• International Journal of Industrial Entomology and Biomaterials
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• v.25 no.2
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• pp.187-193
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• 2012

Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5- and 3- end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1778-1783
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• 2006

Event-specific PCR detection methods are the primary trend in genetically modified (GM) plant detection owing to their high specificity based on the flanking sequence of the exogenous integrant. Therefore, this study describes a real-time PCR system for event-specific GM canola GT73, consisting of a set of primers, TaqMan probe, and single target standard plasmid. For the specific detection of GT73 canola, the 3'-integration junction sequence between the host plant DNA and the integrated specific border was targeted. To validate the proposed method, test samples of 0, 1, 3, 5, and 10% GT73 canola were quantified. The method was also assayed with 15 different plants, and no amplification signal was observed in a real-time PCR assay with any of the species tested, other than GT73 canola.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• Journal of Microbiology
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• v.42 no.2
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• pp.139-142
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• 2004

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.230-230
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• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Molecules and Cells
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• v.23 no.3
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• pp.349-356
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• 2007

Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5'-flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with peri-centromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa.

• Korean Journal of Veterinary Service
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• v.22 no.4
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• pp.363-370
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• 1999

Isolation of PRRSV was attempted from 646 swine sera collected from swine farms. The MARC-145 cell, which is highly permissive to PRRSV, was used for virus isolation, propagation, IFA test, and VN test. Total 36 cytopathic viruses to MARC-145 cells were isolated. The virus isolates were identified as a PRRSV by the IFA test and VN test using the reference sera prepared by experimental infection of reference PRRSV CNV-1 into 30 day-old pig. In addition to serological conformation, ORF5 of genomic RNA of 6 selected cytopathic viruses were amplified by the RT-PCR. The resulting PCR products were examined by electrophoresis in 1.2% agarose gel. An appropriate bands of about 680bp including the flanking sequence of total 80bp were seen on agarose gel.

• Korean journal of applied entomology
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• v.38 no.2
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• pp.145-149
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• 1999

To develop the baculovirus expression vector system (BEVS) adopting p10 gene promoter of Spodoptera exigua nucleopolyhedrovirus (SeNPV), we characterized the p10 gene of SeNPV. The nucleotide sequence of 545 bases including the coding region of p10 gene was determined. Compared with the previously reported SeNPV p10 gene (Zuidema et al., 1993), 4 bases were different in the 5' and 3' flanking region but no difference was found in the coding region. The p10 gene was located within Xho I 1.5 Kb, Sph 1 2.4 Kb and Cla I 4.0 Kb fragments by Southern hybridization analysis. Also, the Sph I 2.4 Kb and the Cla I 4.0 Kb fragments were cloned and their restriction enzyme maps were determined.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.134-140
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• 1992

Teh $polA^{+}$ gene can be transducted in a multicopy mini-Mu plasmid, but not cloned because the product of this gene is lethal when overproduced. Although, we obtained one surviving cell, in which the ColEl-derived mini-Mu plasmid suffered a spontaneous deletion exactly at the region where the $polA^{+}$ gene was cloned. The $PolA^{+}$ unstream flanking sequence containing the promoter and pribnow-box was delected in vivo ; consequently this gene is not able to be expressed.