scholarly journals Characterization of triosephosphate isomerase mutants with reduced enzyme activity in Mus musculus.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 837-844 ◽  
Author(s):  
S Merkle ◽  
W Pretsch
Keyword(s):  
Enzyme Activity ◽  
Triosephosphate Isomerase ◽  
Hematological Parameters ◽  
Glucose Consumption ◽  
Dominant Mode ◽  
Metabolic Energy ◽  
Wild Type ◽  
Total Lack ◽  
Mutant Lines

Abstract Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.

scholarly journals Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael J Prigge ◽  
Matthieu Platre ◽  
Nikita Kadakia ◽  
Yi Zhang ◽  
Kathleen Greenham ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Early Embryo ◽  
Wild Type ◽  
The Family ◽  
Dependent Inhibition ◽  
Root Gravitropism ◽  
Specialized Function ◽  
Mutant Lines

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

scholarly journals The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism

2007 ◽  
Vol 406 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Mark A. Hooks ◽  
James E. Turner ◽  
Elaine C. Murphy ◽  
Katherine A. Johnston ◽  
Sally Burr ◽  
...  
Keyword(s):  
Glyoxylate Cycle ◽  
Sequence Length ◽  
Acetate Metabolism ◽  
Soluble Carbohydrate ◽  
Wild Type ◽  
Abc Protein ◽  
Atp Binding Cassette Transporter ◽  
Mutant Lines ◽  
Simple Sequence

The Arabidopsis acn (acetate non-utilizing) mutants were isolated by fluoroacetate-resistant germination and seedling establishment. We report the characterization of the acn2 mutant. Physiological analyses of acn2 showed that it possessed characteristics similar to those of the mutants cts (COMATOSE)-1 and pxa [peroxisomal ABC (ATP-binding-cassette) transporter]1. The acn2 locus was mapped to within 3 cM of the CTS gene on the bottom arm of chromosome IV using CAPS (cleavage amplification polymorphism) and SSLP (simple sequence-length polymorphism) markers. Crossing acn2 and cts-1 failed to restore the fluoroacetate-sensitive phenotype, suggesting that these mutations were allelic. Sequencing of the ACN2 locus revealed a C→T nonsense mutation in exon 13, which would have resulted in the elimination of the C-terminal hemitransporter domain of the encoded protein. Neither the full-length CTS protein nor the truncated protein was detected on immunoblots using either C-terminal- or N-terminal-specific anti-CTS antibodies respectively, demonstrating the absence of the entire CTS protein in acn2 mutants. Emerged seedlings of both cts-1 and pxa1 alleles displayed increased resistance to FAc (monofluoroacetic acid) compared with the corresponding wild-type seedlings. Complementation studies showed that mutation of the CTS gene was responsible for the FAc-resistant phenotype, as when the wild-type protein was expressed in both the cts-1 and pxa1 mutant lines, the strains became FAc-sensitive. Feeding studies confirmed that both acn2 and cts-1 mutants were compromised in their ability to convert radiolabelled acetate into soluble carbohydrate. These results demonstrate a role for the ABC protein CTS in providing acetate to the glyoxylate cycle in developing seedlings.

scholarly journals Triosephosphate isomerase activity-deficient mice show haemolytic anaemia in homozygous condition

2009 ◽  
Vol 91 (1) ◽  
pp. 1-4 ◽  
Author(s):  
WALTER PRETSCH
Keyword(s):  
Enzyme Activity ◽  
Triosephosphate Isomerase ◽  
Haemoglobin Concentration ◽  
Spleen Weight ◽  
Functional Domain ◽  
Residual Enzyme Activity ◽  
Wild Type ◽  
Exon 2 ◽  
Isomerase Activity ◽  
Tissue Extracts

SummaryA triosephosphate isomerase (TPI) mutant, Tpi1a-m6Neu, with approximately 57% residual enzyme activity in blood compared with wild-type was detected among offspring of triethylenemelamine-treated male mice. Homozygous mutants with about 13% residual enzyme activity were recovered in progeny of inter se matings of heterozygotes. The loss of TPI activity was evident both in blood and in other tissue extracts. Values for haematocrit, haemoglobin, number of red blood cells (RBC), mean corpuscular volume of RBC, mean corpuscular haemoglobin concentration and spleen weight show significant differences between wild-type animals and homozygous mutants. Sequence analysis revealed a substitution (c.A149G) in the Tpi1 gene. This mutation results in an Asp to Gly substitution at codon 49 in exon 2 at a highly conserved position located in the functional domain of the TPI protein which is responsible for the correct dimerization of the subunits. As a potential animal model, Tpi1a-m6Neu represents the only available TPI-deficient homozygous viable mouse mutation.

scholarly journals Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

2022 ◽  
Vol 82 ◽  
Author(s):  
Fernanda Ghenov ◽  
Edileusa Cristina Marques Gerhardt ◽  
Luciano Fernandes Huergo ◽  
Fabio Oliveira Pedrosa ◽  
Roseli Wassem ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Glutamine Synthetase ◽  
Affinity Chromatography ◽  
Azospirillum Brasilense ◽  
Nitrogen Assimilation ◽  
Atp Hydrolysis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
E Coli

Abstract Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

scholarly journals Spontaneous Root-Nodule Formation in the Model Legume Lotus japonicus: A Novel Class of Mutants Nodulates in the Absence of Rhizobia

2006 ◽  
Vol 19 (4) ◽  
pp. 373-382 ◽  
Author(s):  
Leïla Tirichine ◽  
Euan K. James ◽  
Niels Sandal ◽  
Jens Stougaard
Keyword(s):  
Root Nodule ◽  
Lotus Japonicus ◽  
Nodule Development ◽  
Nodule Formation ◽  
Wild Type ◽  
Model Legume ◽  
Isolation And Characterization ◽  
Mutant Lines ◽  
Spontaneous Nodules

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.

scholarly journals Genetic Analysis of Organ Fusion in Arabidopsis thaliana

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 607-619
Author(s):  
Susan J Lolle ◽  
Wendy Hsu ◽  
Robert E Pruitt
Keyword(s):  
Reproductive Development ◽  
Floral Organ ◽  
Growth Responses ◽  
Wild Type ◽  
Multiple Alleles ◽  
Aerial Parts ◽  
Complementation Groups ◽  
Pollen Growth ◽  
Mutant Lines

Abstract Postgenital organ fusion occurs most commonly during reproductive development and is important in many angiosperms during genesis of the carpel. Although a number of mutants have been described that manifest ectopic organ fusion, little is known about the genes involved in regulating this process. In this article we describe the characterization of a collection of 29 Arabidopsis mutants showing an organ fusion phenotype. Mapping and complementation analyses revealed that the mutant alleles define nine different loci distributed throughout the Arabidopsis genome. Multiple alleles were isolated for the four complementation groups showing the strongest organ fusion phenotype while the remaining five complementation groups, all of which show only weak floral organ fusion, have a single representative allele. In addition to fusion events between aerial parts of the shoot, some mutants also show abnormal ovule morphology with adjacent ovules joined together at maturity. Many of the fusion mutants isolated have detectable differences in the rate at which chlorophyll can be extracted; however, in one case no difference could be detected between mutant and wild-type plants. In three mutant lines pollen remained unresponsive to contact with the mutant epidermis, demonstrating that organ fusion and pollen growth responses can be genetically separated from one another.

scholarly journals Characterization of wheat lacking B-type starch granules

2021 ◽  
Author(s):  
Benedetta Saccomanno ◽  
Pierre Berbezy ◽  
Kim Findlay ◽  
Jennifer Shoesmith ◽  
Cristobal Uauy ◽  
...  
Keyword(s):  
Starch Granule ◽  
Starch Content ◽  
Starch Granules ◽  
Wild Type ◽  
Bread Making ◽  
Wheat Varieties ◽  
Alcohol Production ◽  
Mutant Lines ◽  
B Granules

ABSTRACTThe physicochemical and agronomical properties of a new form of bread wheat, lacking B-type starch granules (BlessT) was assessed. Three BlessT mutant lines, made by combining homoeologous deletions of BGC1, a gene responsible for the control of B-granule content were compared with two sibling lines with normal starch phenotype and the parent line, cv. Paragon. Quantification of starch granule size and number in developing grain confirmed the lack of small, B-type starch granules throughout development in BlessT. Most starch, flour, grain and loaf characteristics did not vary between BlessT and the wild type sibling controls. However, BlessT starches had higher water absorption, reduced grain hardness and higher protein content, and dough made from BlessT flour required more water and had increased elasticity. Despite the lack of B-granules, BlessT lines do not display a significant decrease in total starch content suggesting that it should be possible to produce commercial wheat varieties that lack B-type starch granules without compromising yield. These findings support the potential utility of this novel type of wheat as a specialist crop in applications ranging from bread making and alcohol production to improved industrial starch products.

scholarly journals Functional characterization of the chlorzoxazone 6-hydroxylation activity of human cytochrome P450 2E1 allelic variants in Han Chinese

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9628
Author(s):  
Ting Wang ◽  
Huihui Du ◽  
Jingsong Ma ◽  
Lu Shen ◽  
Muyun Wei ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Genetic Polymorphisms ◽  
Han Chinese ◽  
Enzyme Function ◽  
Wild Type ◽  
Coding Region ◽  
Allelic Variants ◽  
Hydroxylation Activity

Backgrounds Cytochrome P450 (P450) 2E1 is one of the primary enzymes responsible for the metabolism of xenobiotics, such as drugs and environmental carcinogens. The genetic polymorphisms of the CYP2E1 gene in promoter and coding regions have been identified previously in the Han Chinese population from four different geographic areas of Mainland China. Methods To investigate whether genetic variants identified in the CYP2E1 coding region affect enzyme function, the enzymes of four single nucleotide polymorphism (SNP) variants in the coding region (novel c.1009C>T, causing p.Arg337X, where X represents the translational stop codon; c.227G>A, causing p.Arg76His; c.517G>A, yielding p.Gly173Ser; and c.1263C>T, presenting the highest allele frequency), two novel alleles (c.[227G>A;1263C>T] and c.[517G>A;1263C>T]), and the wild-type CYP2E1 were heterologously expressed in COS-7 cells and functionally characterized in terms of expression level and chlorzoxazone 6-hydroxylation activity. The impact of the CYP2E1 variant sequence on enzyme activity was predicted with three programs: Polyphen 2, PROVEAN and SIFT. Results The prematurely terminated p.Arg337X variant enzyme was undetectable by western blotting and inactive toward chlorzoxazone 6-hydroxylation. The c.1263C>T and c.[517G>A;1263C>T] variant enzymes exhibited properties similar to those of the wild-type CYP2E1. The CYP2E1 variants c.227G>A and c.[227G>A;1263C>T] displayed significantly reduced enzyme activity relative to that of the wild-type enzyme (decreased by 42.8% and 32.8%, respectively; P < 0.01). The chlorzoxazone 6-hydroxylation activity of the c.517G>A transfectant was increased by 31% compared with the wild-type CYP2E1 enzyme (P < 0.01). Positive correlations were observed between the protein content and enzyme activity for CYP2E1 (P = 0.0005, r2 = 0.8833). The characterization of enzyme function allelic variants in vitro was consistent with the potentially deleterious effect of the amino acid changes as determined by prediction tools. Conclusions These findings indicate that the genetic polymorphisms of CYP2E1, i.e., c.1009C>T (p.Arg337X), c.227G>A (p.Arg76His), and c.517G>A (p.Gly173Ser), could influence the metabolism of CYP2E1 substrates, such as chlorzoxazone.

Identification of root morphology mutants in barley

2011 ◽  
Vol 9 (2) ◽  
pp. 357-360 ◽  
Author(s):  
Riccardo Bovina ◽  
Valentina Talamè ◽  
Matteo Ferri ◽  
Roberto Tuberosa ◽  
Beata Chmielewska ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Root Morphology ◽  
Forward Genetics ◽  
Seedling Stage ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Mutant Lines ◽  
Scanning Electron

In this study, a forward-genetics analysis was performed on a portion of TILLMore, a chemically mutagenized population of barley cv. ‘Morex’ (http://www.distagenomics.unibo.it/TILLMore/), to identify root morphology alterations by comparison with ‘Morex’ wild-type. For this purpose, a simple paper-roll approach was performed to identify phenotypic variants at the seedling stage. The analysis of c. 1000 M4 families allowed us to identify c. 70 lines with altered root morphology. A more accurate phenotypic characterization of a portion of the mutant lines has been performed using stereomicroscopy and a scanning electron microscopy approach.

Characterization of genes involved in erythritol catabolism in Rhizobium leguminosarum bv. viciae

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2061-2074 ◽  
Author(s):  
Christopher K. Yost ◽  
Amber M. Rath ◽  
Tanya C. Noel ◽  
Michael F. Hynes
Keyword(s):  
Rhizobium Leguminosarum ◽  
Triosephosphate Isomerase ◽  
Genetic Locus ◽  
High Homology ◽  
Wild Type ◽  
High Sequence Identity ◽  
Catabolic Genes ◽  
Wide Range ◽  
Genomic Dna Sequence

A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. A Tn5-B22 mutant (19B-3) unable to grow on erythritol was isolated from a mutant library of R. leguminosarum strain VF39SM. The mutated gene eryF was cloned and partially sequenced, and determined to have a high homology to permease genes of ABC transporters. A cosmid complementing the mutation (pCos42) was identified and was shown to carry all the genes necessary to restore the ability to grow on erythritol to a VF39SM strain cured of pRleVF39f. In the genomic DNA sequence of strain 3841, the gene linked to the mutation in 19B-3 is flanked by a cluster of genes with high homology to the known erythritol catabolic genes from Brucella spp. Through mutagenesis studies, three distinct operons on pCos42 that are required for growth on erythritol were identified: an ABC-transporter operon (eryEFG), a catabolic operon (eryABCD) and an operon (deoR-tpiA2-rpiB) that encodes a gene with significant homology to triosephosphate isomerase (tpiA2). These genes all share high sequence identity to genes in the erythritol catabolism region of Brucella spp., and clustalw alignments suggest that horizontal transfer of the erythritol locus may have occurred between R. leguminosarum and Brucella. Transcription of the eryABCD operon is repressed by EryD and is induced by the presence of erythritol. Mutant 19B-3 was impaired in its ability to compete against wild-type for nodulation of pea plants but was still capable of forming nitrogen-fixing nodules.

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