scholarly journals A Genetic Screen To Identify Genes Influencing the Secondary Redox Couple NADPH/NADP+ in the Yeast Saccharomyces cerevisiae

2019 ◽  
Vol 10 (1) ◽  
pp. 371-378 ◽  
Author(s):  
Shambhu Yadav ◽  
Tejasvinee Atul Mody ◽  
Archi Sharma ◽  
Anand Kumar Bachhawat
Keyword(s):  
Mitochondrial Gene ◽  
Pentose Phosphate ◽  
Genetic Screen ◽  
Yeast Saccharomyces Cerevisiae ◽  
Link Type ◽  
New Genes ◽  
Gene Knockouts ◽  
Low Levels ◽  
Reduced Forms ◽  
Glyoxylate Reductase

NADPH is an important cofactor in the cell. In addition to its role in the biosynthesis of critical metabolites, it plays crucial roles in the regeneration of the reduced forms of glutathione, thioredoxins and peroxiredoxins. The enzymes and pathways that regulate NADPH are thus extremely important to understand, and yet are only partially understood. We have been interested in understanding how NADPH fluxes are altered in the cell. We describe here both an assay and a genetic screen that allows one to discern changes in NADPH levels. The screen exploits the secondary redox property of NADPH. At low levels of glutathione we show that the redox contributions of NADPH become critical for growth, and we have used this to develop a genetic screen for genes affecting NADPH homeostasis. The screen was validated in pathways that both directly (pentose phosphate pathway) and indirectly (glycolytic pathway) affect NADPH levels, and was then exploited to identify mitochondrial genes that affect NADPH homeostasis. A total of 239 mitochondrial gene knockouts were assayed using this screen. Among these, several genes were predicted to play a role in NADPH homeostasis. This included several new genes of unknown function, and others of poorly defined function. We examined two of these genes, FMP40 which encodes a protein required during oxidative stress and GOR1, glyoxylate reductase. Our studies throw new light on these proteins that appear to be major consumers of NADPH in the cell. The genetic screen is thus predicted to be an exceedingly useful tool for investigating NADPH homeostasis.

scholarly journals The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 300
Author(s):  
Camilla Ceccatelli Berti ◽  
Giulia di Punzio ◽  
Cristina Dallabona ◽  
Enrico Baruffini ◽  
Paola Goffrini ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mitochondrial Diseases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genome Data ◽  
New Genes ◽  
Eukaryotic Organism ◽  
Novel Variants ◽  
Therapeutic Molecules ◽  
Nuclear Mutations

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

Rhizobium paknamense sp. nov., isolated from lesser duckweeds (Lemna aequinoctialis)

2013 ◽  
Vol 63 (Pt_10) ◽  
pp. 3823-3828 ◽  
Author(s):  
Chokchai Kittiwongwattana ◽  
Chitti Thawai
Keyword(s):  
Dna Hybridization ◽  
Type Species ◽  
Sequence Similarity ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Low Levels ◽  
Sequence Similarity Analysis ◽  
Ph Range

A Gram-stain-negative, rod-shaped bacterium was isolated and designated strain L6-8T during a study of endophytic bacterial communities in lesser duckweed (Lemna aequinoctialis). Cells of strain L6-8T were motile with peritrichous flagella. The analysis of the nearly complete 16S rRNA gene sequence indicated that strain L6-8T was phylogenetically related to species of the genus Rhizobium . Its closest relatives were Rhizobium borbori DN316T (97.6 %), Rhizobium oryzae Alt 505T (97.3 %) and Rhizobium pseudoryzae J3-A127T (97.0 %). The sequence similarity analysis of housekeeping genes recA, glnII, atpD and gyrB showed low levels of sequence similarity (<91.5 %) between strain L6-8T and other species of the genus Rhizobium with validly published names. The pH range for growth was 4.0–9.0 (optimum 6.0–7.0), and the temperature range for growth was 20–45 °C (optimum 30 °C). Strain L6-8T tolerated NaCl up to 2 % (w/v) (optimum 1 % NaCl). The predominant components of cellular fatty acids were C19 : 0 cyclo ω8c (31.32 %), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 25.39 %) and C16 : 0 (12.03 %). The DNA G+C content of strain L6-8T was 60.4 mol% (T m). nodC and nifH were not amplified in strain L6-8T. DNA–DNA relatedness between strain L6-8T and R. borbori DN316T, R. oryzae Alt505T and R. pseudoryzae J3-A127T was between 11.2 and 18.3 %. Based on the sequence similarity analyses, phenotypic, biochemical and physiological characteristics and DNA–DNA hybridization, strain L6-8T could be readily distinguished from its closest relatives and represents a novel species of the genus Rhizobium , for which the name Rhizobium paknamense sp. nov. is proposed. The type strain is L6-8T ( = NBRC 109338T = BCC 55142T).

scholarly journals Fermentación de la almendra de copoazú (Theobroma grandiflorum [Willd. ex Spreng.] Schum.): evaluación y optimización del proceso

2010 ◽  
Vol 11 (2) ◽  
pp. 107 ◽  
Author(s):  
Jenifer Criollo ◽  
Dagoberto Criollo ◽  
Angélica Sandoval Aldana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Process Optimization ◽  
Fermentation Process ◽  
Raw Material ◽  
Sensory Panel ◽  
Yeast Saccharomyces Cerevisiae ◽  
Theobroma Grandiflorum ◽  
Cutting Test ◽  
Maximum Range ◽  
Low Levels

<p>La almendra de copoazú como producto promisorio para la industria de cosméticos, chocolate, bebidas, licores y conservas, se evaluó el proceso de fermentación variando el tiempo de remoción de la masa (24 y 48 horas) y la pulpa inicial (30 y 100%). Se tuvieron en cuenta las condiciones de los productores en el acceso a equipos de despulpado. Se cuantificó la temperatura de la masa en tres puntos (superior, medio e inferior), acidez, pH, humedad, prueba de corte y análisis sensorial. Se encontró bajo desarrollo de la temperatura de fermentación en los tratamientos con 100% de pulpa y se registraron las máximas temperaturas entre 35 y 36°C que indican deficiencias en el proceso; no se alcanzó los 40°C requeridos para la muerte del embrión. El 30% de pulpa inicial y la remoción cada 24 horas por 9 días, fueron las mejores condiciones encontradas. La optimización con 0,1% de levadura (Saccharomyces cerevisiae) aumentó la temperatura de fermentación hasta 44°C, los granos fermentados hasta 56,14% y el mayor desarrollo de sabores frutales con intensidad de 4, mostrando un mejor proceso de fermentación. El panel sensorial mostró que los licores de copoazú tienen notas frutales destacadas y bajos valores de otros sabores evaluados. Los resultados son semejantes a los cacaos criollos, conocidos en el mundo como materia prima de licores finos y de aroma.</p><p> </p><p><strong>Fermentation of the copoazu kernel (Theobroma grandiflorum [Willd. ex Spreng.] Schum.): Assessmente and process optimization</strong></p><p>The fermentation of copoazu kernels (a promising product for the cosmetics industry, chocolate, beverages, liquors and preserves) was evaluated varying the time of mass removal (24 and 48 hours) and the initial pulp (30 and 100%). This study took into account the degree of access the producers had to pulping equipment. We quantified temperature of the mass at three points (top, middle and bottom), acidity, pH, moisture, cutting test and sensory analysis. The observed temperatures during fermentation in the treatments with 100% pulp reached a maximum range between 35 and 36°C which indicated deficiencies in the process as the 40°C required for the death of the seed was not attained. Thirty percent initial pulp with removal every 24 hours for 9 days yielded the best results. Optimization with 0.1% yeast (Saccharomyces cerevisiae) increased the fermentation temperature to 44°C, augmented fermented beans to 56.14% and saw a development of fruit flavors with an intensity of 4, demonstrating a better fermentation process. The sensory panel showed that copoazu liquors have outstanding fruity notes and low levels of other evaluated flavors. The results are similar to the criollo cacao, known worldwide as a raw material for fine liquors and fragrances.</p>

scholarly journals Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

2018 ◽  
Author(s):  
Jeremy G. Bird ◽  
Urmimala Basu ◽  
David Kuster ◽  
Aparna Ramachandran ◽  
Ewa Grudzien-Nogalska ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Promoter Sequence ◽  
Mitochondrial Rna ◽  
Sensors And Actuators ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Transcripts ◽  
Rna Capping ◽  
Reduced Forms

AbstractBacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

scholarly journals Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Bama Charan Mondal ◽  
Jiwon Shim ◽  
Cory J Evans ◽  
Utpal Banerjee
Keyword(s):  
Drosophila Melanogaster ◽  
Blood Cells ◽  
Lymph Gland ◽  
Genetic Screen ◽  
Signal Control ◽  
Vegf Receptor ◽  
Interacting Protein ◽  
Hematopoietic Development ◽  
New Genes ◽  
Progenitor Maintenance

Blood progenitors within the lymph gland, a larval organ that supports hematopoiesis in Drosophila melanogaster, are maintained by integrating signals emanating from niche-like cells and those from differentiating blood cells. We term the signal from differentiating cells the ‘equilibrium signal’ in order to distinguish it from the ‘niche signal’. Earlier we showed that equilibrium signaling utilizes Pvr (the Drosophila PDGF/VEGF receptor), STAT92E, and adenosine deaminase-related growth factor A (ADGF-A) (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). Little is known about how this signal initiates during hematopoietic development. To identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling, we performed a genetic screen that identified bip1 (bric à brac interacting protein 1) and Nucleoporin 98 (Nup98) as additional regulators of the equilibrium signal. We show that the products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr.

scholarly journals Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion

2020 ◽  
Vol 8 (4) ◽  
pp. 546 ◽  
Author(s):  
Jürgen J. Heinisch ◽  
Johannes Knuesting ◽  
Renate Scheibe
Keyword(s):  
Kluyveromyces Lactis ◽  
Expression System ◽  
Disulfide Bridge ◽  
Pentose Phosphate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Physiological Properties ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Synthetic Media ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

Halobaculum magnesiiphilum sp. nov., a magnesium-dependent haloarchaeon isolated from commercial salt

2013 ◽  
Vol 63 (Pt_3) ◽  
pp. 861-866 ◽  
Author(s):  
Hirokazu Shimoshige ◽  
Tomoaki Yamada ◽  
Hiroaki Minegishi ◽  
Akinobu Echigo ◽  
Yasuhiro Shimane ◽  
...  
Keyword(s):  
Type Species ◽  
Lipid Analysis ◽  
Halophilic Archaea ◽  
Single Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Link Type ◽  
Physiological And Biochemical Characteristics ◽  
Low Levels

Two extremely halophilic archaea, strains MGY-184T and MGY-205, were isolated from sea salt produced in Japan and rock salt imported from Bolivia, respectively. Both strains were pleomorphic, non-motile, Gram-negative and required more than 5 % (w/v) NaCl for growth, with optimum at 9–12 %, in the presence of 2 % (w/v) MgCl2 . 6H2O. In the presence of 18 % (w/v) MgCl2 . 6H2O, however, both strains showed growth even at 1.0 % (w/v) NaCl. Both strains possessed two 16S rRNA genes (rrnA and rrnB), and they revealed closest similarity to Halobaculum gomorrense JCM 9908T, the single species with a validly published name of the genus Halobaculum , with similarity of 97.8 %. The rrnA and rrnB genes of both strains were 100 % similar. The rrnA genes were 97.6 % similar to the rrnB genes in both strains. DNA G+C contents of strains MGY-184T and MGY-205 were 67.0 and 67.4 mol%, respectively. Polar lipid analysis revealed that the two strains contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester derived from C20C20 archaeol. The DNA–DNA hybridization value between the two strains was 70 % and both strains showed low levels of DNA–DNA relatedness (48–50 %) with Halobaculum gomorrense JCM 9908T. Physiological and biochemical characteristics allowed differentiation of strains MGY-184T and MGY-205 from Halobaculum gomorrense JCM 9908T. Therefore, strains MGY-184T and MGY-205 represent a novel species of the genus Halobaculum , for which the name Halobaculum magnesiiphilum sp. nov. is proposed; the type strain is MGY-184T ( = JCM 17821T = KCTC 4100T).

scholarly journals Retroposed New Genes Out of the X in Drosophila

2002 ◽  
Vol 12 (12) ◽  
pp. 1854-1859
Author(s):  
Esther Betrán ◽  
Kevin Thornton ◽  
Manyuan Long
Keyword(s):  
Population Genetics ◽  
Molecular Mechanisms ◽  
Sequence Data ◽  
Evolutionary Process ◽  
Significant Excess ◽  
Link Type ◽  
New Genes ◽  
Asymmetric Pattern ◽  
Unpublished Information

New genes that originated by various molecular mechanisms are an essential component in understanding the evolution of genetic systems. We investigated the pattern of origin of the genes created by retroposition in Drosophila. We surveyed the wholeDrosophila melanogaster genome for such new retrogenes and experimentally analyzed their functionality and evolutionary process. These retrogenes, functional as revealed by the analysis of expression, substitution, and population genetics, show a surprisingly asymmetric pattern in their origin. There is a significant excess of retrogenes that originate from the X chromosome and retropose to autosomes; new genes retroposed from autosomes are scarce. Further, we found that most of these X-derived autosomal retrogenes had evolved a testis expression pattern. These observations may be explained by natural selection favoring those new retrogenes that moved to autosomes and avoided the spermatogenesis X inactivation, and suggest the important role of genome position for the origin of new genes.[The sequence data from this study have been submitted to GenBank under accession nos. AY150701–AY150797. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: M.-L. Wu, F. Lemeunier, and P. Gibert.]

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