Cloning and functional characterization of a monoterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2015 ◽  
Vol 69 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Kuan-Feng Huang ◽  
Yi-Ru Lee ◽  
Yen-Hsueh Tseng ◽  
Sheng-Yang Wang ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Monoterpene Synthase ◽  
Young Leaves ◽  
Rapid Amplification

AbstractEleutherococcus trifoliatusalso known as the three-leavedEleutherococcus, a member of the Araliaceae (ginseng) family, is commonly used in traditional Chinese medicine. Recently, many studies have demonstrated the bioactivities of the secondary metabolites inE. trifoliatus. In this study, a monoterpene synthase fromE. trifoliatushas been characterized. A pair of degenerate primers was designed and a fragment with conserved region of terpene synthase (TPS) was obtained. After 5′- and 3′-rapid amplification of cDNA ends (RACE), the full-length cDNA was obtained. The gene designatedEtLIMcontains an open reading frame of 1752 bp with a predicated molecular mass of 67.3 kDa. It was expressed in young leaves, stems, and drupes. The product ofEtLIMhas been identified by gas chromatography/mass spectrometry (GC/MS) as limonene.

Identification and functional characterization of a sesquiterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2012 ◽  
Vol 66 (2) ◽  
Author(s):  
Chi-Hsiang Wen ◽  
Yen-Hsueh Tseng ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Mass Spectrometry Analysis ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Sesquiterpene Synthase ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Authentic Standard ◽  
The Common

Abstract In the present study, one sesquiterpene synthase gene in Eleutherococcus trifoliatus was identified and characterized. Full-length cDNA was obtained from stems. It contained an open reading frame of 1671 bp (EtCop) with a predicted molecular mass of 64.5 kDa. The amino acid sequence of EtCop contained the common terpene synthase family motifs RR(x)8W, RxR and DDxxD. The recombinant protein from Escherichia coli was incubated with farnesyl diphosphate in order to identify the function of EtCop. The product of EtCop could be identified as an α-copaene by means of gas chromatography-mass spectrometry analysis and comparison with an authentic standard.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

Cloning and expression of a sesquiterpene synthase gene from Taiwania cryptomerioides

Holzforschung ◽  
2015 ◽  
Vol 69 (9) ◽  
pp. 1041-1048 ◽  
Author(s):  
Hui-Ling Hsieh ◽  
Li-Ting Ma ◽  
Sheng-Yang Wang ◽  
Fang-Hua Chu
Keyword(s):  
Major Product ◽  
Cloning And Expression ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Degenerate Primers ◽  
Sesquiterpene Synthase ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Young Leaves ◽  
Taiwania Cryptomerioides

Abstract Taiwania (Taiwania cryptomerioides Hayata) is a conifer species native to Taiwan, which is known for several bioactive secondary metabolites extracted from it. In this study, a sesquiterpene synthase (TPS) gene isolated from Taiwania was in focus. First, a pair of degenerate primers was designed for reverse transcription-polymerase chain reaction based on the total RNA extracted from the leaves of a mature tree. A DNA fragment with the conserved region of TPS gene was obtained. After 5′- and 3′-end amplification, the full-length gene was obtained, which contains an open reading frame of 1791 bp and encodes a predicted molecular mass of 70.2-kDa protein. The gene was highly expressed in young leaves, female flowers, and cones. The expression in leaves was enhanced by salicylic acid. To identify the function of TPS, the recombinant protein from Escherichia coli (Migula) Castellani & Chalmers was incubated with farnesyl diphosphate. Gas chromatography/mass spectrometry analysis and retention time as well as mass spectrum matching with authentic standards revealed that the major product of TPS is sesquiterpene α-gurjunene. The gene was, therefore, designated as Tc-Gur.

scholarly journals Functional Characterization of Alanine Racemase fromSchizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

2001 ◽  
Vol 183 (7) ◽  
pp. 2226-2233 ◽  
Author(s):  
Takuma Uo ◽  
Tohru Yoshimura ◽  
Naotaka Tanaka ◽  
Kaoru Takegawa ◽  
Nobuyoshi Esaki
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Source ◽  
Functional Characterization ◽  
Open Reading Frame ◽  
Alanine Racemase ◽  
Sole Nitrogen Source ◽  
Recombinant Yeast ◽  
Reading Frame ◽  
Putative Protein

ABSTRACT Schizosaccharomyces pombe has an open reading frame, which we named alr1 +, encoding a putative protein similar to bacterial alanine racemase. We cloned thealr1 + gene in Escherichia coli and purified the gene product (Alr1p), with an M rof 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparentKm and V max values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that ofl-alanine, respectively. S. pombe usesd-alanine as a sole nitrogen source, but deletion of thealr1 + gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway forl-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but notd-alanine as a sole nitrogen source. Moreover,d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1 + gene enabled S. cerevisiae to grow efficiently ond-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
Author(s):  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Shuttle Vector ◽  
Open Reading Frame ◽  
Cosmid Library ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Degradation Activity ◽  
Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

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