scholarly journals Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta

2019 ◽  
Author(s):  
Hayato Suzuki ◽  
Ery Odette Fukushima ◽  
Yuko Shimizu ◽  
Hikaru Seki ◽  
Yukiko Fujisawa ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Lotus Japonicus ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Loss Of Function ◽  
In Planta ◽  
Metabolic Intermediate ◽  
Oxidation Activity ◽  
Model Legume

Abstract Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

scholarly journals A Novel Cyclic AMP-Dependent Epac-Rit Signaling Pathway Contributes to PACAP38-Mediated Neuronal Differentiation

2006 ◽  
Vol 26 (23) ◽  
pp. 9136-9147 ◽  
Author(s):  
Geng-Xian Shi ◽  
Holger Rehmann ◽  
Douglas A. Andres
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Neuronal Differentiation ◽  
Function Analysis ◽  
Small Gtpase ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Stimulation Of

ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of Gsα protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of Gsα/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.

Chemically inducible gene expression in seeds before testa rupture

2015 ◽  
Vol 25 (3) ◽  
pp. 345-352 ◽  
Author(s):  
Mariko Nonogaki ◽  
Taira Sekine ◽  
Hiroyuki Nonogaki
Keyword(s):  
Gene Expression ◽  
Function Analysis ◽  
Expression System ◽  
Basic Research ◽  
Inducible Expression ◽  
Upstream Sequence ◽  
Dependent Manner ◽  
Inducible Gene Expression ◽  
Inducible Expression System ◽  
Inducible Gene

AbstractImpermeability of the testa hinders efficient penetration of some small chemicals, such as transcriptional inhibitors, through the endosperm and the embryo during seed experiments. InArabidopsisseeds, 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid, a substrate for β-glucuronidase, did not permeate through the endosperm and embryo efficiently at the stages before testa rupture. TheArabidopsistesta also limited efficient entry of methoxyfenozide, a chemical ligand that was used for inducible gene expression experiments, into seeds. While the detection of a reporter gene at the early imbibitional stages could be replaced by reverse transcription-polymerase chain reaction (RT-PCR), the interference of entry of the chemical ligand into seeds by the testa was still problematic to gene induction experiments. To develop an efficient inducible expression system for gene function analysis in seeds, an inducible expression system with nitrate, which is a testa-permeable ligand, was examined. The vector containing the 2.1-kb upstream sequence ofNITRITE REDUCTASE 1was able to cause expression of a test gene (long non-coding RNA) in imbibed seeds at the stage before testa rupture in a nitrate-dependent manner. This system can be used not only for characterization of genes associated with seed dormancy and germination in basic research, but also for the development of germination recovery or enhancement technologies for agricultural applications.

scholarly journals Gene Silencing by Expression of Hairpin RNA in Lotus japonicus Roots and Root Nodules

2003 ◽  
Vol 16 (8) ◽  
pp. 663-668 ◽  
Author(s):  
Hirotaka Kumagai ◽  
Hiroshi Kouchi
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Rna Silencing ◽  
Root Nodules ◽  
Lotus Japonicus ◽  
Loss Of Function ◽  
Coding Region ◽  
Hairpin Rna ◽  
Model Legume ◽  
Root Transformation

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express β-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

scholarly journals Comparison of the Transcript Profiles from the Root and the Nodulating Root of the Model Legume Lotus japonicus by Serial Analysis of Gene Expression

2005 ◽  
Vol 18 (5) ◽  
pp. 487-498 ◽  
Author(s):  
Erika Asamizu ◽  
Yasukazu Nakamura ◽  
Shusei Sato ◽  
Satoshi Tabata
Keyword(s):  
Gene Expression ◽  
Expressed Sequence Tag ◽  
Early Stage ◽  
Regulation Of Gene Expression ◽  
Lotus Japonicus ◽  
Dependent Manner ◽  
Transcript Analysis ◽  
Model Legume ◽  
Root Nodulation ◽  
Transcript Profiles

We performed a comprehensive transcript analysis on the early stage of root nodulation in the model legume Lotus japonicus by serial analysis of gene expression (SAGE). SAGE libraries were made from uninfected roots and nodulating roots abundant in nodule primordia, and 85,482 and 80,233 SAGE tags were recovered, respectively. Comparison of the tag frequency identified 407 tag species that appeared in significantly greater numbers in the nodulating root than in the uninfected root, and the converse was found for 428 tag species. Gene identification of the tags was performed by matching them to L. japonicus expressed sequence tag sequences. We made several novel findings by applying SAGE to transcript analysis of legume root nodulation. A gene that showed the most significant increase in tag number upon nodulation has not been described previously. Different levels of transcription induction among leghemoglobin gene paralogs were found, indicating the effectiveness of SAGE in discriminating different gene family members. We identified genes for 44 unknown tags by means of reverse SAGE. We found 11 antisense tags that increased during nodulation, indicating that regulation of gene expression by antisense transcripts may occur in an organ-dependent manner.

An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL–driven leukemogenesis

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2200-2210 ◽  
Author(s):  
Corinna Albers ◽  
Anna L. Illert ◽  
Cornelius Miething ◽  
Hannes Leischner ◽  
Melanie Thiede ◽  
...  
Keyword(s):  
Drug Targets ◽  
Function Analysis ◽  
Expression System ◽  
Hematopoietic Cells ◽  
Mouse Strains ◽  
Loss Of Function ◽  
Tumor Models ◽  
Murine Tumor

Abstract Genetic loss-of-function studies in murine tumor models have been essential in the analysis of downstream mediators of oncogenic transformation. Unfortunately, these studies are frequently limited by the availability of genetically modified mouse strains. Here we describe a versatile method allowing the efficient expression of an oncogene and simultaneous knockdown of targets of interest (TOI) from a single retroviral vector. Both oncogene and TOI-specific miR30-based shRNA are under the control of the strong viral long terminal repeat promoter, resulting in a single shared RNA transcript. Using this vector in a murine syngeneic BM transplantation model for BCR-ABL–induced chronic myeloid leukemia, we find that oncogene expression and target knockdown in primary hematopoietic cells with this vector is efficient both in vitro and in vivo, and demonstrate that Raf1, but not BRAF, modulates BCR-ABL–dependent ERK activation and transformation of hematopoietic cells. This expression system could facilitate genetic loss-of-function studies and allow the rapid validation of potential drug targets in a broad range of oncogene-driven murine tumor models.

scholarly journals UGT84F9 is the major flavonoid UDP-glucuronosyltransferase in Medicago truncatula

2021 ◽  
Author(s):  
Olubu A Adiji ◽  
Maite L Docampo-Palacios ◽  
Anislay Alvarez-Hernandez ◽  
Giulio M Pasinetti ◽  
Xiaoqiang Wang ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Phase Ii ◽  
Loss Of Function ◽  
In Planta ◽  
Model Legume ◽  
Flavonoid Compounds ◽  
Wide Range ◽  
Udp Glucuronosyltransferase ◽  
Phase Ii Metabolites

Abstract Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

A Cuticle Collagen Encoded by the lon-3 Gene May Be a Target of TGF-β Signaling in Determining Caenorhabditis elegans Body Shape

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1631-1639
Author(s):  
Yo Suzuki ◽  
Gail A Morris ◽  
Min Han ◽  
William B Wood
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Shape ◽  
Small Body ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Cellular Mechanisms ◽  
C Elegans ◽  
Gene Expression Analyses ◽  
Dose Dependent

Abstract The signaling pathway initiated by the TGF-β family member DBL-1 in Caenorhabditis elegans controls body shape in a dose-dependent manner. Loss-of-function (lf) mutations in the dbl-1 gene cause a short, small body (Sma phenotype), whereas overexpression of dbl-1 causes a long body (Lon phenotype). To understand the cellular mechanisms underlying these phenotypes, we have isolated suppressors of the Sma phenotype resulting from a dbl-1(lf) mutation. Two of these suppressors are mutations in the lon-3 gene, of which four additional alleles are known. We show that lon-3 encodes a collagen that is a component of the C. elegans cuticle. Genetic and reporter-gene expression analyses suggest that lon-3 is involved in determination of body shape and is post-transcriptionally regulated by the dbl-1 pathway. These results support the possibility that TGF-β signaling controls C. elegans body shape by regulating cuticle composition.

scholarly journals The Parkinson’s Disease-Associated Protein DJ-1 Protects Dictyostelium Cells from AMPK-Dependent Outcomes of Oxidative Stress

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1874
Author(s):  
Suwei Chen ◽  
Sarah J. Annesley ◽  
Rasha A. F. Jasim ◽  
Paul R. Fisher
Keyword(s):  
Oxidative Stress ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Respiration ◽  
Cysteine Residue ◽  
Reduced Form ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Cellular Pathology

Mitochondrial dysfunction has been implicated in the pathology of Parkinson’s disease (PD). In Dictyostelium discoideum, strains with mitochondrial dysfunction present consistent, AMPK-dependent phenotypes. This provides an opportunity to investigate if the loss of function of specific PD-associated genes produces cellular pathology by causing mitochondrial dysfunction with AMPK-mediated consequences. DJ-1 is a PD-associated, cytosolic protein with a conserved oxidizable cysteine residue that is important for the protein’s ability to protect cells from the pathological consequences of oxidative stress. Dictyostelium DJ-1 (encoded by the gene deeJ) is located in the cytosol from where it indirectly inhibits mitochondrial respiration and also exerts a positive, nonmitochondrial role in endocytosis (particularly phagocytosis). Its loss in unstressed cells impairs endocytosis and causes correspondingly slower growth, while also stimulating mitochondrial respiration. We report here that oxidative stress in Dictyostelium cells inhibits mitochondrial respiration and impairs phagocytosis in an AMPK-dependent manner. This adds to the separate impairment of phagocytosis caused by DJ-1 knockdown. Oxidative stress also combines with DJ-1 loss in an AMPK-dependent manner to impair or exacerbate defects in phototaxis, morphogenesis and growth. It thereby phenocopies mitochondrial dysfunction. These results support a model in which the oxidized but not the reduced form of DJ-1 inhibits AMPK in the cytosol, thereby protecting cells from the adverse consequences of oxidative stress, mitochondrial dysfunction and the resulting AMPK hyperactivity.

scholarly journals H3K27 modifiers regulate lifespan in C. elegans in a context-dependent manner

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Abigail R. R. Guillermo ◽  
Karolina Chocian ◽  
Gavriil Gavriilidis ◽  
Julien Vandamme ◽  
Anna Elisabetta Salcini ◽  
...  
Keyword(s):  
Lifespan Extension ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Animal Cells ◽  
C Elegans ◽  
Specific Effects ◽  
Aberrant Gene Expression ◽  
Lysine Methyltransferase ◽  
Epigenetic Signatures ◽  
Aberrant Gene

Abstract Background Evidence of global heterochromatin decay and aberrant gene expression in models of physiological and premature ageing have long supported the “heterochromatin loss theory of ageing”, which proposes that ageing is aetiologically linked to, and accompanied by, a progressive, generalised loss of repressive epigenetic signatures. However, the remarkable plasticity of chromatin conformation suggests that the re-establishment of such marks could potentially revert the transcriptomic architecture of animal cells to a “younger” state, promoting longevity and healthspan. To expand our understanding of the ageing process and its connection to chromatin biology, we screened an RNAi library of chromatin-associated factors for increased longevity phenotypes. Results We identified the lysine demethylases jmjd-3.2 and utx-1, as well as the lysine methyltransferase mes-2 as regulators of both lifespan and healthspan in C. elegans. Strikingly, we found that both overexpression and loss of function of jmjd-3.2 and utx-1 are all associated with enhanced longevity. Furthermore, we showed that the catalytic activity of UTX-1, but not JMJD-3.2, is critical for lifespan extension in the context of overexpression. In attempting to reconcile the improved longevity associated with both loss and gain of function of utx-1, we investigated the alternative lifespan pathways and tissue specificity of longevity outcomes. We demonstrated that lifespan extension caused by loss of utx-1 function is daf-16 dependent, while overexpression effects are partially independent of daf-16. In addition, lifespan extension was observed when utx-1 was knocked down or overexpressed in neurons and intestine, whereas in the epidermis, only knockdown of utx-1 conferred improved longevity. Conclusions We show that the regulation of longevity by chromatin modifiers can be the result of the interaction between distinct factors, such as the level and tissue of expression. Overall, we suggest that the heterochromatin loss model of ageing may be too simplistic an explanation of organismal ageing when molecular and tissue-specific effects are taken into account.

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