (37), which can in turn determine further mtDNA damage (38,39).

To evaluate whether the mitochondrial mutability consequent to POLG mutations could result from ROS damage on mtDNA, we analysed the effect of a ROS scavenger, dihydrolipoic acid. Exposure to dihydrolipoic acid caused a 6-fold decrease in the petite mutability of the heterozygous MIP1/mip1 diploid carrying the mip1Y757C dominant allele and of the {Delta}mip1 haploid strain carrying the mip1G224A recessive allele. However, we could not establish whether the polymerase mutations increase the amount of ROS or whether they make the mtDNA more sensitive to ROS.

We have previously reported that the presence of ROS scavengers reduced significantly the consequences of a mutation equivalent to an arPEO mutation in the human ANT1 gene (11). Likewise, the data presented in this paper ushers in the development of a rational anti-ROS strategy for patients with pol {gamma}A mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast strains and media
Yeast strains are listed in Table 3. YP medium contained 1% Bacto-yeast extract and 2% Bacto-peptone (Difco). Minimal medium (YNB) contained 7 g/l yeast nitrogen base without amino acids (Difco) supplemented with appropriate amino acids and bases for auxotrophy. Various carbon sources were added at the indicated concentrations. N1 medium contained 1% peptone, 1% yeast extract, 2% ethanol in 50 mM phosphate buffer at pH 6.3, supplemented with 4 g/l erythromycin (SIGMA).


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  		Table 3. List of yeast strains

 
Construction of yeast strains carrying mip1 mutations
Saccharomyces cerevisiae strain W303-1B (40) was used for the construction of a null {Delta}mip1 mutant by one step gene disruption using KanMX expression cassette (41). MIP1 ORF was completely deleted and substituted by the Kanr marker, whose correct target of at MIP1 locus was verified by PCR. Oligonucleotides used for MIP1 disruption and verification are listed in Table 3. {Delta}mip1 mutant strain (strain OF1), {rho}0, was then crossed with the isogenic strain W303-1A (40), giving rise to the diploid DWM MIP1/{Delta}mip1 {rho}+. This strain was transformed with wt MIP1 or mip1Y757C or mip1G224A mutant alleles, cloned in the centromeric vector pFL38 or with the empty plasmid. Diploid clones obtained will be named: MIP1/{Delta}mip1//MIP1, MIP1/{Delta}mip1//mip1Y757C, MIP1/{Delta}mip1//mip1G224A, MIP1/{Delta}mip1//pFL38, respectively.

Absence of MIP1 determines the complete and irreversible loss of mtDNA. For this reason, we maintained wt MIP1 expressed by a plasmid during the construction of the haploid mutant strains and eliminated it by plasmid shuffling once the mutant allele was already into the cell. For the construction of haploid strains, DWM strain transformed with the pFL38MIP1 plasmid was sporulated. By tetrad analysis the haploid spore DWM-5A, carrying the {Delta}mip1 disruption and the pFL38MIP1, and therefore {rho}+, was selected. This strain was the host for wt MIP1 or mip1Y757C or mip1G224A mutant allele, cloned in the pFL39 centromeric vector. By plasmid shuffling in the presence of 5-fluoro orotic acid (5FOA, SIGMA), it was then possible to isolate DWM-5A strain devoid of pFL38MIP1. Haploid clones obtained will be named: {Delta}mip1//MIP1, {Delta}mip1//mip1Y757C, {Delta}mip1//mip1G224A respectively.

Strains carrying {Delta}sml1 mutation were derived from strain YG855, kindly obtained from Rodney Rothstein. This strain was crossed with DWM-5A ({Delta}mip1) thus obtaining the diploid YO81 heterozygous at {Delta}sml1 and {Delta}mip1 loci. By sporulation and tetrad analysis of this strain, we selected the spores YO81-3A ({Delta}sml1) and YO81-4B ({Delta}mip1{Delta}sml1). These clones were then crossed to obtain the diploid DYY MIP1/{Delta}mip {Delta}sml1/{Delta}sml1, used as the host for wt MIP1 or mip1Y757C or mip1G224A mutant alleles cloned in pFL38. For the construction of haploid strains, DYY diploid strain, transformed with the pFL38MIP1 plasmid, was sporulated. By tetrad analysis, the haploid spore DYY-4C {Delta}mip1 {Delta}sml1 carrying pFL38MIP1, therefore being {rho}+, was selected. This strain was transformed by mip1G224A mutant allele, cloned in the centromeric vector pFL39. By plasmid shuffling in the presence of 5-fluoro orotic acid, it was then possible to isolate the DYY-4C strain ({Delta}mip1 {Delta}sml1) carrying mipG224A allele and devoid of pFL38MIP1. The same has been done also with MIP1 wt allele in order to obtain strains transformed with the same plasmid pFL39 and consequently carrying the same selectable marker, TRP1.

Construction of mutant alleles
The mip1G224A and mip1Y757C mutant alleles were produced by site-directed mutagenesis, using the QuikChange Kit (STRATAGENE). The template DNA was the wt MIP1 cloned in pUC19 vector. To obtain this construction, we PCR-amplified a DNA fragment of 5110 bp, containing the ORF and the 5' and 3' flanking regions (851 and 441 bp, respectively), using genomic DNA of strain W303-1B as template and the forward primer MIP1C (containing a SacI site at the 5' end) and the reverse primer MIP1E (containing a SalI site at the 5' end).

The list of base changes, and corresponding modified primers used to generate mutated vectors, is reported in Table 4. In order to maximize the expression of these variants, the preferred yeast codons (42) were used in the oligonucleotide sequences used for mutagenesis: GCT for Alanine and TGT for Cysteine. Mutagenized inserts for each different construct were sequence-verified on both strands. The sequences of the oligonucleotide primers used for cloning and sequencing MIP1 are also listed in Table 3.


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  		Table 4. List of oligonucleotides used

 
Plasmids
MIP1 gene and mutant alleles were cloned in both the centromeric vectors pFL38 and pFL39 (43) at SacI and SalI sites. These vectors carry URA3 and TRP1 markers, respectively. RNR1 gene, cloned in the multicopy vector pRS425 (plasmid pWJ841), was kindly obtained from Rodney Rothstein.

Mitochondrial DNA mutation frequency
Analysis of petite frequencies
Diploid and haploid strains transformed with wt or mutated mip1 alleles were pregrown overnight in YNB medium supplemented with 2% ethanol and then inoculated in the same medium supplemented with 2% glucose. After 15 generations of growth at 36°C, cells were plated on YNB agar plates supplemented with 2% ethanol and 0.25% glucose at a dilution that gave approximately 200 cells/plate. Petite frequencies were defined as the percentage of colonies showing the petite phenotype after a 5 day incubation at 28°C.

To test the effect of dihydrolipoic acid, cells were grown in YNB medium supplemented with 2% glucose in the presence or in the absence of 30 µM dihydrolipoic acid (SIGMA) for 15 generations at 28°C. These experiments were performed at 28°C because lipoic acid at 37°C determined high level of cells lethality.

Evaluation of the nature of petite clones
In order to evaluate the nature of the petite mutants produced by pathologic mutations, petite clones were crossed with mit– strains harbouring point mutations in genes encoding respiratory proteins. The four mit– mutations used for this analysis map in the first and in the sixth exons of cob gene, in the cox2 gene and in the cox3 gene, respectively. This choice was based on the observation that in the petite {rho}– mutants, the fragments that are most frequently retained encompass cytochrome b, cox2 and cox3 genes (44,45). The petite clones unable to complement any of the mit– mutants could be either {rho}0 (no mtDNA) or {rho}– carrying rearranged mtDNA molecules which lack the three mutant genes. To distinguish between these two possibilities, we evaluated the presence/absence of mtDNA in a set of these clones by Southern analysis. Mitochondrial DNA was extracted by rapid mitochondrial preparation (46) from clones unable to complement any of the mit–, grown in YNB supplemented with 2% glucose. Aliquots of 1 µg of DNA were digested with EcoRV (Amersham). Southern-blot analysis was carried out as previously described (47). mtDNAs extracted from three {rho}+, three {rho}– and from three {rho}0 independent strains were also hybridized as a control. Hybridization was performed by standard methods, with a 5'-[{gamma}-32P]ATP end-labelled yeast mtDNA-specific sequence repeat (5'-CTCCTTTCGGGGTTCCGGCTCCCG TGGCCGGGCCCCGG-3') as a probe. Among the 25 putative {rho}o clones that were analysed with this method, no mtDNA was observed in all but two clones, in which the mtDNA was likely not to encompass any of the mit– mutations (Fig. 3). These results indicate that the absence of mit– complementation is a good indicator of a {rho}0 condition.

Erythromycin resistance assays
Fifteen independent colonies from each strain were inoculated in separate 10 ml cultures of YNB+glucose and allowed to reach the stationary phase. After 24 h, a small sample was removed to determine the total number of respiration-competent cells by plating onto YP supplemented with 2% ethanol. The remainder of each culture was plated onto solid N1 medium containing 4 mg/ml erythromycin (SIGMA) and grown at 28°C for 8 days until drug-resistant colonies formed. The experiment was carried out in duplicate. The mutation frequency was calculated as number of EryR colonies/total number of colonies.

Miscellaneous
Transformation of yeast strain was obtained by the lithium chloride method (48). Restriction enzyme digestions, E. coli transformation and plasmid extractions were performed using standard methods (47). Cytochrome spectra and respiration were determined as described previously (10).


   ACKNOWLEDGEMENTS
 
We are indebted to Luigi Palmieri for suggestions in the experimental design and procedure. We thank Françoise Foury for critical discussion; Antonietta Cirasolo and Roberto Silva for their skilful technical assistance; Rodney Rothstein for the generous gift of strain YG855 and plasmid pWJ841. This work was funded by grants from Fondazione Telethon-Italy no. GGP030039, Fondazione Pierfranco and Luisa Mariani, and the European Community's sixth Framework Programme for Research (EUMITOCOMBAT consortium, contract no. LSHM-CT-2004-503116).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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