Success rates of Hall technique crowns in primary molars: a retrospective pilot study.

The purpose of this retrospective observational study was to assess success rates, both clinical and radiographic, of stainless steel crowns (SSCs) placed on primary molars using the Hall technique. A retrospective analysis was performed on recorded data of patients with any primary molar treated with an SSC that was placed using the Hall technique at the University of Iowa College of Dentistry during 2011-2015. The primary outcome measure was the success or failure of the SSCs placed with the Hall technique. These outcomes were categorized as either clinical and radiographic success or failure. Clinical failure was defined as the need for pulp therapy or extraction following crown placement. Radiographic failure was defined as the presence of any pathological condition-including external or internal root resorption, bifurcation radiolucency, widened periodontal ligament, or ectopic eruption of permanent first molar adjacent to the Hall crown-following crown placement. Records indicated that 100 boys received a total of 179 crowns (61.1% of all Hall crowns placed), and 64 girls received 114 crowns. The mean age of the patients was 5.1 years (SD, 2.4 years). Of 293 SSCs included in the study, 180 received at least 1 follow-up examination after a mean of 9.9 months (SD, 6.5 months). At the first follow-up visit, 178 (98.9%) of 180 SSCs placed using the Hall technique were clinically successful. Of 87 crowns with radiographs available, 85 (97.7%) were radiographically successful. At the second follow-up visit (after a mean of 20.1 months), 74 of 76 (97.4%) were rated as clinically successful, and 37 of 39 (94.9%) were radiographically successful. Results of this study provide evidence of high clinical and radiographic success rates for SSCs placed on primary molars with the Hall technique.

Mutations on the N-terminal edge of the DELSEED loop in either the α or ß subunit of the mitochondrial F1-ATPase enhance ATP hydrolysis in the absence of the central γ rotor.

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)ß(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)ß(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)ß(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and ß subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the ß subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the ß subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or ß subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)ß(3) subcomplex.

The biogenesis of mitochondria in Saccharomyces cerevisiae. A comparison between cytoplasmic respiratory-deficient mutant yeast and chlormaphenicol-inhibited wild type cells.

The effects of chloramphenicol on S. cerevisiae and on a cytoplasmic respiratory-deficient mutant derived from the same strain are compared. In the normal yeast, high concentrations of chloramphenicol in the growth medium completely inhibit the formation of cytochromes a, a(3), b, and c(1) and partially inhibit succinate dehydrogenase formation, whereas they do not affect cytochrome c synthesis. This has been correlated with the marked reduction of mitochondrial cristae formation in the presence of the drug. In glucose-repressed normal yeast, chloramphenicol has little effect on the formation of outer mitochondrial membrane, or on the synthesis of malate dehydrogenase and fumarase. However, both these enzymes, as well as the number of mitochondrial profiles, are markedly decreased when glucose de-repressed yeast is grown in the presence of chloramphenicol. The antibiotic did not appear to affect the cytoplasmic respiratory-deficient mutant. The results have been interpreted to indicate that chloramphenicol inhibits the protein-synthesizing system characteristic of the mitochondria. Since the drug does not prevent the formation of cytochrome c, of several readily solubilized mitochondrial enzymes, or of outer mitochondrial membrane, it is suggested that these are synthesized by nonmitochondrial systems.

Unveiling the mystery of mitochondrial DNA replication in yeasts.

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing.

sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

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.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

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.

Studying mitochondria in an attractive model: Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe, widely used for studies of cell cycle control and differentiation, provides an alternative and complementary model to the budding yeast Saccharomyces cerevisiae for studies of nucleo-mitochondrial interactions. There are striking similarities between S. pombe and mammalian cells, in both their respiratory physiology and their mitochondrial genome structure. This technical review briefly lists the general and specific properties that are helpful to know when starting to use fission yeast as a model system for mitochondrial studies. In addition, advice is given for cell growth and genetic techniques, tips for disruption of genes involved in respiration are presented. and a basic differential centrifugation protocol is provided for the isolation of purified mitochondria that are suitable for diverse applications such as subfractionation and in vitro import.

RRP20, a component of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase.

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.

The petite mutation in yeasts: 50 years on.

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.

Mutations in MGI genes convert Kluyveromyces lactis into a petite-positive yeast.

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.

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase.

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.

Abnormal mitochondrial genomes in yeast restored to respiratory competence.

When crosses are performed between newly arisen, spontaneous petite mutants of Saccharomyces cerevisiae, respiratory competent (restored) colonies can form. Some of the restored colonies are highly sectored and produce large numbers of petite mutants. The high-frequency petite formation trait is inherited in a non-Mendelian manner, and elimination of mitochondrial DNA from these strains results in the loss of the trait. These results indicate that abnormal mitochondrial genomes are sometimes formed during restoration of respiratory competence. It is hypothesized that these abnormalities result either from recombination between mitochondrial DNA fragments to produce molecules having partial duplications contained on inverted or transposed sequences, or else recombinational "hot spots" have been expanded.

Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis.

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.

Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence. II. Organization of mitochondrial genomes in strains having high and moderate frequencies of petite mutant formation.

Restriction enzyme analysis of aberrant mtDNA molecules in restored strains of Saccharomyces cerevisiae that displays an elevated level of petite formation has shown the occurrence of novel junction fragments and nonstoichiometric amounts for some unaltered bands. Five aberrant mitochondrial genomes from high-frequency petite-forming (hfp) strains (greater than 60% petites per generation) contain like-oriented duplications and single copy regions. High-frequency petite formation is postulated to arise from increased intramolecular recombination between duplicated segments. Mitochondrial DNA structures in two other hfp strains cannot be easily interpreted and might arise from intramolecular recombination. Mitochondria DNA from moderate-frequency petite-forming (mfp) strains (5-16% petites per generation) contains inverted duplications in two cases. The elevated petite formation is postulated to arise from homologous recombination between directly repeated sequences. In mtDNA from one mfp strain, deletion end-points have been shown to overlap. Such deletion endpoint overlap is postulated to be required for the maintenance of the tandem duplication in hfp strains. Two regions of the wild-type mtDNA (between cyb and oli2 and between SrRNA and oxi2) appear to be dispensable for mitochondrial function.

The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA.

The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.

Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence. I. Association of both high and moderate frequencies of petite mutant formation with the presence of aberrant mitochondrial DNA.

When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5-16%) and those with a high frequency (greater than 60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome. Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.

Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts.

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.

Kinetic properties of F1-ATPase influence the ability of yeasts to grow in anoxia or absence of mtDNA.

A mechanism for hypoxia survival by eukaryotic cells is suggested from studies on the petite mutation of yeasts. Previous work has shown that mutations in the alpha, beta and gamma subunit genes of F1-ATPase can suppress lethality due to loss of the mitochondrial genome from the petite-negative yeast Kluyveromyceslactis. Here it is reported that suppressor mutations appear to increase the affinity of F1-ATPase for ATP. Extension of this study to other yeasts shows that petite-positive species have a higher affinity for ATP in the hydrolysis reaction than petite-negative species. Possession of a F1-ATPase with a low K(m) for ATP is considered to be an adaptation for hypoxic growth, enabling maintenance of the mitochondrial inner membrane potential, deltapsi, by enhanced export of protons through F1F0-ATP synthase connected to increased ATP hydrolysis at low substrate concentration.