Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.     

Purification and characterization of a DNA helicase from Saccharomyces cerevisiae

A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.     

Audiologic findings in patients with a point mutation at nucleotide 3,243 of mitochondrial DNA

In preparation for the introduction of Project 2000 throughout Scotland, a need was identified to monitor the quality of the learning environment for student nurses in the practice setting. No mechanism existed to measure quality and standards, and a group was formed to devise, pilot and implement an educational audit tool which could address these requirements, since limited information was available in the literature. Using this tool, the group subsequently examined the learning opportunities available, concentrating on the physical environment, the learning climate/environment, and the students' perceptions of the learning environment within the practice setting, across hospital and community sectors, including residential accommodation and nurseries, in the wide geographical area covered by the health board. This paper describes the process by which the tool was formulated, tested and implemented, the problems encountered, and the improvements in communications between teaching and clinical staff, leading to improved opportunities for the provision of excellence in patient care.     

Purification and characterization of DNA helicase III from the yeast Saccharomyces cerevisiae

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.     

Absence of association between a mitochondrial DNA mutation at nucleotide position 3243 and schizophrenia in Japanese

PURPOSE: To evaluate the effect that increased numbers of women medical school graduates have had on the composition of orthopedic surgery residencies, and to evaluate trends over time in the likelihood of women medical students to select orthopedic residencies. METHOD: The author analyzed JAMA's "Reports on Graduate and Undergraduate Medical Education" for the years 1977 to 1996, calculating the numbers of women and men in orthopedic surgery and other surgery residencies, and medical school composition. RESULTS: Although there have been modest gains in the number of women in orthopedic surgery training programs in the United States, women continue to choose orthopedics only one-seventh as often as do men. CONCLUSION: Orthopedics remains an unattractive career choice for women medical students compared with their men counterparts. Biases and stereotypes about women and about orthopedic surgery may account for this difference.     

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome

OBJECTIVES: To report the features of malignancies responsible for a chest wall mass and involving the sternum, the sternocostal and/or sternoclavicular joints, the chondrocostal junction and/or the adjacent soft tissues. METHODS: The medical records of patients with a chest wall mass due to malignant disease were reviewed retrospectively. The following data were abstracted from each record: characteristics of the pain and mass, constitutional symptoms, physical findings, laboratory test results, findings from imaging studies (plain radiographs, computed tomography and magnetic resonance imaging of the chest, radionuclide bone scan), histologic features of the biopsy specimen from the chest wall mass and origin of the mass. RESULTS: Seven men and three women with a mean age of 53.1 years were included in the study. A single patient had a history of malignant disease (lymphoma); in the remaining nine patients the chest wall mass was the first manifestation of the malignancy. All ten patients had pain with a mixed time pattern. The mass was located on the sternum in half the patients and in a parasternal location in the other half. Erythrocyte sedimentation rate elevation was found in seven patients, an increased serum level of lactate dehydrogenase in one and a monoclonal immunoglobulin in three. Sternal lesions were visible on plain radiographs in four patients. Computed tomography of the chest consistently disclosed sternal or sternocostal lytic lesions with spread to the adjacent soft tissues; in five cases, enlarged lymph nodes were visible in the anterior part of the mediastinum. Magnetic resonance imaging of the chest did not add to the information provided by computed tomography. Radionuclide uptake on the bone scan was increased, decreased, or normal at the site of the lesion. The cause was Hodgkin's disease in two cases, non-Hodgkin's lymphoma in three, metastatic bone disease in two (from an adenocarcinoma of the lung and a hepatocarcinoma, respectively), multiple myeloma in one, and solitary plasmacytoma in two. CONCLUSION: A chest wall mass can be caused by a known or as yet undiagnosed malignancy. Chest wall involvement due to malignant disease in rare, however. The specific features of sternal metastases, lymphomas involving the sternum, and sternal plasmacytomas are discussed. Nonmalignant chest wall lesions that can manifest as a bulging or swelling of the chest wall are reviewed.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.     

Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage

Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a 'step-arrest' mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells.     

Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow micro-aerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.     

In situ characterization of islets in diabetes with a mitochondrial DNA mutation at nucleotide position 3243

Changes in the pancreas of diabetic patients with the A-to-G mitochondrial DNA (mtDNA) mutation at nucleotide position 3243 base pair (bp) have not previously been described. The clinical phenotypes of diabetes associated with the mtDNA 3243 mutation range from NIDDM to IDDM. We sought the presence of the mutation and studied volume of beta-, alpha-, and delta-cells, mitochondrial enzyme activity, and presence of apoptosis in diabetic pancreases obtained at autopsy. Pancreases were obtained from 16 patients with IDDM, from 18 patients with NIDDM, and from 11 nondiabetic patients. Mitochondrial enzyme activity was determined for cytochrome c oxidase (COX), the subunits of which are partially encoded by mtDNA, and for succinate dehydrogenase (SDH), the subunits of which are solely encoded by nuclear DNA. The volumes of islet beta-, alpha-, and delta-cells were estimated by computerized morphometry. Pancreatic cells were examined for apoptosis by an in situ end-labeling procedure. The mtDNA 3243 mutation was detected in 1 of 16 (6%) pancreases from the IDDM patients; none of the pancreases from 18 NIDDM patients and 11 nondiabetic patients had the mutation. The single patient with the mtDNA 3243 mutation was a 56-year-old woman with IDDM, aged 39 years at diabetes onset, whose mother was diagnosed with NIDDM. The patient had a history of secondary failure of oral hypoglycemic agents and had a marked decrease in the number of beta-cells. The islet beta-cells and non-beta-cells of the patient showed extremely decreased COX enzyme activity. The islet cells in the patient showed a high activity when examined for SDH. Some pancreatic exocrine cells also showed decreased COX activity with high SDH activity. In IDDM, NIDDM, and nondiabetic patients without the mtDNA 3243 mutation, only weak staining for SDH of the islet cells showed. The percentage of heteroplasmy of the mtDNA 3243 mutation in pancreatic micropunched islet specimens was 63 +/- 5% (mean +/- SD) in the islets, 32 +/- 3% in the exocrine pancreas, and 8 +/- 1% in peripheral polymorphonuclear cells. Apoptotic cells were not observed in the IDDM pancreas in the patient with the mtDNA 3243 mutation. The fact that higher levels of mutated mtDNA at 3243 bp were found in affected islets rather than in other tissue suggests that the distribution of the mutant may determine the effect on islet function. A characteristic decrease in the mitochondrial enzyme with COX activity and accelerated SDH activity of the affected islets may provide new insights into the pathogenesis of mitochondrial diabetes.     

Partial purification of Pde1 from Saccharomyces cerevisiae: enzymatic redundancy for the repair of 3'-terminal DNA lesions and abasic sites in yeast

Earlier work indicates that the major DNA repair phosphodiesterase (PDE) in yeast cells is the well-characterized Apn1 protein. Apn1 demonstrates both Mg2+-independent PDE activity and Mg2+-independent class II apurinic/apyrimidinic (AP) endonuclease activity and represents greater than 90% of the activity detected in crude extracts from wild-type yeast cells. Apn1 is related to Echerichia coli endonuclease IV, both in its enzymatic properties and its amino acid sequence. In this work, we report the partial purification of a novel yeast protein, Pde1, present in Apn1-deficient cells. Pde1 is purified by sequential BioRex-70, PBE118, and MonoS chromatography steps using a sensitive and highly specific 3'-phosphoglycolate-terminated oligonucleotide-based assay as a measure of PDE activity. Mg2+-stimulated PDE and Mg2+-stimulated class II AP endonuclease copurify during this procedure. These results indicate that yeast, like many other organisms studied to date, has enzymatic redundancy for the repair of 3'-blocking groups and abasic sites.     

Comparison of oxidative base damage in mitochondrial and nuclear DNA

In 1994-1995, a child and five dogs from villages located between Jerusalem and Tel-Aviv, Israel were diagnosed with visceral leishmaniasis (VL). Based on these findings, the distribution of VL in domestic and wild canids in central Israel was examined. In the two villages where canine index cases were identified, a substantial proportion (11.5%, 14 of 122) of the dogs examined were seropositive. However, the rate of infection in five neighboring villages was only 1% (1 of 99). Parasites were cultured from 92% (12 of 13) of the seropositive dogs biopsied and the strains were characterized as Leishmania infantum by a clamped polymorphic-polymerase chain reaction, monoclonal antibodies, and/or excreted factor serology. The discovery of VL close to major urban centers is an important public health issue. The disease appears to have emerged recently in this area, and it is unclear whether the parasite was re-introduced or was continuously present at low levels in this region. The presence of seropositive wild canids, jackals (7.6%, 4 of 53) and red foxes (5%, 1 of 20), in central Israel, and the reappearance of the jackal population after near extinction suggests that wild canids may play a role in spreading this disease.     

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.     

Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae

We recently showed the involvement of the L-arginine/nitric oxide (NO) pathway in acid-induced duodenal mucosal bicarbonate secretion in rats. The aim of the present study was to confirm this observation in pigs by direct measurements of NO production. Experiments were performed on 16 anaesthetized pigs of both sexes treated with guanethidine (6 mg kg-1, intravenously). A duodenal segment, devoid of pancreaticobiliary influxes, was perfused with saline and the duodenal mucosal bicarbonate secretion was calculated from continuous measurements of pH and PCO2. The perfusate contents of NO and its oxidative product nitrite were determined by chemiluminescence, after reduction of nitrite to NO. Luminal acidification with 30 mM hydrochloric acid increased the output of bicarbonate as well as NO to the perfusate, by 195 +/- 45% and 106 +/- 10%, respectively. These responses to acid were markedly inhibited by adding the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.3 mM) to the perfusate. The inhibitory effect of L-NMMA could be reversed by administration of L-arginine (3 mM). The study presents simultaneous measurements of bicarbonate and NO outputs to a duodenal luminal perfusate. The results strongly support the view that the L-arginine/NO pathway is involved in the acid-induced duodenal mucosal bicarbonate secretory response.     

Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae

Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.