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Introduction: The aim of this study was to evaluate white spot lesion (WSL) remineralization and fluoride uptake by the application of fluoride varnishes directly onto artificial WSLs in vitro., Methods: MI varnish containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and 2.26% fluoride and Duraphat varnish containing 2.26% fluoride (no added calcium) were compared with a placebo varnish (no added calcium or fluoride). Two WSLs were prepared in enamel slabs and varnish applied to cover one of the two lesions. Each slab was immersed in artificial saliva for 14 days at 37°C. Mineral content was determined using transverse microradiography and fluoride uptake using electron probe microanalysis. The data were statistically analysed using a linear mixed model., Results: Both MI and Duraphat varnishes significantly remineralized the covered and uncovered WSLs when compared with the placebo varnish (P < 0.001). The WSLs covered with varnish showed greater remineralization than those uncovered. MI varnish produced the highest level of remineralization and significantly greater fluoride uptake (0.44 ± 0.08 wt%) compared with Duraphat (0.24 ± 0.03 wt%) and the placebo varnish (0.06 ± 0.05 wt%)., Conclusion: Varnish containing fluoride and CPP-ACP was superior to varnish containing fluoride alone in promoting WSL remineralization and fluoride uptake., (© 2020 Australian Dental Association.)

Background: The aim of this study was to investigate the effect of treatment with the saliva biomimetic, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and SnF 2 /NaF compared with SnF 2 /NaF alone on coronal surface caries progression in head-and-neck cancer patients undergoing radiotherapy., Methods: Twenty-four participants were randomized into two groups. Both groups used 0.4% SnF 2 gel and a 0.32% NaF toothpaste; the test group also applied a crème containing 10% CPP-ACP three times daily while the control group used an identical crème without CPP-ACP (placebo). Resting saliva flow rate and saliva fluoride concentrations were determined. Caries status was assessed using ICDASII at baseline and 12-weeks postradiotherapy. Data were statistically analysed using a linear mixed effects model., Results: Both groups showed significantly reduced resting saliva flow rate (P < 0.001) postradiotherapy. There were no significant differences in flow rates and fluoride concentration between groups. The CPP-ACP group exhibited a significant (P < 0.05) 51% reduction in coronal surface caries progression compared with the placebo group., Conclusion: Resting salivary flow rate was significantly reduced in head-and-neck cancer patients following radiotherapy and use of CPP-ACP with SnF 2 /NaF significantly lowered caries progression compared with SnF 2 /NaF alone., (© 2018 Australian Dental Association.)

Dental caries is associated with plaque dysbiosis, leading to an increase in the proportions of acidogenic and aciduric bacteria at the expense of alkali-generating commensal species. Stannous fluoride (SnF 2 ) slows the progression of caries by remineralization of early lesions but has also been suggested to inhibit glycolysis of aciduric bacteria. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) promotes fluoride remineralization by acting as a salivary biomimetic that releases bioavailable calcium and phosphate ions, and the peptide complex has also been suggested to modify plaque composition. We developed a polymicrobial biofilm model of caries using 6 bacterial species representative of supragingival plaque that were cultured on sound human enamel and pulsed with sucrose 4 times a day to produce a high cariogenic challenge. We used this model to explore the mechanisms of action of SnF 2 and CPP-ACP. Bacterial species in the biofilms were enumerated with 16S rRNA gene sequence analyses, and mineral loss and lesion formation were determined in the enamel directly under the polymicrobial biofilms via transverse microradiography. The model tested the twice-daily addition of SnF 2 , CPP-ACP, or both. SnF 2 treatment reduced demineralization by 50% and had a slight effect on the composition of the polymicrobial biofilm. CPP-ACP treatment caused a similar inhibition of enamel demineralization (50%), a decrease in Actinomyces naeslundii and Lactobacillus casei abundance, and an increase in Streptococcus sanguinis and Fusobacterium nucleatum abundance in the polymicrobial biofilm. A combination of SnF 2 and CPP-ACP resulted in a greater suppression of the acidogenic and aciduric bacteria and a significant 72% inhibition of enamel demineralization.

Background: To evaluate changes in xerostomia status, salivary characteristics and gland volumes 2 years following radiotherapy in nasopharyngeal carcinoma patients., Methods: Xerostomia scores, salivary flow rates, pH and buffering capacity were measured at pre-radiotherapy, mid-radiotherapy, 2 weeks, 3 months and 2 years post-radiotherapy. Salivary gland volumes and their correlation with radiation dose were also assessed., Results: Mean radiation dose to oral cavity, parotid and submandibular glands (SMG) was 44.5, 65.0 and 38.6 Gy respectively. Parotid and SMG volumes decreased 33% at 3 months post-radiotherapy; volumes at 2 years post-radiotherapy were 84% and 51% of pre-radiotherapy levels, respectively. Correlations were observed between parotid gland volume per cent reduction and its radiation dose and between resting salivary flow rate reduction and post-radiotherapy/pre-radiotherapy SMG volume ratio. Salivary flow rates and resting saliva pH remained significantly low at 2 years post-radiotherapy (both flow rates, P = 0.001; resting saliva pH, P = 0.005). Similarly, xerostomia scores remained significantly higher compared with pre-radiotherapy levels., Conclusions: Submandibular gland volumetric shrinkage persisted 2 years after radiotherapy. Xerostomia scores remained significantly higher, and salivary flow rates and resting saliva pH remained significantly lower, suggesting that study participants were still at risk for hyposalivation-related oral diseases., (© 2018 Australian Dental Association.)

Background: Dental erosion is an increasingly prevalent problem associated with frequent consumption of acidic foods and beverages. The aim of this study was to measure the food acid content and the erosive potential of a variety of sugar-free confections., Methods: Thirty sugar-free confections were selected and extracts analysed to determine pH, titratable acidity, chemical composition and apparent degree of saturation with respect to apatite. The effect of the sugar-free confections in artificial saliva on human enamel was determined in an in vitro dental erosion assay using change in surface microhardness., Results: The change in surface microhardness was used to categorize the confections as high, moderate or low erosive potential. Seventeen of the 30 sugar-free confections were found to contain high concentrations of food acids, exhibit low pH and high titratable acidity and have high erosive potential. Significant correlations were found between the dental erosive potential (change in enamel surface microhardness) and pH and titratable acidity of the confections. Ten of these high erosive potential confections displayed dental messages on the packaging suggesting they were safe for teeth., Conclusions: Many sugar-free confections, even some with 'Toothfriendly' messages on the product label, contain high contents of food acids and have erosive potential., (© 2017 Australian Dental Association.)

Background: The aim of this study was to evaluate the ability of calcium phosphate and fluoride containing varnishes to inhibit enamel demineralization., Methods: Six varnishes were selected for analysis: (1) Enamel Pro containing amorphous calcium phosphate; (2) Clinpro White containing functionalized tricalcium phosphate (fTCP); (3) MI Varnish containing casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP); (4) Duraphat (first no added calcium control); (5) Profluorid (second no added calcium control); and (6) placebo (no added calcium or fluoride control). Human enamel slabs (36) were each cut into half-slabs and covered with one of the six dental varnishes to create a window. The half-slabs were then individually immersed in a polyacrylate demineralization buffer pH 4.8 for four days at 37 °C with a change of solution each day. Mineral content was determined using transverse microradiography., Results: All fluoride-containing varnishes significantly inhibited enamel demineralization when compared with the placebo varnish. However, out of the calcium phosphate and fluoride containing varnishes only MI Varnish, containing fluoride and CPP-ACP was superior to the fluoride-alone varnishes. MI Varnish also released the highest levels of calcium, phosphate and fluoride ions., Conclusions: MI Varnish containing fluoride and CPP-ACP was superior to the other varnishes in protecting against enamel demineralization., (© 2015 Australian Dental Association.)

Background: Quantitative light-induced fluorescence (QLF) and digital photography (DP) have been proposed as clinical methods for measuring changes in enamel mineral content. The aim of this study was to compare the ability of QLF and DP with the in vitro gold standard transverse microradiography (TMR) to measure the remineralization of enamel subsurface lesions., Methods: Subsurface lesions were formed in enamel (n = 40) and exposed to remineralization solutions for 10 days. Changes were analysed by DP, QLF and TMR to determine percentage changes in luminescence (%L), fluorescence (%F) and mineral content (%R), respectively and correlation between these parameters determined., Results: The correlations between TMR and QLF (r = 0.63), TMR and DP (r = 0.59), and DP and QLF (r = 0.64) were all moderate but statistically significant (p < 0.001). The variability in %L and, to a lesser extent, %F values significantly impacted on the potential role of DP and QLF as methods by which mineral content changes produced by remineralization treatments could be accurately measured., Conclusions: Both QLF and DP provided data that correlated moderately with TMR data. QLF images were easier to analyse, free of glare and had less variability compared with those produced using DP., (© 2012 Australian Dental Association.)

Background: Dental erosion is an increasingly prevalent problem in Australia, with the consumption of sports beverages suggested as a risk factor. The aim of this study was to compare the erosive potential of Australian sports beverages., Methods: Ten beverages were selected and analysed to determine their pH, titratable acidity and apparent degree of saturation with respect to apatite. The erosive potential of the beverages was measured by human enamel surface loss and surface softening following a 30-minute exposure. A taste testing panel was established to determine the palatability of the sports beverages., Results: All sports beverages except Sukkie and Endura produced substantial surface loss and surface softening. Compared with the other sports beverages, Sukkie and Endura had a higher pH, lower titratable acidity and higher calcium content. However, Sukkie and Endura were deemed to be less palatable than the other more acidic sports beverages., Conclusions: The majority of the sports beverages tested produced dental erosion in this in vitro model. However, two new products Sukkie and Endura have lower erosive potential but also lower palatability., (© 2012 Australian Dental Association.)

Remineralisation has been shown to be an effective mechanism of preventing the progression of enamel caries. The aim of this double-blind, randomised, cross-over in situ study was to compare enamel remineralisation by chewing sugar-free gum with or without casein phosphopeptide amorphous calcium phosphate (CPP-ACP) where the enamel lesions were exposed to dietary intake and some were covered with gauze to promote plaque formation. Participants wore removable palatal appliances containing 3 recessed enamel half-slabs with subsurface lesions covered with gauze and 3 without gauze. Mineral content was measured by transverse microradiography, and plaque composition was analysed by real-time polymerase chain reaction. For both the gauze-free and gauze-covered lesions, the greatest amount of remineralisation was produced by the CPP-ACP sugar-free gum, followed by the gum without CPP-ACP and then the no-gum control. Recessing the enamel in the appliance allowed plaque accumulation without the need for gauze. There was a trend of less remineralisation and greater variation in mineral content for the gauze-covered lesions. The cell numbers of total bacteria and streptococci were slightly higher in the plaque from the gauze-covered enamel for 2 of the 3 treatment legs; however, there was no significant difference in Streptococcus mutans cell numbers. In conclusion, chewing sugar-free gum containing CPP-ACP promoted greater levels of remineralisation than a sugar-free gum without CPP-ACP or a no-gum control using an in situ remineralisation model including dietary intake irrespective of whether gauze was used to promote plaque formation or not., (Copyright © 2012 S. Karger AG, Basel.)

Background: To investigate, in vitro, the effect on enamel erosion of the addition of 0.2% w/v casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) to four commercially-available soft drinks, two of which were carbonated., Methods: Enamel specimens (n=27) were sectioned from sound extracted human third molar teeth and polished to a mirror finish. Exposed enamel windows of 1 mm2 were created by painting the surface with acid-resistant nail varnish. Four citric flavoured soft drinks (pH range 2.2 to 2.4) and distilled deionized water (DDW) were tested. Each drink was tested with and without 0.2% CPP-ACP w/v. The specimens were placed into 50 mL of solution at 37 °C for 30 minutes, rinsed and varnish removed. The samples were profiled with a white light profilometer and erosive depths recorded., Results: All soft drinks tested caused enamel erosion but adding 0.2% w/v CPP-ACP significantly reduced (p<.05) erosive depth in all test solutions in comparison with the solutions without CPP-ACP. The erosive depths for all solutions with 0.2% CPP-ACP did not differ significantly from those of DDW., Conclusions: Adding CPP-ACP at 0.2% w/v significantly decreased the erosivity of all four soft drinks. The erosivity of the soft drinks with 0.2% CPP-ACP added did not differ significantly from that of distilled water.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been demonstrated to exhibit anticariogenic activity in randomized, controlled clinical trials of sugar-free gum and a tooth cream. Two randomized, double-blind, crossover studies were conducted to investigate the potential of CPP-ACP added to hard candy confections to slow the progression of enamel subsurface lesions in an in situ model. The confections studied were: (1) control sugar (65% sucrose + 33% glucose syrup); (2) control sugar-free; (3) sugar + 0.5% (w/w) CPP-ACP; (4) sugar + 1.0% (w/w) CPP-ACP; (5) sugar-free + 0.5% (w/w) CPP-ACP. Participants (10 and 14 in study 1 and 2) wore a removable palatal appliance containing enamel half-slabs with subsurface lesions, except for meals and oral hygiene procedures, and consumed 1 confection 6 times a day for 10 days. The enamel half-slabs were inset to allow the development of plaque on the enamel surface. Participants rested for 1 week before crossing over to another confection. The appliances were stored in a humid container at 37 degrees C when not in the mouth. After each treatment period, the enamel half-slabs were removed, paired with their demineralized control half-slabs, embedded, sectioned and then analysed using transverse microradiography. In both studies consumption of the control sugar confection resulted in significant demineralization (progression) of the enamel subsurface lesions. However, consumption of the sugar confections containing CPP-ACP did not result in lesion progression, but in fact in significant remineralization (regression) of the lesions. Remineralization by consumption of the sugar + 1.0% CPP-ACP confection was significantly greater than that obtained with the sugar-free confection., (Copyright 2010 S. Karger AG, Basel.)

Background: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is an anticariogenic agent that is suitable to be added to foods. The aim of this double-blind, three-way crossover randomized study was to investigate the capacity of CPP-ACP, when added to bovine milk, to remineralize enamel subsurface lesions in situ., Methods: Ten subjects drank 100 mL of bovine milk containing no added CPP-ACP (control milk), 0.2% (w/v) CPP-ACP or 0.3% (w/v) CPP-ACP, for 30 seconds once daily for 15 days, whilst wearing removable appliances with attached slabs of enamel containing subsurface enamel lesions. After each treatment and a one-week washout period, subjects crossed over to another treatment and this was repeated until they had consumed each of the three milk products. At the completion of each treatment the enamel slabs were removed and remineralization was determined using microradiography., Results: The results demonstrated that all three milk samples remineralized enamel subsurface lesions in situ. However, the two milk samples containing added CPP-ACP each produced significantly greater remineralization than the control milk., Conclusions: The remineralizing effect of CPP-ACP in milk was dose-dependent with milk containing 0.2% CPP-ACP and 0.3% CPP-ACP producing an increase in mineral content of 81% and 164%, respectively, relative to the control milk.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) slows the progression of caries and remineralizes enamel subsurface lesions. The aim of this study was to determine the ability of CPP-ACP to increase the incorporation of fluoride into plaque and to promote enamel remineralization in situ. Randomized, double-blind, cross-over studies involved mouthrinses and dentifrices containing CPP-ACP and fluoride. The mouthrinses were used for 60 sec, three times/day for 5 days, and supragingival plaque was collected and analyzed for F. The dentifrices were rinsed as a water slurry for 60 sec four times/day for 14 days in an in situ model. The addition of 2% CPP-ACP to the 450-ppm-F mouthrinse significantly increased the incorporation of fluoride into plaque. The dentifrice containing 2% CPP-ACP produced a level of remineralization similar to that achieved with a dentifrice containing 2800 ppm F. The dentifrice containing 2% CPP-ACP plus 1100 ppm F was superior to all other formulations.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to investigate the effects of CPP-ACP in a fruit-flavoured sugar-free chewing gum containing citric acid on enamel remineralization, and acid resistance of the remineralized enamel, using an in situ remineralization model. The study utilized a double-blind, randomized, crossover design with three treatments: (i) sugar-free gum (2 pellets) containing 20 mg citric acid and 18.8 mg CPP-ACP, (ii) sugar-free gum containing 20 mg citric acid alone, (iii) sugar-free gum not containing CPP-ACP or citric acid. Ten subjects were instructed to wear removable palatal appliances, with 4 half-slab insets of human enamel containing demineralized subsurface lesions and to chew gum (2 pellets) for 20 min 4 times per day for 14 days. At the completion of each treatment the enamel half-slabs were removed and half of the remineralized lesion treated with demineralization buffer for 16 h in vitro. The enamel slabs (remineralized, acid-challenged and control) were then embedded, sectioned and subjected to microradiography to determine the level of remineralization. Chewing with gum containing citric acid and CPP-ACP resulted in significantly higher remineralization (13.0 +/- 2.2%) than chewing with either gum containing no CPP-ACP or citric acid (9.4 +/- 1.2%) or gum containing citric acid alone (2.6 +/- 1.3%). The acid challenge of the remineralized lesions showed that the level of mineral after acid challenge was significantly greater for the lesions exposed to the gum containing CPP-ACP., (2007 S. Karger AG, Basel)

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes incorporated into sugar-free chewing gum have been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to compare the ability of CPP-ACP, with that of other forms of calcium, to be retained in supragingival plaque and remineralize enamel subsurface lesions in situ when delivered in a mouthrinse or sugar-free gum in randomized, double-blind trials. In the mouthrinse study, only the CPP-ACP-containing mouthrinse significantly increased plaque calcium and inorganic phosphate levels, and the CPP were immunolocalized to the surfaces of bacterial cells as well as the intercellular matrix. In the chewing gum studies, the gum containing the CPP-ACP, although not containing the most calcium per piece of gum, produced the highest level of enamel remineralization independent of gum-chewing frequency and duration. The CPP could be detected in plaque extracts 3 hrs after subjects chewed the CPP-ACP-containing gum. The results showed that CPP-ACP were superior to other forms of calcium in remineralizing enamel subsurface lesions.

Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.

The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.

The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated K1L23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a "petite-positive" strain carrying a rho o-lethality suppressor atp mutation rendered cells unable to grow on a nonfermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis.

Fifty years ago it was reported that baker's yeast, Saccharomyces cerevisiae, can form "petite colonie" mutants when treated with the DNA-targeting drug acriflavin. To mark the jubilee of studies on cytoplasmic inheritance, a review of the early work will be presented together with some observations on current developments. The primary emphasis is to address the questions of how loss of mtDNA leads to lethality (rho 0-lethality) in petite-negative yeasts and how S. cerevisiae tolerates elimination of mtDNA. Recent investigation have revealed that rho 0-lethality can be suppressed by specific mutations in the alpha, beta, and gamma subunits of the mitochondrial F1-ATPase of the petite-negative yeast Kluyveromyces lactis and by the nuclear ptp alleles in Schizosaccharomyces pombe. In contrast, inactivation of genes coding for F1-ATPase alpha and beta subunits and disruption of AAC2, PGS1/PEL1, and YME1 genes in S. cerevisiae convert this petite-positive yeast into a petite-negative form. Studies on nuclear genes affecting dependence on mtDNA have provided important insight into the functions provided by the mitochondrial genome and the maintenance of structural and functional integrity of the mitochondrial inner membrane.

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (rho-) and can survive complete loss of the organellar genome (rho(o)), the genetic factor(s) that permit(s) survival of rho- and rho(o) 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 alpha or beta subunit, but not the gamma, delta, or epsilon subunit of F1, renders cells petite-negative. The F1 complex, or a subcomplex composed of the alpha and beta subunits only, is essential for survival of rho(o) cells and those impaired in electron transport. The activity of F1 that suppresses rho(o) 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 rho(o) lethality has been achieved by simultaneous expression of S. cerevisiae F1 alpha and gamma 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 rho- or rho(o) mutations in S. cerevisiae.

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.

The nucleotide sequences of two segments of 6,737 ntp and 258 nto of the 18.4-kb circular mitochondrial (mt) DNA molecule of the soft coral Sarcophyton glaucum (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) have been determined. The larger segment contains the 3' 191 ntp of the gene for subunit 1 of the respiratory chain NADH dehydrogenase (ND1), complete genes for cytochrome b (Cyt b), ND6, ND3, ND4L, and a bacterial MutS homologue (MSH), and the 5' terminal 1,124 ntp of the gene for the large subunit rRNA (1-rRNA). These genes are arranged in the order given and all are transcribed from the same strand of the molecule. The smaller segment contains the 3' terminal 134 ntp of the ND4 gene and a complete tRNA(f-Met) gene, and these genes are transcribed in opposite directions. As in the hexacorallian anthozoan, Metridium senile, the mt-genetic code of S. glaucum is near standard: that is, in contrast to the situation in mt-genetic codes of other invertebrate phyla, AGA and AGG specify arginine, and ATA specifies isoleucine. However, as appears to be universal for metazoan mt-genetic codes, TGA specifies tryptophan rather than termination. Also, as in M. senile the mt-tRNA(f-Met) gene has primary and secondary structural features resembling those of Escherichia coli initiator tRNA, including standard dihydrouridine and T psi C loop sequences, and a mismatched nucleotide pair at the top of the amino-acyl stem. The presence of a mutS gene homologue, which has not been reported to occur in any other known mtDNA, suggests that there is mismatch repair activity in S. glaucum mitochondria. In support of this, phylogenetic analysis of MutS family protein sequences indicates that the S. glaucum mtMSH protein is more closely related to the nuclear DNA-encoded mitochondrial mismatch repair protein (MSH1) of the yeast Saccharomyces cerevisiae than to eukaryotic homologues involved in nuclear function, or to bacterial homologues. Regarding the possible origin of the S. glaucum mtMSH gene, the phylogenetic analysis results, together with comparative base composition considerations, and the absence of an MSH gene in any other known mtDNA best support the hypothesis that S. glaucum mtDNA acquired the mtMSH gene from nuclear DNA early in the evolution of octocorals. The presence of mismatch repair activity in S. glaucum mitochondria might be expected to influence the rate of evolution of this organism's mtDNA.

Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

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Specific mutations in nuclear MGI genes encoding the alpha, beta and gamma subunits of the mitochondrial inner membrane F1-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F0 component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly- 3. 9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1.

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MG12 and MG15 encoding the alpha- and gamma-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost. In this study we show that wild-type K. lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned the K. lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase. These observations suggest that mtDNA in K. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of Fo.

We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits.

The present study was undertaken to examine how osteoarthritis affects the expression of type-X collagen, a hypertrophic chondrocyte-specific collagen in articular cartilage. A well characterized sheep polyclonal antiserum, as well as three mouse monoclonal antibodies against canine type-X collagen, was used to immunolocalize type-X collagen in human and canine joints. Its expression in osteoarthritic cartilage was altered in several locations. In the canine osteoarthritic joints, type-X collagen increased in and just above the zone of calcified cartilage and was present diffusely throughout the calcified matrix. In both the human and canine cartilage, type-X collagen was localized around cell clones in the transitional zone of cartilage. This is surprising, since that region of the cartilage does not calcify and one of the proposed roles of type-X collagen is in mineralization. Thus, the osteoarthritic process may damage the matrix in the superficial layer and induce changes leading to the expression of the hypertrophic chondrocyte phenotype.

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

Mapping the 23-kb circular mitochondrial DNA from the yeast Kluyveromyces thermotolerans has shown that only one change occurs in the gene order in comparison to the 19-kb mtDNA of Candida (Torulopsis) glabrata. Sequence analysis of the mitochondrially encoded cytochrome oxidase subunit 2 gene reveals that despite their conserved gene order, the two small genomes are more distantly related than larger mtDNA molecules with multiple rearrangements. This result supports a previous observation that larger mitochondrial genomes are more prone to rearrange than smaller forms and suggests that the architecture of the two small molecules is likely to represent the structure of an ancestor.

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A nuclear mutation, mgm101, results in temperature sensitive loss of mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae. The corresponding gene, MGM101, was isolated from a genomic DNA library by complementation. Sequence analysis shows that MGM101 encodes a positively charged protein of 269 amino acids with a calculated molecular weight of 30 kDa. This analysis also reveals that MGM101 is adjacent to the ribosomal protein gene RPS7A on chromosome X and hybridization indicates it occurs in single copy. Creation of a null mutant by targeted disruption showed that the gene has no essential cellular function, aside from its participation in mitochondrial genome maintenance. As no counterpart has been identified in databases it is a novel protein whose role has yet to be determined. Expression of MGM101 is low on glucose medium but on galactose there is a two-fold increase in the level of the transcript.

Mitochondrial genomes from yeasts in the Dekkera/Brettanomyces/Eeniella group vary in size from 28 to 101 kb. Mapping of genes has shown that the three smallest genomes, of 28-42 kb, have the same gene order, whereas the three larger mitochondrial DNAs of 57-101 kb are rearranged relative to the smaller molecules and between themselves. To examine the relationships between these genomes, a phylogenetic tree has been constructed by sequence comparison of the mitochondrial-encoded cytochrome oxidase subunit gene (COX2) from the six species. Contrary to expectation, the tree shows that the larger rearranged genomes are more closely related than the smaller mtDNAs. This result indicates that the gene order of the smaller mtDNAs (28-42 kb) is ancestral and that larger mtDNA molecules (57-101 kb) are more prone to rearrangement than smaller forms.

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.

By employing pulsed-field gel electrophoresis we have determined the size of the rDNA cluster in wild-type yeast strains representing genera of Candida, Kluyveromyces, Pachysolen, Schizosaccharomyces and Torulaspora. Although the genome size of the examined species is similar (12.3-13.9 Mb), at least a four-fold variation has been observed between the lowest amount of rDNA repeats in P. tannophilus (28) and the highest in C. glabrata and S. poombe (> 115). In two species the rDNA cluster is represented by two loci, residing either in one (S. pombe) or two chromosomes (C. glabrata).

Migratory behaviour of mitochondrial DNA (mtDNA) from fungal species belonging to Plectomycetes, Loculoascomycetes and the zoosporic moulds, Oomycetes, has been studied by Pulsed Field Gel Electrophoresis (PFGE). Electrophoretic profiles demonstrate that long, linear molecules of a heterogenous size are the prevailing in-vivo form of organelle DNA in all examined species. These profiles are consistent with the presence of the rolling-circle mode of mtDNA replication that occurs in all branches of true fungi and zoosporic moulds.

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 baker's 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.

Base 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.

The 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, 1.5 a, and 1.5 b 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 cox1.5 a and K1 cox1.4/Sc cox1.5 b 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.

Mitochondrial intron content is variable in the yeast Kluyveromyces lactis. Strains can be divided into three classes depending on the structure of the cytochrome oxidase subunit 1 (COX1) gene: (1) those containing intron K1 cox1.1, (2) those containing K1 cox1.2, 3 and 4 and, (3) those that contain all four introns. In addition, strains belonging to the first class (designated Type B strains), have an altered mitochondrial gene order relative to strains from classes (2) and (3) (Type A, Hardy et al. 1989). Crossing experiments reveal that K1 cox1.1 (a group II intron) transfers at high frequency (89%) to mitochondrial genomes lacking this intron. By contrast, the mobility of the remaining introns (all group I) is of the order of 7%.

A spontaneously arising mitochondrial DNA (mtDNA) variant of Saccharomyces cerevisiae has been formed by two extra copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA sequence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400-500 bp whereas differences in 1-2 bp occur approximately every 60 bp. Some polymorphisms are associated with optional G + C-rich sequences (GC clusters). Two such optional GC clusters and one A + T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity in S. cerevisiae mtDNA are discussed.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.