• BMB Reports
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• v.38 no.4
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• pp.457-467
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• 2005

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk-1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80% amino acid identity.

• BMB Reports
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• v.34 no.4
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• pp.371-378
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• 2001

The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.870-875
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• 2001

A DNA fragment, pCKB13, containing two genes encoding Coenzyme a transferase, was isolated from a genomic DNA library of S. marcescens KCTC 2172. The complete nucleotide sequence of the 2,081-bp BamHI fragment on pCKB13 was determined. Sequencing of the fragment led to the identification of two open reading frames showing high homology with two Coenzyme A (CoA) transferases, Acetoacetyl-CoA transferase (Acot) and Succinyl-CoA transferase (Scot), enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The enzyme activity of Coenzyme A transferase increased after introducing the multicopy of the cloned gene in E. coli. The recombinant protein, overexpressed by multicopy and induction with IPTG, was a polypeptide of 42 kDa, as confirmed by SDS-PAGE. The protein was purified to homogeneity through three sequential chromatographic procedures including ion-exchanged DEAE-sepharose, CM-sepharose, and Mono Q.

• Biomedical Science Letters
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• v.11 no.2
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• pp.227-235
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• 2005

It has been reported that endurance performance is influenced by various environmental and genetic factors. In view of an important role of human mitochondrial DNA (mtDNA) as a candidate for endurance performance, this study focused on the relationships between $VO_{2max}$ value as a measure of endurance performance or other associated phenotypes and four mtDNA restriction fragment length polymorphisms (RFLPs) (Bam HI, Hinc II1, Hinc II2 and Nci I) in the NADH dehydrogenase subunit 5 and one (Kpn I) in the D-loop region of mtDNA. MtDNA was purified from buffy coat in human peripheral blood, and PCR-RFLP analysis was performed to estimate the allele frequencies of each polymorphism in the mtDNA. There were no significant differences in allele distributions of all polymorphisms studied between male athletes and controls, respectively (P>0.05). However, the Kpn I polymorphism was significantly associated with diastolic blood pressure level in male athletes, respectively (P<0.05). Therefore, our results suggest that this polymorphism might be one of the factors modifying inter-individual difference in cardiovascular risk. Further studies using larger sample size will be required to generalize these results from the study described herein.

• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.237-242
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• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• Korean Journal of Veterinary Research
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• v.32 no.4
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• pp.597-603
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• 1992

To elucidate the molecular genetical properties of the canine parvoviruses isolated from the diseased puppies in the regions of Kyunggi and Chungnam provinces, the replicative form (RF) DNA of four field isolates were compared with those of two attenuated vaccine strains and a reference strains of CPV by restriction endonuclease analysis (REA). REA by Hinf I showed that three CPV isolates except CPV-V15 had an identical banding pattern with two vaccine strains, one standard strain and feline panleukopenia virus (FPLV). In CPV-V15 strain the fourth fragment of DNA with 800 bp was deleted. REA by Bgl II and Pst I indicated that CPV-V15 and FPLV had a bigger second fragment than those of the other strains of CPV. Meanwhile REA by Bam HI revealed that all the field isolates and vaccine strains used in this experiment showed similar banding patterns.

• Animal cells and systems
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• v.12 no.3
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• pp.149-155
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• 2008

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21(DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase(GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

• Journal of Plant Biology
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• v.38 no.1
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• pp.107-113
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• 1995

pRLYS1 containing intact rbcL gene of maize (Zea mays L. cv Golden X Bantam T-51; Zm-A) was digested with several restriction enzymes to construct subcones carrying promoter region of rbcL. The DNA fragments of 0.20, 0.19, 0.92 and 1.55 kb among the EcoRI digests, the EcoRI-DdeI digests, the AvaI digests and the EcoRI-BamHI digests of pRLYS1 were subcloned into pBluscriptSK＋and named pRLPS2, pRLPS3, pRLPS14 and pRLPS35, respectively. Four subclones contain the 1.92 kb portion from 136 nucleotide downstream to 1780 nucleotide upstream from the ATG initiation codon of rbcL gene. pRLPS2 (-29 to -229) and pRLPS3 (-239 to -420 from the ATG) were sequenced. When nucleotide sequence of Zm-A was compared with sequence of rbcL promoter region of a different cultivar of maize (Zea mays L. cv WFG TMS X BS7; Zm-B), the difference rate between two cultivars was 4.3%. The mean of sequence divergence between Zm-A and three grass species in the same tribe, Andropogoneae, in the upstream region from 29 to 420 of ATG was 1.8%, whereas between Zm-B and above-mentioned three species was 5.4%. Therefore, Zm-A seems to evolutionarily closer to three other species in Andropogoneae tribe than Zm-B is.

• KSBB Journal
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• v.5 no.1
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• pp.49-58
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• 1990

We constructed hybrid plasmids to allow controlled and extracellular production of human alpha-interferon in Escherichia coli. The hybrid plassmids were constructed by transferring alpha-lFN gene from plasmid Hif-2h which has the alpha-lFN gene at PstI restriction site of pBR322, to plasmids pIN -IIIB3 and pIN-IIIC3 at restriction sites between HindIII and BamHI. Plasmids pIN-IIIB3 and pIN-IIIC3 carry E. coli lipoprotein promoter, lac promoter and operator in tandem. The plasmids also have lacl genes which encode for lac repressors, which allows controlled expression of genes cloned to the plasmids by using of inducer IPTG. Lipoprotein signal sequence is located just ahead of cloning sites of the plasmids, which helps cells to excrete or secrete cloned gene products. Plasmid pUC9 was used as a intermediate vector for transferring of alpha-lFN gene from Hif-2h to pIN vectors in order to solve the problem of different restriction sites between Hif-2h and pIN vectors.

• Journal of agriculture & life science
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• v.46 no.2
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• pp.129-137
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• 2012

A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli $DH5{\alpha}$. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli $DH5{\alpha}$ exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about $50^{\circ}C$ for its enzymatic activity.