Cotranscription of the wild-type chloroplast atpE gene encoding the CF1/CF0 epsilon subunit with the 3′ half of the rps7 gene in Chlamydomonas reinhardtii and characterization of frameshift mutations in atpE

1990 ◽  
Vol 221 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Dominique Robertson ◽  
John E. Boynton ◽  
Nicholas W. Gillham
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Wild Type ◽  
Gene Encoding ◽  
Frameshift Mutations ◽  
Epsilon Subunit

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

2002 ◽  
Vol 205 (9) ◽  
pp. 1209-1219 ◽  
Author(s):  
Natalie Perzov ◽  
Vered Padler-Karavani ◽  
Hannah Nelson ◽  
Nathan Nelson
Keyword(s):  
Sucrose Gradient ◽  
Ph Sensor ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Immunological Assays ◽  
Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

scholarly journals Biochemical Characterization of Wild-Type and Mutant Isoamylases of Chlamydomonas reinhardtii Supports a Function of the Multimeric Enzyme Organization in Amylopectin Maturation

2001 ◽  
Vol 125 (4) ◽  
pp. 1723-1731 ◽  
Author(s):  
David Dauvillée ◽  
Christophe Colleoni ◽  
Gregory Mouille ◽  
Matthew K. Morell ◽  
Christophe d'Hulst ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Biochemical Characterization ◽  
Wild Type ◽  
Enzyme Organization

Characterization of mutant alleles of myospheroid, the gene encoding the beta subunit of the Drosophila PS integrins.

Genetics ◽  
1992 ◽  
Vol 132 (2) ◽  
pp. 519-528 ◽  
Author(s):  
T A Bunch ◽  
R Salatino ◽  
M C Engelsgjerd ◽  
L Mukai ◽  
R F West ◽  
...  
Keyword(s):  
Chromosome Pairing ◽  
Protein Product ◽  
Beta Subunit ◽  
Wild Type ◽  
Muscle Attachment ◽  
Negative Interaction ◽  
Gene Encoding ◽  
Attachment Sites ◽  
Muscle Attachment Sites

Abstract This paper presents the characterization of nine alleles of myospheroid, which encodes the beta PS subunit of the Drosophila PS integrins. On Southern blots, the mysXB87, mysXN101 and mysXR04 genes yield restriction digest patterns similar to that seen for wild-type chromosomes, however the mys1 and mysXG43 genes contain detectable deletions. mys1, mysXB87 and mysXG43 make little or no stable protein product, and genetically behave as strong lethal alleles. For the mysXN101 mutation, protein product is seen on immunoblots and a reduced amount of beta PS protein is seen at muscle attachment sites of embryos; this mutant protein retains some wild-type function, as revealed by complementation tests with weak alleles. Protein is also seen on immunoblots from mysXR04 embryos, and this allele behaves as an antimorph, being more deleterious in some crosses than the complete deficiency for the locus. mysts2 and mysnj42 are typically lethal in various combinations with other alleles at high temperatures only, but even at high physiological temperatures, neither appears to eliminate gene function completely. The complementation behaviors of mysts1 and mysts3 are quite unusual and suggest that these mutations involve regulatory phenomena. For mysts3, the data are most easily explained by postulating transvection effects at the locus. The results for mysts1 are less straightforward, but point to the possibility of a chromosome pairing-dependent negative interaction.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

scholarly journals Characterization of the norB Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium Synechocystis sp. Strain PCC6803

2002 ◽  
Vol 68 (2) ◽  
pp. 668-672 ◽  
Author(s):  
Andrea B�sch ◽  
B�rbel Friedrich ◽  
Rainer Cramm
Keyword(s):  
Nitric Oxide ◽  
Reductase Activity ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Subunit ◽  
Transcriptional Fusions ◽  
Synechocystis Sp ◽  
Negative Mutant

ABSTRACT A norB gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier Ralstonia eutropha. Heterologous complementation of a nitric oxide reductase-negative mutant of R. eutropha with norB from Synechocystis restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of Synechocystis wild-type cells. Virtually no nitric oxide reductase activity was present in a norB-negative mutant of Synechocystis. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for Synechocystis when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor norB expression in Synechocystis. Transcription of norB was not enhanced by the addition of the NO-generating agent sodium nitroprusside.

scholarly journals Isolation and Characterization of Burkholderia cenocepacia Mutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability

2004 ◽  
Vol 186 (2) ◽  
pp. 270-277 ◽  
Author(s):  
Kate L. Farmer ◽  
Mark S. Thomas
Keyword(s):  
Wild Type Strain ◽  
Opportunistic Pathogen ◽  
Burkholderia Cenocepacia ◽  
Wild Type ◽  
Iron Starvation ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Putative Operon ◽  
Green Fluorescent

ABSTRACT The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

scholarly journals Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1483
Author(s):  
Jun Morishita ◽  
Ryutaro Tokutsu ◽  
Jun Minagawa ◽  
Toru Hisabori ◽  
Ken-ichi Wakabayashi
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Regulatory Mechanism ◽  
Model Organism ◽  
Negative Phototaxis ◽  
Light Conditions ◽  
Wild Type ◽  
Positive Phototaxis ◽  
Unicellular Green Alga ◽  
Cellular Factors

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

scholarly journals Cytochrome cM downscales photosynthesis under photomixotrophy in Synechocystis sp. PCC 6803

2019 ◽  
Author(s):  
Daniel Solymosi ◽  
Dorota Muth-Pawlak ◽  
Lauri Nikkanen ◽  
Duncan Fitzpatrick ◽  
Ravendran Vasudevan ◽  
...  
Keyword(s):  
Photosynthetic Apparatus ◽  
Wild Type ◽  
Gene Encoding ◽  
Transport Chain ◽  
Similar Phenotype ◽  
Photosynthetic Microorganisms ◽  
Photosynthesis And Respiration ◽  
Pcc 6803

AbstractPhotomixotrophy is a metabolic state, which enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we show via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual reduction of the plastoquinone pool in wild-type Synechocystis, which fully downscales photosynthesis over three days of growth. This process is circumvented by deleting the gene encoding cytochrome cM (CytM), a cryptic c-type heme protein widespread in cyanobacteria. ΔCytM maintained active photosynthesis over the three day period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of Photosystem II and Photosystem I. Overall, this resulted in a higher growth rate than wild-type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a similar phenotype under photomixotrophy to ΔCytM, demonstrating that CytM is not transferring electrons to these complexes, which has previously been suggested. In summary, the obtained data suggests that CytM may have a regulatory role in photomixotrophy by reducing the photosynthetic capacity of cells.One sentence summaryThe cryptic, highly conserved cytochrome cM completely blocks photosynthesis in Synechocystis under three days of photomixotrophy, possibly by suppressing CO2 assimilation.

Characterization of a norflurazon-resistant mutant of Chlamydomonas reinhardtii

Weed Science ◽  
1997 ◽  
Vol 45 (3) ◽  
pp. 374-377 ◽  
Author(s):  
Varsha Vartak ◽  
Sujata Bhargava
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Mode Of Action ◽  
Resistant Mutant ◽  
Phytoene Desaturase ◽  
Wild Type ◽  
Cross Tolerance ◽  
In The Wild ◽  
Similar Mode ◽  
Target Enzyme

A norflurazon-resistant mutant has been isolated from Chlamydomonas reinhardtii that showed a three-fold factor of resistance over wild type cultures. In comparison to wild type cultures, the mutant showed better retention of chlorophylls and carotenoids when grown in light in the presence of norflurazon. When grown in the dark, chlorophyll losses were similar, while carotenoid losses were lower than in the wild type cultures. Higher levels of phytoene accumulated in the wild type cultures in the presence of norflurazon than in the resistant cultures. The resistant cultures also showed cross tolerance to EMD-IT 5914, a herbicide with a similar mode of action. Norflurazon resistance in this alga appears to arise from alterations in the target enzyme phytoene desaturase.

Sign in / Sign up

Export Citation Format

Share Document