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Objective::To clone the complementary deoxyribonucleic acid (cDNA) of auxin/indole acetic acid protein (Aux/IAA) from Glycyrrhiza glabra (GgARPI) and analyze its sequence by bioinformatics. Method::RNA was extracted from fresh root of G. glabra, the cDNA sequence of GgARPI gene was cloned by reverse transcription polymerase chain reaction (RT-PCR), then sequencing and bioinformatic analysis were performed. Result::The GgARPI cDNA sequence with the full length of 686 bp was obtained from G. glabra. The full open reading frame (ORF) was 585 bp, encoding 194 amino acid residues. The bioinformatic analysis showed that the protein coded by GgARPI was a stable hydrophilic protein, with a relative molecular weight of 21.95 kDa and isoelectric point of 6.85.It contained no signal peptides or transmembrane domain. Its secondary structure mainly consisted of random coil. An Aux/IAA superfamily was included in the conversed domain. Homology analysis indicated that it had a close evolutionary relationship with leguminous plants, and a distant evolutionary relationship with monocotyledon, such as Setaria italica. Conclusion::GgARPI cDNA sequence is successfully cloned from G. glabra for the first time, which will lay a foundation for studying the function of GgARPI and explaining the molecular regulatory mechanism of biosynthesis of glycyrrhizic acid in G. glabra.
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ObjectiveThe role of human tumor-related calcium signal transductor 2 (TACSTD2) in promoting tumorigenesis has been noticed recently. We predicted the molecular structure and biological function of TACSTD2 by bioinformatic methods, in order to provide reference for further study of TACSTD2.MethodsThe homo sapiens TACSTD2 mRNA and protein amino acid sequences were obtained from the National Center for Biotechnology Information (NCBI) database. The bioinformatic methods were used to analyze the open reading frame(ORF) of TACSTD2, physicochemical properties, signal peptide and protein localization, subcellular localization, and prediction of transmembrane structure and secondary structure, tertiary structure, potential protein modification sites, domains, protein modification sites proteins, protein interacting with TACSTD2, biological functions of TACSTD2, and expression of TACSTD2 in human normal tissues and certain tumor types.ResultsAccording to the mRNA sequence of TACSTD2, there are 12 ORFs, and the longest is ORF1, with a total of 972bp, encoding 323 amino acids. The hydrophilic amino acid of TACSTD2 is more than that of hydrophobic amino acid, indicating that TACSTD2 belongs to hydrophilic protein. TACSTD2 is a highly conserved alkaline secreted protein with a transmembrane region both inside and outside the cytoplasm. The presence of nuclear localization signal(NLS) in the amino acid sequence of TACSTD2 suggests that TACSTD2 can locate in cell nucleus. TACSTD2 mainly distribute in cytoplasmic membrane, extracellular, nucleus and cytoplasm. The secondary structure prediction results showed that the main structure of TACSTD2 was random coil, followed by a α helix. TACSTD2 has 15 serine modification sites, 17 threonine modification sites, and 8 tyrosine modification sites. The TACSTD2 has protein interactions with Claudin(CLDN) protein family; and participating in signaling pathway such as cell surface receptor, cell proliferation, negative regulation of epithelial cell migration, and so on. Comparing with normal human tissues, its mRNA expression is up-regulated in most tumor types such as cervical cancer, lung cancer, thyroid cancer, uterine cancer, liver cancer, colorectal cancer.ConclusionAccording to the analysis of the structure and function of TACSTD2, TACSTD2 is highly-expression in multiple malignancies. It can participate in the process of proliferation, migration and adhesion of malignant tumor cells through cell surface receptor signaling pathways. This study provide reference for the further research about the function of TACSTD2.
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ObjectiveThe role of human tumor-related calcium signal transductor 2 (TACSTD2) in promoting tumorigenesis has been noticed recently. We predicted the molecular structure and biological function of TACSTD2 by bioinformatic methods, in order to provide reference for further study of TACSTD2.MethodsThe homo sapiens TACSTD2 mRNA and protein amino acid sequences were obtained from the National Center for Biotechnology Information (NCBI) database. The bioinformatic methods were used to analyze the open reading frame(ORF) of TACSTD2, physicochemical properties, signal peptide and protein localization, subcellular localization, and prediction of transmembrane structure and secondary structure, tertiary structure, potential protein modification sites, domains, protein modification sites proteins, protein interacting with TACSTD2, biological functions of TACSTD2, and expression of TACSTD2 in human normal tissues and certain tumor types.ResultsAccording to the mRNA sequence of TACSTD2, there are 12 ORFs, and the longest is ORF1, with a total of 972bp, encoding 323 amino acids. The hydrophilic amino acid of TACSTD2 is more than that of hydrophobic amino acid, indicating that TACSTD2 belongs to hydrophilic protein. TACSTD2 is a highly conserved alkaline secreted protein with a transmembrane region both inside and outside the cytoplasm. The presence of nuclear localization signal(NLS) in the amino acid sequence of TACSTD2 suggests that TACSTD2 can locate in cell nucleus. TACSTD2 mainly distribute in cytoplasmic membrane, extracellular, nucleus and cytoplasm. The secondary structure prediction results showed that the main structure of TACSTD2 was random coil, followed by a α helix. TACSTD2 has 15 serine modification sites, 17 threonine modification sites, and 8 tyrosine modification sites. The TACSTD2 has protein interactions with Claudin(CLDN) protein family; and participating in signaling pathway such as cell surface receptor, cell proliferation, negative regulation of epithelial cell migration, and so on. Comparing with normal human tissues, its mRNA expression is up-regulated in most tumor types such as cervical cancer, lung cancer, thyroid cancer, uterine cancer, liver cancer, colorectal cancer.ConclusionAccording to the analysis of the structure and function of TACSTD2, TACSTD2 is highly-expression in multiple malignancies. It can participate in the process of proliferation, migration and adhesion of malignant tumor cells through cell surface receptor signaling pathways. This study provide reference for the further research about the function of TACSTD2.
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Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.
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Plant Diseases , Ascomycota/genetics , Ascomycota/pathogenicity , Transcription Factors/isolation & purification , Ascomycota/metabolism , Stress, Physiological , Transcription Factors/genetics , Transcription Factors/metabolism , Carrier Proteins , Gene Expression , Blotting, Western , Open Reading Frames , Zinc Fingers , Cloning, Molecular , Zea mays , Escherichia coli , Helminthosporium , EpitopesABSTRACT
Objective To obtain the key enzyme gene involving in the monoterpenoid biosynthesis pathway, an iridoid oxidase gene (GrIDO) and its promoter were cloned from the leaves of Gentiana rigescens, and its bioinformatics analysis were also performed. Methods The gene specific primers were designed according to the GrIDO gene of transcriptome in G. rigescens. The open reading frame (ORF) of GrIDO gene was cloned by RT-PCR method. The bioinformation of GrIDO gene was analyzed by online softwares. Meanwhile, the gene specific primers were designed according to the cloned GrIDO gene, and the promoter of GrIDO gene was amplified by PCR method. Its sequence analysis was also performed. Results The length of GrIDO gene ORF (GenBank accession number KP722034) was 1 557 bp, which encoded a protein with 518 amino acids. Its relative molecular weight was 58 920 with the theoretical isoelectric point of 8.40. GrIDO was the member of cytochrome P450 superfamily and may localize in chloroplast. GrIDO was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (51.07%) and loops (42.69%). GrIDO protein had a high similarity with CrIDO of Catharanthus roseus (85.83%) and their genetic relationship was close. The cloned GrIDO promoter had a length of 720 bp (GenBank accession number KT428570), which possessed cis-regulatory elements TATA-box and CAAT-box, and had cis-acting elements involved in the abscisic acid and MeJA responsiveness, part of a light responsive element and MYB transcription factor binding site. Conclusion The expression of GrIDO gene were regulated by multifactors. The GrIDO gene and its promoter were cloned from G. rigescens. This study will lay foundations for the functional research of GrIDO gene in monoterpenoid biosynthesis pathway.
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Objective To analysis the DNA sequence of HEV ORF3 cDNA and express in HepG2 cell .Methods Recombinant vector carrying HEV ORF3 cDNA was constructed and identified by DNA sequencing .Expression of recombinant vector in HepG2 cell was determined by immunofluorescence technique .Results The recombinant vector was constructed and expressed in HepG2 cell successfully .Conclusion The research laid experimental foundation for exploring the biological function of ORF3 protein in the pathogenesis of viral hepatitis E .
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In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007-2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates-HH08, DY, and YN-2011-were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.
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Twenty-nine thyroid tissue samples were collected from patients with thyroid nodules.The total RNA were extracted,the gene expressions of TFF3,HMGA2,C1orf24,and DDIT3 were detected by RT-PCR.In comparison with normal thyroid tissues,the expression of C1orf24 mRNA was decreased in the follicular thyroid adenoma (FTA) group,but increased in the follicular thyroid carcinomas (FTC) group.The expressions of DDIT3 and HMGA2 mRNA were increased in both FTA and FTC groups,and were even higher in the latter.The expressions of TFF3 mRNA level were decreased in FTA and FTC.The data suggested that molecular markers C1ort24,DDIT3,HMGA2,and TFF3 in thyroid tissue seem to be helpful in the differential diagnosis between follicular adenomas and carcinomas.
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The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.
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Animals , Animal Husbandry , Genes, Viral , Genetic Variation , Lung/virology , Lymph Nodes/virology , Open Reading Frames , Phylogeny , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/chemistry , Republic of Korea , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Sequence Analysis, Protein/veterinary , SwineABSTRACT
Aims: To study the genetic and transcript profiling of the genes specifying cytosolic HSP90s in Triticum aestivum. Study Design: Random sampling. Place and Duration of Study: Indian Agricultural Research Institute, New Delhi, India, between August to December, 2011. Methodology: We include C-306 (thermotolerant) and PBW343 (thermosusceptible) cultivars of wheat for the study. Total RNA was isolated using Trizol method and gene was identified and isolated using RT-PCR. In silico characterization was done using different bioinformatic tools. Quantitative real time PCR was carried out using BioRad CFX96 platform and Pfaffl’s method was used for the comparative change in fold expression of the gene. Results: Here, we report cloning of an HSP90 gene from C-306 wheat cultivar having an ORF of 700 amino acids. Genome Blast identified 3 different clusters of reference sequence on chromosome no 4, 8 and 9 having LOC 100125696 and showing maximum homology with HSP90 reported from Triticum aestivum. Pure amino acid composition revealed highest composition of glutamic acid followed by lysine and leucine whereas, cysteine composition was lowest. Protein characterization showed the existence of 10 networks of coevolved amino acids. Quantitative real time PCR showed 1.5, 4.5, 5 & 7.4 fold increase in expression of HSP90 in case of C-306 compared to 2.5, 6.4, 6.9 & 5.6 fold increase in case of PBW343 at vegetative (root & shoot), pollination and milky dough stage. Multiple co-chaperones of HSP90 were observed by immunoblot assay in response to differential heat shock. Conclusion: This investigation proves that HSP90 is one of the key components of defense mechanism in wheat against heat stress which requires the formation of cochaperone complexes with HSP70 for its functional activity. There is a need to exploit the transcription factors associated with HSP90 and its regulation and differential expression in order to use it for developing thermotolerant wheat cultivars.
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Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.
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Amino Acid Sequence , Baculoviridae , Blotting, Western , Clone Cells , DNA, Complementary , Electrophoresis , Genome, Viral , Glutathione Transferase , Glycoproteins , Korea , Open Reading Frames , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Sequence Analysis , Sf9 Cells , Sodium , Swine , VirusesABSTRACT
The primary clue for locating protein-coding regions is the open reading frame and the determination of ORFs (Open Reading Frames) is the first step toward the gene prediction, especially for prokaryotes. In this respect, we have developed a web-based ORF search tool called ORF Miner. The ORF Miner is a graphical analysis utility which determines all possible open reading frames of a selectable minimum size in an input sequence. This tool identifies all open reading frames using alternative genetic codes as well as the standard one and reports a list of ORFs with corresponding deduced amino acid sequences. The ORF Miner can be employed for sequence annotation and give a crucial clue to determination of actual protein-coding regions.
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Animals , Amino Acid Sequence , Ecthyma, Contagious , Genetic Code , Open Reading Frames , Resin CementsABSTRACT
Objective To investigate whether glucosyltransfernse(GTF) and open reading frame 4 (ORF4) coded by fap1-orf4 gene locus of Streptococcus parasanguis was involved in the regulation of Fap1 glycosylation, and mature and to determine whether there was interaction between GTF and ORF4. Methods A gene replacement strategy was adapted to construct gtf and orf4 allele replace mutant of S. Parasanguis. Complementation assay and Western blot were used to test fap1 expression levels. Yeast two-hybrid analysis and GST pull down assays were adapted to determine the interaction between GTF and ORF4. Results (1) Compared with wild S. Parasanguis, mature Fapl (Mr about 220×103) disappeared and were substituted with high molecular weight Fapl (Mr about 360×103) in gtf or orf4 alleie replace mutants of S. Parasan-guis. Complementation assay showed that pVPT-GFP-gtf and pVPT-GFP-orf4 restored mature fap1 expression in gtf or orf4 alleie replace mutants, respectively. (2) With Yeast two-hybrid analysis, the eotransformants, AH109/pAD-Gtf+pBD-orf4 and AHlOg/pAD-orf4+pBD-gtf growed on SD-LTHA selective ngar plate after streaked, reversely, the eotransformants, AH109/pAD+pBD-orf4,AH109/pAD+pBD-gtf、AH109/pBD+ pAD-orf4、AH109/pBD+pAD-gtf did not grow on SD-LTHA selective agar plate, furthermore, the cotrans-formants, AH109/pAD+pBD-orf4 and AH109/pAD-orf4+pBD-gtfshowed blue during X-α-gal assay. (3) GST pull down assay confirmed the direct interaction between GTF and ORF4. Conclusion There is inter-action between GTF and ORF4 coded byfapl-orf4 gene locus of S. Parasangnis and the formation of the GTF and ORF4 complex was required for the glycosylation and mature of Fapl in S. Parasanguis.
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Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group Ⅱ NPV. The HearNPV vfgftranscripts were detected from 18 to 96 h post-infection (hpi) of Hz-AMI cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM 1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.
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Dual coding is a phenomenon which refers to a mature mRNA that contains two open reading frames(ORFs) in the same direction to code two different proteins.This phenomenon is quite common in prokaryote,bacteriophages and viruses as an economical and effective way to present the genome information.However,recent reports suggest that the dual coding phenomenon does also occur in eukaryote.Dual coding will help us to further understand the complicated regulation of eukaryotic genes or even the law of molecular evolution.
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Objective:To construct the eukaryotic expression vector of open reading frame of KH gene(KH-ORF),an unknown gene obtained from K562 cells,and investigate its function.Methods:The KH-ORF cDNA was cleaved from plasmid expression vector cut with the same restriction endonucleases.pCI-KH-ORF,the pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCl-neo mammalian recombinant expression plasmid,was transfected into K562 cells with liposome and screened by G418.KH-ORF expression was tested by RT-PCR.The effect of expression product on cell proliferation was tested by growth-curve and LDH detection.Results:KH-ORF was expressed in K562 cells.The effects of increased proliferation were significant.Conclusion:The product of KH-ORF may be a functional protein that is related to the proliferation of K562 cells,and KH gene may be a functional unknown leukemia gene.
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No abstract available.
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Objective:To construct the prokaryotic expression system of open reading frame of KH gene (KH-ORF),an unknown gene in K562 cells.Methods:The 225bp KH-ORF cDNA was cleaved from plasmid pGEM-T-easy-KH-ORF with EcoRI and SalI and subcloned into the pCI-neo Mammalian Expression Vector cutted with the same restriction endonucleases.The recombinant expression plasmid was transfected into BL21 (DE3) plysS by hot-shock assay.Induced by IPTG, the recombinant protein product was tested by SDS-PAGE.Results:24kD KH-ORF product was obtained as inclusion bodies in BL21 (DE3) plysS after the expression vector had been induced by IPTG for 30 minutes Conclusion :The construction of prokaryotic expression system of KH- ORF is successful.