scholarly journals Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

2021 ◽  
Vol 43 (4) ◽  
pp. 119-128
Author(s):  
Nguyen Van Giang ◽  
Luu Han Ly ◽  
Pham Le Bich Hang ◽  
Le Thi Thu Hien
Keyword(s):  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Ginsenoside Biosynthesis ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
As Stress ◽  
Root Tissues

Panax vietnamensis Ha et Grushv. is a species of the genus Panax native to Central Vietnam, containing a family of triterpene saponins named ginsenosides. This group of biomolecules possesses valuable therapeutic properties against cancer, hepatitis, diabetes, inflammation as well as stress and anxiety. Farnesyl diphosphate synthase (FPS) is a key enzyme participating in the ginsenoside biosynthesis pathway. In this study, a FPS gene from P. vietnamensis (PvFPS) was isolated and characterized. The PvFPS cDNA contained an open reading frame of 1032 bp, encoding a polypeptide chain of 342 amino acid residues. Nucleotide sequence comparison showed that FPS was highly conserved among most species, with two Aspartate-rich motifs responsible for product chain length determination strongly sustained. PvFPS was closely related to those of the same genera and order and differed from those from other kingdoms. PvFPS expression was detected at a greater level in root tissues than in leaves in all ages. Our findings provided information concerning the properties of a crucial gene in the ginsenoside biosynthesis, thus enhancing our understanding of this important pathway.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

scholarly journals Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts

1987 ◽  
Vol 245 (2) ◽  
pp. 595-603 ◽  
Author(s):  
G J Price ◽  
P Jones ◽  
M D Davison ◽  
B Patel ◽  
I C Eperon ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Fusion Protein ◽  
Tyrosine Kinases ◽  
Consensus Sequence ◽  
Coli Strain ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Internal Repeat ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3′ sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

2004 ◽  
Vol 36 (4) ◽  
pp. 303-308 ◽  
Author(s):  
Guo-Qing Dong ◽  
Xiao-Ling Yuan ◽  
Ya-Jun Shan ◽  
Zhen-Hu Zhao ◽  
Jia-Pei Chen ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Fibrinolytic Enzyme ◽  
Eisenia Foetida ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Native Form ◽  
Reading Frame ◽  
Fibrin Plate ◽  
Earthworm Fibrinolytic Enzyme ◽  
High Degree

Abstract The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

scholarly journals Cloning and Expression Analysis of a Farnesyl Diphosphate Synthase (FPPS) Gene from Chamaemelum nobile

2017 ◽  
Vol 45 (2) ◽  
pp. 358-364 ◽  
Author(s):  
Xiao-Meng LIU ◽  
Ting-Ting TAO ◽  
Xiang-Xiang MENG ◽  
Wei-Wei ZHANG ◽  
Jie CHANG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Condensation Reaction ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Farnesyl Diphosphate ◽  
Cloning And Expression ◽  
Farnesyl Diphosphate Synthase ◽  
Reading Frame ◽  
Chamaemelum Nobile ◽  
Multiple Alignment Analysis

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene (CnFPPS) was cloned from Chamaemelum nobile. The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile. Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla. The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile.

scholarly journals Characterization of a flavocytochrome that is induced during the anaerobic respiration of Fe3+ by Shewanella frigidimarina NCIMB400

1999 ◽  
Vol 342 (2) ◽  
pp. 439-448 ◽  
Author(s):  
Paul S. DOBBIN ◽  
Julea N. BUTT ◽  
Anne K. POWELL ◽  
Graeme A. REID ◽  
David J. RICHARDSON
Keyword(s):  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Nucleotide Sequencing ◽  
Amino Acid Residues ◽  
Sequencing Data ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Reading Frame ◽  
Ferric Pyrophosphate ◽  
Reduction Potentials

A 63.9 kDa periplasmic tetrahaem flavocytochrome c3, designated Ifc3, was found to be expressed in Shewanellafrigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates. Ifc3 was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c3-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV. A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc3, ifcA, did not lead to a significant change in the rate of Fe3+ reduction in batch culture. However, during such growth the Ifc3-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane β-barrel protein of 324 amino acid residues.

scholarly journals Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family

1992 ◽  
Vol 287 (1) ◽  
pp. 261-267 ◽  
Author(s):  
C T Dolphin ◽  
E A Shephard ◽  
S Povey ◽  
R L Smith ◽  
I R Phillips
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Chromosome 1 ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Primary Sequence ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Distinct Member ◽  
Human Chromosome 1

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

scholarly journals Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol biosynthesis.

2005 ◽  
Vol 52 (1) ◽  
pp. 221-232 ◽  
Author(s):  
Kariona Grabińska ◽  
Grazyna Sosińska ◽  
Jacek Orłowski ◽  
Ewa Swiezewska ◽  
Thierry Berges ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Relationships ◽  
Dolichyl Phosphate ◽  
Preferential Synthesis

In the yeast Saccharomyces cerevisiae the RER2 and SRT1 genes encode Rer2 and Srt1 proteins with cis-prenyltransferase (cis-PT-ase) activity. Both cis-PT-ases utilize farnesyl diphosphate (FPP) as a starter for polyprenyl diphosphate (dolichol backbone) formation. The products of the Rer2 and Srt1 proteins consist of 14-17 and 18-23 isoprene units, respectively. In this work we demonstrate that deletion or overexpression of SRT1 up-regulates the activity of Rer2p and dolichol content. However, upon overexpression of SRT1, preferential synthesis of longer-chain dolichols and a decrease in the amount of the shorter species are observed. Furthermore, overexpression of the ERG20 gene (encoding farnesyl diphosphate synthase, Erg20p) induces transcription of SRT1 mRNA and increases the levels of mRNA for RER2 and DPM1 (dolichyl phosphate mannose synthase, Dpm1p). Subsequently the enzymatic activity of Rer2p and dolichol content are also increased. However, the amount of Dpm1p or its enzymatic activity remain unchanged.

scholarly journals Pneumococcal Bacteriophage Cp-1 Encodes Its Own Protease Essential for Phage Maturation

1998 ◽  
Vol 72 (4) ◽  
pp. 3491-3494 ◽  
Author(s):  
Ana C. Martín ◽  
Rubens López ◽  
Pedro García
Keyword(s):  
Escherichia Coli ◽  
Major Capsid Protein ◽  
Open Reading Frame ◽  
Posttranslational Processing ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Gram Positive Bacteria ◽  
First Case ◽  
Key Enzyme ◽  
Head Protein

ABSTRACT The major capsid protein of the pneumococcal phage Cp-1 that accounts for 90% of the total protein found in the purified virions is synthesized by posttranslational processing of the product of the open reading frame (ORF) orf9. Cloning of different ORFs of the Cp-1 genome in Escherichia coli and Streptococcus pneumoniae combined with Western blot analysis of the expressed products led to the conclusion that the product oforf13 is an endoprotease that cleaves off the first 48 amino acid residues of the major head protein. This protease appears to be a key enzyme in the morphopoietic pathway of the Cp-1 phage head. To our knowledge, this is the first case of a bacteriophage infecting gram-positive bacteria that encodes a protease involved in phage maturation.

scholarly journals Isolation and characterization of SUA5, a novel gene required for normal growth in Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 791-801 ◽  
Author(s):  
J G Na ◽  
I Pinto ◽  
M Hampsey
Keyword(s):  
Transcription Initiation ◽  
State Level ◽  
Normal Growth ◽  
Gene Encoding ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
New Gene ◽  
Type Gene ◽  
Data Banks

Abstract We have identified the sua5 locus as a suppressor of an aberrant ATG codon located in the leader region of the cyc1 gene. The sua5-1 allele enhances the iso-1-cytochrome c steady state level in the cyc1-1019 mutant from 2% to approximately 60% of normal (Cyc+) and also confers a marked slow growth (Slg-) phenotype. Suppression is not a consequence of altered transcription initiation at the cyc1 locus. The SUA5 wild-type gene was isolated and sequenced, revealing an open reading frame (ORF) encoding a potential protein of 46,537 Da. SUA5 transcript analyses were consistent with expression of the predicted ORF and Sua5 antisera detected a protein with an apparent molecular mass of 44 kDa. SUA5 was mapped to chromosome VII, immediately adjacent to the PMR1 gene. Hybridization analysis revealed the presence of a related gene on chromosome XII. Neither the SUA5 DNA sequence nor deduced amino acid sequence showed homology to any sequences in the data banks. Disruption of SUA5 conferred the same Cyc+ and Slg- phenotypes as the sua5-1 suppressor, which is the result of a missense mutation, encoding a Ser107----Phe replacement. In addition, sua5 null mutants lack cytochrome a.a3 and fail to grow on lactate or glycerol medium. These results define SUA5 as a new gene encoding a novel protein that is necessary for normal cell growth.

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