• Journal of Microbiology
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• v.42 no.4
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.1
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• pp.89-93
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• 2004

Expression of a fusion protein between B. thuringiensis crystal protein, Cry1Ac1 and a scorpion insect toxin (AaIT, Androctonus australis Hector insect toxin) in acrystalliferous B. thuringiensis strain (Cry-B strain) was examined. The cry 1Ac1 gene was cloned in B. thuringiensis-E coli shuttle vector, pHT3101, under the control of the native cry 1Ac1 gene promoter (pProAc) and a gene encoding AaIT was inserted in XhoI site in the middle of the cry 1Ac1 gene (pProAc-ScoR). B. thuringiensis Cry-B strain carrying pProAc-ScoR (PyoAc-ScoR/CB) produced an inclusion body of irregular shape and the expressed fusion protein is approximately 65 kDa in size. Sporulated cells and spore-crystal mixtures of ProAc-ScoR/CB had insecticidal activity against Plutella xylostella larvae, showing $LT_50$ of ProAc-ScoR/CB (22.59 hrs) lower than that of ProAc/CB (30.06 hrs) at $1{\times}{10^7} {CEU/cm^2}$. These results suggest that the fusion protein including a B. thuringiensis crystal protein and an AaIT may be functionally expressed in B. thupingiensis. Moreover, we verified the additive toxicity of AaIT, which is a new feasible candidate for insect control.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.118-125
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• 2006

The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.1-7
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• 1992

We cloned a gene cluster encoding phenanthrene-degrading enzymes on a 6.8-kb Xhol fragment from the Pseudomonas sp. DJ77 chromosomal DNA into the vector pBLUESCRIPT SIC(+). The resultant clone, containing the recombinant plilsmid pHENX7, was able to convert 3-methylcatechol to a yellow mela-cleavage compound. Since the pHENX7R in which the DNA insert was cloned in the opposite orientation lacked extradiol dioxygenase activity. the direction of transcription was established. Four polypeptides, PhnC (24 kDa). PhnD (31 kDa), PhnE (34 kDa). and PhnF (15 kDa), were identified in E coli JM101 transformed with several pHENX7-derived plasmids. The locations and extents of ~ndividual genes were determined by subcloning. The gene order was phnC-phnD-phnE-phnF-phnG, and phnC, phnD, phnE, and phnG genes encoded glutathione S-transferase, mrta-cleavage compound hydrolase, extradiol dioxygenase, mera-cleavage compound dehydrogenase, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.2
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• pp.217-223
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• 2004

A cathepsin L-like cysteine protease, v-cath, encoded by the baculovirus has been shown to playa role in host liquefaction. We have identified a v-cath gene in the silkworm virus, Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. The 969 bp v-cath has an open reading frame of 323 amino acids. A putative cleavage site and catalytic sites were conserved in BmNPV-K1 v-cath. The predicted three-dimensional structure of BmNPV-K1 v-cath revealed that the overall fold of BmNPV-K1 v-cath is similar to that of other proteases of the papain family. The deduced amino acid sequence of BmNPV-K1 v-cath showed 98% and 97% protein sequence identity to BmNPV T3 strain and to Autographa californica nuclear polyhedrosis virus, respectively. The BmNPV-K1 v-cath differed at 4 amino acid positions from BmNPV T3. The v-cath gene in BmNPV-K1 genome is located on the EcoRV 6 kb and XhoI 9 kb fragments. Northern hybridization analysis of BmNPV K1 v-cath gene revealed that it is expressed late in infection.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.2
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• pp.196-203
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• 2003

The application of sealants is a highly technique-sensitive procedure, requiring an extremely dry field prior to placement. Moisture contamination of the etched enamel surface before sealant placement is cited as the main reason for sealant failure. The purpose of this study was to evaluate the effects of different methods of sealant application on the shear bond strength of sealants to enamel. In groups 1, 2, 3, 4 Teethmate(unfilled sealant) was used, while Ultraseal XTplus(filled sealant) was used in groups 5, 6, 7, 8. Groups 1 and 5(control) were acid etched for 15 seconds using 35% phosphoric acid, washed and then dried. In groups 2, 6 drying agents were applied, and in groups 3, 7 bonding agents were applied and light cured. In groups 4 and 8 both drying agent and bonding agent were applied. Then sealant was cured to the specimen using molds 3mm in diameter and 2mm in height. Thermocycling was performed and shear bond strength was finally measured. The following results were obtained : 1. Groups using filled sealant(groups 5, 6, 7, 8) showed higher shear bond strengths compared to groups using unfilled sealant(groups 1, 2, 3, 4). 2. Among groups using unfilled sealant(groups 1, 2, 3, 4), groups 2, 3, 4 showed significantly higher shear bond strength compared to group 1(p<0.05). There were no significant differences among groups 2, 3 and 4. 3. There were no significant differences(p>0.05) among groups using filled sealant(groups 5, 6, 7, 8). 4. When modes of fracture were examined, cohesive failure was observed in groups 2, 3 and 4.

• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.296-299
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• 2015

Cowpea mild mottle virus (CPMMV) has a wide range of hosts, such as the pea family and tomato. CPMMV is a non-reported virus in Korea, and is domestically designated as a controlled virus associated with plant quarantine. In this study, a rapid diagnostic method for the detection of CPMMV at quarantine sites was developed. For the development of a user-based system, the PCR compositions and conditions use existing methods of quarantine for the viruses. Two sets of RT-PCR and nested PCR were developed in this study that could be amplified from 579 → 298 dp and 638 → 252 bp, respectively. Furthermore, a sequence inserted positive control plasmid was developed, which is able to identify false-positives resulting from laboratory contamination. The findings of this study are important for the diagnosis of CPMMV in imported crops held in plant quarantine.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.53-59
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• 1992

Four bacterial strains capable of catabolizing 4-chlorobiphen!;l (4CB) were isolated from the industrial waste waters. The bacterial isolates designated as PO$. P20, P27, and P1242. respectively, were examined for their catabolic activities. And in order to examine molecular homology of the 4CB catabolizing genes of these bacterial isolates. Southern hybridization was conducted with bphABC genes of P. p.srudoalculigrnrs KF707 as a DNA probe. The metabolites of 2-hydroxy-6-0x0-6-(4'-chlorophenyl)hexa-2 .4-dienoic acid and Cchlorobenzoate were detected to be produced by the isolatc:~ in the MM2 liquid cultures. But Cchlorobenzoate was further catabolized to produce 4.-hydroxybenzoate by DJ-12, P08. and P27. but not by P20 and P1242. As results of hybridization, homologous regions were commonly observed in Xhol fragments of 2.2 and 1.8 kb and in EcoRl fragment of 11 kb in the DJ- 12. P08, and P27 isolates. But in any restriction enzyme digests ot the P20 and PI242 isolates. homologous region was not detected. The cbp genes of the bactcria capable of catabolizing 4CB in nature could be divided into two groups by divergence<

• Research in Plant Disease
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• v.14 no.1
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• pp.15-20
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• 2008

Three isolates of Cucumber mosaic virus (CMV) were isolated from weed hosts showing typical mosaic symptoms, and some properties of the viruses were investigated. CMV isolates, designated as Is-CMV, Jd-CMV and Pla-CMV from Isodon inflexus, Jeffersonia dubia and Phryma leptostachya var. asiatica, respectively, were identified and characterized by biological reaction in several host plants, serological property, dsRNA analysis, reverse transcription-polymerase chain reaction (RT-PCR), restriction fragment-length polymorphism (RFLP). All isolates systemically infected in Nicotiana benthamiana, Cucurbita pepo cv. Black beauty and Cucumis sativus, and did not reveal any differences in these host plants between the isolates. However, remarkable difference in the symptoms was found between the CMVs in Capsicum annuum. Is-CMV induced an asymptomatic symptoms, while Jd-CMV and Pla-CMV produced severe mosaic symptoms in C. annuum plants. In dsRNA analysis, all isolates revealed four major bands with estimated molecular size of 3.4, 3.2, 2.1 and 1.0 kbp. The cDNAs of coat protein gene of the isolates were amplified by RT-PCR using a genus-specific single pair primers that designed to amplify a DNA fragment of approximately ranging from 938 to 966 bp. By restriction mapping analysis using RFLP of the RT-PCR products as well as by serological properties of gel diffusion test, the CMV isolates belong to a typical members of CMV subgroup IA. This is the first report on the occurrence of CMV in the three weed hosts.