scholarly journals Pir1p Mediates Translocation of the Yeast Apn1p Endonuclease into the Mitochondria To Maintain Genomic Stability

2001 ◽  
Vol 21 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
Ratsavarinh Vongsamphanh ◽  
Pierre-Karl Fortier ◽  
Dindial Ramotar
Keyword(s):  
Mitochondrial Genome ◽  
Fold Increase ◽  
Dna Lesions ◽  
Mitochondrial Mutations ◽  
Abasic Sites ◽  
Cell Wall Constituent ◽  
In The Wild ◽  
Functional Consequences ◽  
Localization Signal ◽  
A Cell

ABSTRACT The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism. This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites. Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Δ mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites. Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal. Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery. pir1Δ mutants displayed a striking (∼3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria. To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1Δ and apn1Δ mutants.pir1Δ and apn1Δ mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.

scholarly journals Quantitative Assessment of Heteroplasmy of Mitochondrial Genome: Perspectives in Diagnostics and Methodological Pitfalls

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Igor A. Sobenin ◽  
Konstantin Y. Mitrofanov ◽  
Andrey V. Zhelankin ◽  
Margarita A. Sazonova ◽  
Anton Y. Postnov ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Clinical Manifestations ◽  
Threshold Level ◽  
Mtdna Mutation ◽  
Mitochondrial Mutations ◽  
Organellar Genome ◽  
Wide Range ◽  
Comprehensive Comparison ◽  
A Cell

The role of alterations of mitochondrial DNA (mtDNA) in the development of human pathologies is not understood well. Most of mitochondrial mutations are characterized by the phenomenon of heteroplasmy which is defined as the presence of a mixture of more than one type of an organellar genome within a cell or tissue. The level of heteroplasmy varies in wide range, and the expression of disease is dependent on the percent of alleles bearing mutations, thus allowing consumption that an upper threshold level may exist beyond which the mitochondrial function collapses. Recent findings have demonstrated that some mtDNA heteroplasmic mutations are associated with widely spread chronic diseases, including atherosclerosis and cancer. Actually, each etiological mtDNA mutation has its own heteroplasmy threshold that needs to be measured. Therefore, quantitative evaluation of a mutant allele of mitochondrial genome is an obvious methodological challenge, since it may be a keystone for diagnostics of individual genetic predisposition to the disease. This review provides a comprehensive comparison of methods applicable to the measurement of heteroplasmy level of mitochondrial mutations associated with the development of pathology, in particular, in atherosclerosis and its clinical manifestations.

scholarly journals Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27

1999 ◽  
Vol 19 (6) ◽  
pp. 4143-4152 ◽  
Author(s):  
Julie Parenteau ◽  
Raymund J. Wellinger
Keyword(s):  
Growth Arrest ◽  
Dna Lesions ◽  
Restrictive Temperature ◽  
Telomeric Dna ◽  
Single Stranded Dna ◽  
Template Strand ◽  
Okazaki Fragments ◽  
A Cell ◽  
Lagging Strand

ABSTRACT The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27cells at 37°C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

scholarly journals Phytic Acid Protects against 6-Hydroxydopamine-Induced Dopaminergic Neuron Apoptosis in Normal and Iron Excess Conditions in a Cell Culture Model

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Qi Xu ◽  
Anumantha G. Kanthasamy ◽  
Manju B. Reddy
Keyword(s):  
Dna Fragmentation ◽  
Phytic Acid ◽  
Caspase 3 ◽  
Fold Increase ◽  
Iron Chelator ◽  
Culture Model ◽  
A Cell ◽  
Induced Apoptosis ◽  
6 Hydroxydopamine ◽  
Stress Dependent

Iron may play an important role in Parkinson's disease (PD) since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6) can protect against 6-hydroxydopamine- (6-OHDA-) induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; ) and 30 μmol/L IP6 pretreatment decreased it by 38% (). Similarly, a 63% protection () against 6-OHDA induced DNA fragmentation was observed with IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity () and a 42% increase in DNA fragmentation () with 6-OHDA treatment were decreased by 41% () and 27% (), respectively, with 30 μmol/L IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD.

scholarly journals Effects of Selection to Diflubenzuron and Bacillus thuringiensis Var. Israelensis on the Overwintering Successes of Aedes albopictus (Diptera: Culicidae)

Insects ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 822
Author(s):  
Charalampos S. Ioannou ◽  
Christos Hadjichristodoulou ◽  
Varvara A. Mouchtouri ◽  
Nikos T. Papadopoulos
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Albopictus ◽  
Selection Process ◽  
Mosquito Species ◽  
Fold Increase ◽  
Development Of Resistance ◽  
Control Programmes ◽  
Invasive Mosquito ◽  
In The Wild ◽  
Successive Generations

Aedes albopictus is an invasive mosquito species responsible for local transmission of chikungunya and dengue viruses in Europe. In the absence of available treatments, insecticides-based control remains one of the most important viable strategies to prevent emerging problems. Diflubenzuron (DFB) and Bacillus thuringiensis var. israelensis (Bti) are among the most commonly used larvicides for Ae. albopictus control with consequent concerns for the potential development of resistance. Studies on the resistance emergence in Ae. albopictus and its persistence in the wild to both DFB and Bti are essential for the efficient and sustainable planning of the control programmes. In this context, larvae from a recently laboratory established population were subjected to increasing selective pressure for nine successive generations using both DFB and Bti. The resistance levels and the overwintering success of the selected populations relative to control (colonies that received no selection) were determined. Results revealed an 8.5- and 1.6-fold increase on the resistance levels following selection with DFB and Bti, respectively. The selection process to both larvicides had no apparent impacts on the overwintering capability relative to control, suggesting the successful persistence of the selected individuals in the wild on an annual base.

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

scholarly journals Detection of Amyotrophic Lateral Sclerosis (ALS) via Acoustic Analysis

2018 ◽  
Author(s):  
Raquel Norel ◽  
Mary Pietrowicz ◽  
Carla Agurto ◽  
Shay Rishoni ◽  
Guillermo Cecchi
Keyword(s):  
Disease Progression ◽  
Acoustic Analysis ◽  
Voice Quality ◽  
Non Invasive ◽  
Frequent Monitoring ◽  
In The Wild ◽  
A Cell ◽  
Speech Features ◽  
Severity Of The Disease ◽  
Lateral Sclerosis

ABSTRACTALS is a fatal neurodegenerative disease with no cure. Experts typically measure disease progression via the ALSFRS-R score, which includes measurements of various abilities known to decline. We propose instead the use of speech analysis as a proxy for ALS progression. This technique enables 1) frequent non-invasive, inexpensive, longitudinal analysis, 2) analysis of data recorded in the wild, and 3) creation of an extensive ALS databank for future analysis. Patients and trained medical professionals need not be co-located, enabling more frequent monitoring of more patients from the convenience of their own homes. The goals of this study are the identification of acoustic speech features in naturalistic contexts which characterize disease progression and development of machine models which can recognize the presence and severity of the disease. We evaluated subjects from the Prize4Life Israel dataset, using a variety of frequency, spectral, and voice quality features. The dataset was generated using the ALS Mobile Analyzer, a cell-phone app that collects data regarding disease progress using a self-reported ALSFRS-R questionnaire and several active tasks that measure speech and motor skills. Classification via leave-five-subjects-out cross-validation resulted in an accuracy rate of 79% (61% chance) for males and 83% (52% chance) for females.

scholarly journals Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuxiao Xie ◽  
Shulin Chen ◽  
Xiaochao Xiong
Keyword(s):  
Yarrowia Lipolytica ◽  
Fold Increase ◽  
Genetically Engineered ◽  
Cell Factory ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Yeast Extract Peptone Dextrose ◽  
Dry Cell Weight ◽  
A Cell ◽  
Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

scholarly journals Avoidance of Autophagy Mediated by PlcA or ActA Is Required for Listeria monocytogenes Growth in Macrophages

2015 ◽  
Vol 83 (5) ◽  
pp. 2175-2184 ◽  
Author(s):  
Gabriel Mitchell ◽  
Liang Ge ◽  
Qiongying Huang ◽  
Chen Chen ◽  
Sara Kianian ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Fold Increase ◽  
Host Cells ◽  
Listeriolysin O ◽  
Wild Type ◽  
Conjugation System ◽  
Content Type ◽  
A Cell ◽  
Gain Access

Listeria monocytogenesis a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy byL. monocytogenesprimarily involves PlcA and ActA and that either one of these factors must be present forL. monocytogenesgrowth in macrophages.

Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Mei-Shiue Kuo ◽  
Kuei-Peng Chen ◽  
Whi Fin Wu
Keyword(s):  
Escherichia Coli ◽  
Lon Protease ◽  
Specific Substrate ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Reporter Construct ◽  
Fusion Construct ◽  
In The Wild ◽  
A Cell ◽  
Cell Division Inhibitor

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for β-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBAD–rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA Red –lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased β-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.

scholarly journals Nuclear constriction segregates mobile nuclear proteins away from chromatin

2016 ◽  
Vol 27 (25) ◽  
pp. 4011-4020 ◽  
Author(s):  
Jerome Irianto ◽  
Charlotte R. Pfeifer ◽  
Rachel R. Bennett ◽  
Yuntao Xia ◽  
Irena L. Ivanovska ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Volume Fraction ◽  
Chromosome 1 ◽  
Blood Capillary ◽  
Cancer Cell Migration ◽  
Nuclear Factors ◽  
Functional Consequences ◽  
A Cell ◽  
Local Compaction ◽  
Chromatin Density

As a cell squeezes its nucleus through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, chromatin density and nuclear factors could in principle be physically perturbed. Here, in cancer cell migration through rigid micropores and in passive pulling into micropipettes, local compaction of chromatin is observed coincident with depletion of mobile factors. Heterochromatin/euchromatin was previously estimated from molecular mobility measurements to occupy a volume fraction f of roughly two-thirds of the nuclear volume, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g., BRCA1, 53BP1) or nucleases (e.g., Cas9, FokI), are depleted within the constriction, approaching 0%. Such losses—compounded by the occasional rupture of the nuclear envelope—can have important functional consequences. Studies of a nuclease that targets a locus in chromosome-1 indeed show that constricted migration delays DNA damage.

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