scholarly journals Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Mareike Busche ◽  
Christopher Acatay ◽  
Stefan Martens ◽  
Bernd Weisshaar ◽  
Ralf Stracke
Keyword(s):  
Recombinant Proteins ◽  
Nutritional Value ◽  
Flavonoid Biosynthesis ◽  
Loss Of Function ◽  
Structural Genes ◽  
In Planta ◽  
Anthocyanidin Synthase ◽  
Functional Characterisation ◽  
Genes Encoding

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

scholarly journals Functional characterisation of banana (Musa spp.) 2-oxoglutarate-dependent dioxygenases involved in flavonoid biosynthesis

2021 ◽  
Author(s):  
Mareike Busche ◽  
Christopher Acatay ◽  
Bernd Weisshaar ◽  
Ralf Stracke
Keyword(s):  
Recombinant Proteins ◽  
Nutritional Value ◽  
Flavonoid Biosynthesis ◽  
Loss Of Function ◽  
Structural Genes ◽  
In Planta ◽  
Anthocyanidin Synthase ◽  
Functional Characterisation ◽  
Genes Encoding

Bananas (Musa) are monocotyledonous, perennial plants that are well-known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3 hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

scholarly journals Generation of strong casein kinase 1 inhibitor of Arabidopsis thaliana

2019 ◽  
Author(s):  
Ami N Saito ◽  
Hiromi Matsuo ◽  
Keiko Kuwata ◽  
Azusa Ono ◽  
Toshinori Kinoshita ◽  
...  
Keyword(s):  
Bromine Atom ◽  
Casein Kinase ◽  
In Planta ◽  
Vitro Assay ◽  
Casein Kinase 1 ◽  
Yeast Fungi ◽  
Genes Encoding ◽  
Period Lengthening

AbstractCasein kinase 1 (CK1) is an evolutionarily conserved protein kinase among eukaryotes. Studies on yeast, fungi, and animals have revealed that CK1 plays roles in divergent biological processes. By contrast, the collective knowledge regarding the biological roles of plant CK1 lags was behind those of animal CK1. One of reasons for this is that plants have more multiple genes encoding CK1 than animals. To accelerate the research for plant CK1, a strong CK1 inhibitor that efficiently inhibits multiple members of CK1 proteins in vivo (in planta) is required. Here, we report a novel strong CK1 inhibitor of Arabidopsis (AMI-331). Using a circadian period-lengthening activity as estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship (SAR) study of PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride), a potent CK1 inhibitor of Arabidopsis, and found that PHA767491 analogues bearing a propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) enhance the period-lengthening activity. The period lengthening activity of a hybrid molecule of AMI-212 and AMI-23 (AMI-331) is about 100-fold stronger than that of PHA767491. An in vitro assay indicated a strong inhibitory activity of CK1 kinase by AMI-331. Also, affinity proteomics using an AMI-331 probe showed that targets of AMI-331 are mostly CK1 proteins. As such, AMI-331 is a strong potent CK1 inhibitor that shows promise in the research of CK1 in plants.

Two chloroplastic PLGG1 isoforms function together to transport photorespiratory glycolate and glycerate in rice

2021 ◽  
Author(s):  
Lili Cui ◽  
Chuanling Zhang ◽  
Zhichao Li ◽  
Tuxiu Xian ◽  
Limin Wang ◽  
...  
Keyword(s):  
Rice Genome ◽  
Direct Interaction ◽  
Crop Productivity ◽  
Chloroplast Envelope ◽  
Starch Accumulation ◽  
Loss Of Function ◽  
In Planta ◽  
In Vivo Experiments

Abstract The photorespiratory pathway is highly compartmentalized. As such, metabolite shuttles between organelles are critical to ensure efficient photorespiratory carbon flux. Arabidopsis PLGG1 has been reported as a key chloroplastic glycolate/glycerate transporter. Two homologous genes OsPLGG1a and OsPLGG1b have been identified in the rice genome, although their distinct functions and relationships remain unknown. Herein, our analysis of exogenous expression in oocytes and yeast shows that both OsPLGG1a and OsPLGG1b have the ability to transport glycolate and glycerate. Furthermore, we demonstrate in planta, that the perturbation of OsPLGG1a or OsPLGG1b expression leads to extensive accumulation of photorespiratory metabolites, especially glycolate and glycerate. Under ambient CO2 conditions, loss-of-function osplgg1a or osplgg1b mutant plants exhibited significant decreases in photosynthesis efficiency, starch accumulation, plant height, and crop productivity. These morphological defects were almost entirely recovered when the mutant plants were grown under elevated CO2 conditions instead. In contrast to osplgg1a, osplgg1b mutant alleles produced a mild photorespiratory phenotype and had reduced accumulation of photorespiratory metabolites. Subcellular localization analysis showed that OsPLGG1a and OsPLGG1b are located in the inner and outer membranes of the chloroplast envelope, respectively. In vitro and in vivo experiments revealed that OsPLGG1a and OsPLGG1b have a direct interaction. Our results indicate that both OsPLGG1a and OsPLGG1b are chloroplastic glycolate/glycerate transporters required for photorespiratory metabolism and plant growth, and that they may function as a singular complex.

scholarly journals mRNAs Encoding Telomerase Components and Regulators Are Controlled by UPF Genes in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Jeffrey N. Dahlseid ◽  
Jodi Lew-Smith ◽  
Michael J. Lelivelt ◽  
Shinichiro Enomoto ◽  
Amanda Ford ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Dna Sequences ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Telomeric Silencing ◽  
Expression Of Genes ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

scholarly journals Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases

2021 ◽  
Vol 12 ◽  
Author(s):  
Hanna Marie Schilbert ◽  
Maximilian Schöne ◽  
Thomas Baier ◽  
Mareike Busche ◽  
Prisca Viehöver ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Gene Family ◽  
Expression Profiles ◽  
Reproductive Organs ◽  
In Planta ◽  
Flavonol Synthase ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Spatio Temporal

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

scholarly journals Tuba1a is uniquely important for axon guidance through midline commissural structures

2020 ◽  
Author(s):  
Georgia Buscaglia ◽  
Jayne Aiken ◽  
Katelyn J. Hoff ◽  
Kyle R. Northington ◽  
Emily A. Bates
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Intracellular Transport ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Loss Of Function ◽  
Developmental Guidance ◽  
Genes Encoding ◽  
Developing Neurons

AbstractDeveloping neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

scholarly journals Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

2000 ◽  
Vol 20 (14) ◽  
pp. 5129-5139 ◽  
Author(s):  
Feng-Qian Li ◽  
Archie Coonrod ◽  
Marshall Horwitz
Keyword(s):  
Myogenic Differentiation ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Myoblast Differentiation ◽  
Loss Of Function ◽  
Lysosomal Protease ◽  
Dominant Negative Form ◽  
Genes Encoding

ABSTRACT Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.

scholarly journals Characterization of the Brassica napus flavonol synthase gene family reveals bifunctional flavonol synthases

2021 ◽  
Author(s):  
Hanna Marie Schilbert ◽  
Maximilian Schoene ◽  
Thomas Baier ◽  
Mareike Busche ◽  
Prisca Viehoever ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Gene Family ◽  
Expression Profiles ◽  
Reproductive Organs ◽  
In Planta ◽  
Flavonol Synthase ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Spatio Temporal

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyses the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent-dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homoelogs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3 hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

scholarly journals Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains

1970 ◽  
Vol 2 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Santosh Dulal ◽  
Bhupal Ban ◽  
Gi Hyoek Yang ◽  
Hyun Ho Jung
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Clostridium Botulinum ◽  
Pathogen Detection ◽  
Recombinant Protein Expression ◽  
Synthetic Genes ◽  
Effective System ◽  
Genes Encoding

The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy. Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity DOI: http://dx.doi.org/10.3126/njb.v2i1.5634 Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 1-15

scholarly journals Dolichyl-Phosphate-Glucose Is Used To Make O-Glycans on Glycoproteins of Trichomonas vaginalis

2008 ◽  
Vol 7 (8) ◽  
pp. 1344-1351 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Sudip K. Ghosh ◽  
Ziqiang Guan ◽  
Jike Cui ◽  
Christian R. H. Raetz ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Trichomonas Vaginalis ◽  
Gene Products ◽  
Gene Duplications ◽  
Gene Encoding ◽  
Dolichyl Phosphate ◽  
Genes Encoding ◽  
Carboxypeptidase N ◽  
Glycosylation Defect

ABSTRACT Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.

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