scholarly journals Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila.

1982 ◽  
Vol 2 (4) ◽  
pp. 378-385 ◽  
Author(s):  
C T Roberts ◽  
J E Lavine ◽  
D E Morse
Keyword(s):  
Gene Expression ◽  
Mutant Allele ◽  
Tetrahymena Thermophila ◽  
Molecular Defect ◽  
Deficient Mutant ◽  
Cellular Processes ◽  
Inhibition Of Growth ◽  
Selection Of ◽  
Enzymatic Defect

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila

1982 ◽  
Vol 2 (4) ◽  
pp. 378-385
Author(s):  
C T Roberts ◽  
J E Lavine ◽  
D E Morse
Keyword(s):  
Gene Expression ◽  
Mutant Allele ◽  
Tetrahymena Thermophila ◽  
Molecular Defect ◽  
Deficient Mutant ◽  
Cellular Processes ◽  
Inhibition Of Growth ◽  
Selection Of ◽  
Enzymatic Defect

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression

2008 ◽  
Vol 77 (5) ◽  
pp. 1063-1071 ◽  
Author(s):  
Hiroaki Tanaka ◽  
Eiji Tamai ◽  
Shigeru Miyata ◽  
Yuki Taniguchi ◽  
Hirofumi Nariya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Perfringens ◽  
Deficient Mutant ◽  
Protease Deficient

Molecular characterization of rat gastric mucosal response to potent acid inhibition

2005 ◽  
Vol 22 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Kristin G. Nørsett ◽  
Astrid Lægreid ◽  
Mette Langaas ◽  
Sara Wörlund ◽  
Reidar Fossmark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Responses ◽  
Clinical Medicine ◽  
Defense Responses ◽  
Acid Inhibition ◽  
Expression Levels ◽  
Cellular Processes ◽  
Gastric Corpus ◽  
Gene Expression Levels

Potent acid inhibition with proton pump inhibitors (PPIs) is widely used in clinical medicine, especially for gastroesophageal reflux disease. PPIs cause profound changes in the intragastric environment with near-neutral pH and increase serum concentration of the gastric secretagogue hormone gastrin. Long-term hypergastrinemia increases mucosal thickness and enterochromaffin-like cell density in gastric corpus mucosa and results in development of gastric carcinoids in experimental animals. Our aim was to study responses to potent acid inhibition by characterizing genome-wide gene expression changes in gastric corpus mucosa in rats dosed with the PPI omeprazole. Nine rats received 400 μmol/kg omeprazole daily for 10 wk. Seven rats received vehicle only. Analysis of gastric corpus with microarrays representing 11,848 genes identified 134 genes with changed gene expression levels in omeprazole-dosed rats. Several of the identified genes were previously known to be affected by potent acid inhibition. Of the 62 genes with known functions that changed gene expression levels after PPI dosing, 27 are known to be involved in proliferation and apoptosis and immune, inflammatory, and stress responses. Our study indicates that microarray analysis can detect relevant gene expression changes in the complex gastric tissue, and that cellular processes involved in cell growth and defense responses are strongly affected by PPI dosing. Many genes are identified that were not previously known to be affected by inhibition of gastric acid secretion or that have unknown biological functions. Characterization of the roles of these genes may give new insight into molecular responses to treatment with PPIs.

scholarly journals A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

2019 ◽  
Author(s):  
Andrew M. Hogan ◽  
A. S. M. Zisanur Rahman ◽  
Tasia J. Lightly ◽  
Silvia T. Cardona
Keyword(s):  
Gene Expression ◽  
Host Range ◽  
Phenylacetic Acid ◽  
Inducible Promoter ◽  
Broad Host Range ◽  
Proof Of Concept ◽  
Genetic Tools ◽  
Cellular Processes ◽  
Inhibition Of Growth ◽  
Silence Gene

AbstractGenetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi tool kit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia’s pathogenic arsenal.Author contributionsSTC conceived the idea and design of the research; AMH designed and cloned the dCas9 constructs; AMH and ASMZ designed the sgRNAs, assessed knockdown phenotypes, processed data, and wrote and edited the manuscript; TJL performed RT-qPCR analysis and edited the manuscript; STC supervised the work and provided financial support.Graphical Abstract

scholarly journals O-linked N-acetylglucosamine transferase (OGT) interacts with the histone chaperone HIRA complex and regulates nucleosome assembly and cellular senescence

2016 ◽  
Vol 113 (23) ◽  
pp. E3213-E3220 ◽  
Author(s):  
Jong-Sun Lee ◽  
Zhiguo Zhang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Complex Formation ◽  
Cellular Senescence ◽  
Human Fibroblasts ◽  
Histone Chaperone ◽  
Deficient Mutant ◽  
Nucleosome Assembly ◽  
Cellular Processes ◽  
Glcnac Transferase

The histone chaperone HIRA complex, consisting of histone cell cycle regulator (HIRA), Ubinuclein1 (UBN1), and calcineurin binding protein 1 (CABIN1), deposits histone variant H3.3 to genic regions and regulates gene expression in various cellular processes, including cellular senescence. How HIRA-mediated nucleosome assembly of H3.3–H4 is regulated remains not well understood. Here, we show that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), an enzyme that catalyzes O-GlcNAcylation of serine or threonine residues, interacts with UBN1, modifies HIRA, and promotes nucleosome assembly of H3.3. Depletion of OGT or expression of the HIRA S231A O-GlcNAcylation–deficient mutant compromises formation of the HIRA–H3.3 complex and H3.3 nucleosome assembly. Importantly, OGT depletion or expression of the HIRA S231A mutant delays premature cellular senescence in primary human fibroblasts, whereas overexpression of OGT accelerates senescence. Taken together, these results support a model in which OGT modifies HIRA to regulate HIRA–H3.3 complex formation and H3.3 nucleosome assembly and reveal the mechanism by which OGT functions in cellular senescence.

Applications of Transmission Electron Microscopy in the Characterization of Metal Machinability

1968 ◽  
Vol 26 ◽  
pp. 442-443 ◽  
Author(s):  
L.E. Murr ◽  
A.B. Draper
Keyword(s):  
Electron Microscopy ◽  
State Physics ◽  
Test Material ◽  
Milling Machine ◽  
Rotary Table ◽  
Solid State Physics ◽  
Materials Properties ◽  
Transmission Electron ◽  
Selection Of

The industrial characterization of the machinability of metals and alloys has always been a very arbitrarily defined property, subject to the selection of various reference or test materials; and the adoption of rather naive and misleading interpretations and standards. However, it seems reasonable to assume that with the present state of knowledge of materials properties, and the current theories of solid state physics, more basic guidelines for machinability characterization might be established on the basis of the residual machined microstructures. This approach was originally pursued by Draper; and our presentation here will simply reflect an exposition and extension of this research.The technique consists initially in the production of machined chips of a desired test material on a horizontal milling machine with the workpiece (specimen) mounted on a rotary table vice. A single cut of a specified depth is taken from the workpiece (0.25 in. wide) each at a new tool location.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

Caracterização da Peneira Média em Clones de Coffea canephora

REVISTA FIMCA ◽  
2018 ◽  
Vol 5 (2) ◽  
pp. 28-31
Author(s):  
Darlan Darlan Sanches Barbosa Alves ◽  
Victor Mouzinho Spinelli ◽  
Marcos Santana Moraes ◽  
Carolina Augusto De Souza ◽  
Rodrigo da Silva Ribeiro ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Block Design ◽  
Coffea Canephora ◽  
Coffee Production ◽  
Randomized Block Design ◽  
Significant Difference ◽  
The Mean ◽  
Four Blocks ◽  
Selection Of

Introdução: O estado de Rondônia se destaca como tradicional produtor de café, sendo o segundo maior produtor brasileiro de C. canephora. No melhoramento genético de C. canephora, a seleção de plantas de elevada peneira média está associada à bebida de qualidade superior. Objetivos: O objetivo desse estudo foi avaliar a variabilidade genética de clones de C. canephora para o tamanho dos grãos, mensurado a partir da avaliação da peneira média (PM). Materiais e Métodos: Para isso, foi conduzido ao longo de dois anos agrícolas experimento no campo experimental da Embrapa no município de Ouro Preto do Oeste-RO, para a avaliação da peneira média de 130 genótipos (clones) com características das variedades botânicas Conilon, Robusta e híbridos intervarietais. O delineamento experimental utilizado foi de blocos ao acaso, com quatro repetições de quatro plantas por parcela. Resultados: Não houve resultados significativos para a interação clones X anos, indicando uma maior consistência no comportamento das plantas ao longo do tempo. Porém foram observadas diferenças significativas para o tamanho dos grãos entre os genótipos avaliados, possibilitando selecionar genótipos superiores. Conclusão: Os genótipos agruparam-se em cinco classes de acordo com o teste de média, subsidiando a caracterização de um gradiente de variabilidade da característica avaliada ABSTRACTIntroduction: Coffea canephora accounts for approximately 35% of the world's coffee production. The state of Rondônia stands out as a traditional coffee producer, being the second largest Brazilian producer of C. canephora. In the classical genetic improvement of C. anephora, the selection of plants of high average sieve is associated with a drink of superior quality. Objectives: The objective of this udy was to evaluate the genetic variability of Coffea canephora clones for the agronomic medium sieve (PM). Materials and Methods: The experiment was conducted in the experimental field of Embrapa, municipality of OuroPreto do Oeste-RO, located at coordinates 10º44'53 "S and 62º12'57". One hundred thirty genotypes (clones) of botanical characteristics Conilon, Robusta and intervarietal hybrids were evaluated in the agricultural years 2013-2014 and 2014-2015. The experimental design was a randomized block design with four blocks and four plants per plot, spacing 3.5 x 1.5 meters between plants. Results: Significant difference was found for the grain size. According to the F test, at 5% probability, the genotypes were grouped into five classes according to the mean test. Conclusion: The results obtained subsidized the characterization of a variability gradient of the evaluated trait.

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