• Korean journal of applied entomology
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• v.36 no.4
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• pp.341-350
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• 1997

We have now constructed a novel recombinant baculovirus producing fusion protein with Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin and Bacillus thuringiensis(Bt) cryIA(c) crystal protein. The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The N-terminal of cryIA(c) gene of Bt subsp. kurstaki HD-73 was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front of intact polyhedrin gene or by insertion into the HindIII site in polyhedrin gene. The recombinant baculoviruses were named as BtrusI or BtrusII, respectively. Although single transcript from the fusion protein gene was apparently observed. BtrusI was produced the two proteins, 92 kDa fusion protein and only polyhedrin. In addition, fusion protein produced by BtrusI did not form polyhedra. Interestingly, however, the cells infected with BtrusII did not show a 33 kDa polyhedrin band as a cells infected with BtrusI. Cells infected with BtrusII were only produced fusion protein, but the polyhedra formed by fusion protein was not observed. To determine the insecticidal toxicity of fusion protein, therefore, Sf9 cells infected with BtrusI were inoculated to Bombyx mori larvae. Sf9 cells infected with BtrusI that expressed the fusion protein caused larval mortality although the insecticidal toxicity was low. In conclusion, our results clearly demonstrated that the fusion protein with polyhedrin and Bt cryIA(c) crystal protein have a insecticida toxicity.

• Korean journal of applied entomology
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• v.48 no.4
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• pp.509-517
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• 2009

Bacillus thuringiensis subsp. aizawai CAB109 isolated in Korea is known active against Spodoptera sp.. Especially, B. thuringiensis aizawai CAB109 isolates showed 100% mortality against Spodoptera litura and Spodoptera exigua. To screen highly active B. thuringiensis, the pathogenicity of B. thuringiensis CAB109 was compared with that of commercialized B. thuringiensis products. $LC_{50}$ values of CAB109, product TB-WP and product SC strains of B. thuringiensis were $1.3{\times}10^5$, $2.3{\times}10^6$ and $5.2{\times}10^5\;cfu/ml$ against the 2nd larva of S. litura and $1.8{\times}10^4$, $1.3{\times}10^6$ and $1.5{\times}10^6\;cfu/ml$ against the 2nd larva S. exigua, respectively. To determine new gene's existence and absence, the plasmid DNA was extracted, and compared to that of B.t. aizawai HD-133. Both B. thuringiensis were not like plasmid DNA pattern. PCR technique was used to predict both plasmid DNA's cry gene. PCR products analysis showed that B.t. CAB109 harbor Cry1Aa, Cry1Ab, Cry1C and Cry1D and B.t. HD-133 has Cry1Aa and Cry1Ab, respectively.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1033-1039
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• 2008

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of otfl was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.

• Microbiology and Biotechnology Letters
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• v.24 no.2
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• pp.173-177
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• 1996

Bacillus thuringiensis subsp. morrisoni PG-14 is a gram-positive soil bacterium producing mosquitocidal parasporal inclusions composed of several crystal proteins. Among these crystal protein genes, cryIVD gene is one of major component which has 72 kDa in size. However, these parasporal inclusions sink quickly from the surface of water where mosquito larval feeding occurred. To develope mosquitocidal cyanobacterium, therefore, we constructed the expression vector, pCYASK 5-1 harboring cryIVD gene. The expression vector, pCYASK5-1 was transformed into the cyanobacterium Syne- chocystis PCC6803 reported as a natural mosquito larval food source and the transformants were selected with kanamycin. Expression of IVD gene in transformant was characterized by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. The mosquitocidal activity of a transformant was determined with Culex tritaeniorhynchus. The results showed that, the transformed cyanobacterium is toxic to mosquito larvae and will be expected as a potential agent that is used for mosquito control.

• Journal of Pharmaceutical Investigation
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• v.28 no.2
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• pp.121-126
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• 1998

The solubility and physicochemical stability of caroverine hydrochloride (CRV), an antispasmodic, in buffered aqueous solutions were studied using a reverse phase high performance liquid chromatography. The solubilty of the drug at pH 2.76-5.40 was similar at the range 31.9-36.2 mg/ml $(34^{circ}C)$, but, at the pH higher than 6.0, markedly decreased. The use of polyethylene glycol 400 as a cosolvent did not increase the solubility at any compositions examined. Moreover. increasing molar concentration of aqueous phosphate buffer from 0 to 0.5 M remarkably decreased the solubility. The degradation of CRY followed the apparent first-order kinetics. The degradation was accelerated with decreasing pH and increasing storage temperature. The half-lives for the degradation of CRY (1.0 mg/ml) at pH 1.28. 4.01 and 5.93 $(45^{\circ}C)$ were 2.8, 31.4 and 124 hr. respectively. The pHs of incubated solutions were to some extent lowered perhaps due to the formation of acidic degradation products. The addition of disodium edetate (0.01%) to the CRY solution (pH 4.95) retarded 2.5 times the degradation rate at $45^{\circ}C$, but the use of sodium bisulfite (0.1%) accelerated 2.9 times the rate. The activation energy for the CRY solution (20 mg/ml. pH 5.4) containing 0.01% EDTA was calculated to be 5.98 kcal/mole. When the solution was stored under nitrogen displacement in ampoule, there was no significant degradation even after 3 months at $40^{\circ}C$, indicating that protection from oxidation by air (oxygen) is essential for the complete stabilization of CRY solution.

• Molecules and Cells
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• v.22 no.3
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• pp.285-290
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• 2006

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.115-122
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• 2000

A Bacillus thuringiensis designated NT0423, belonging to B. thuringiensis subsp. aizawai (H 7), was isolated from samples of dust and soil of sericultural farms. B. thuringiensis NT0423 having dualspecificity against Lepidoptera and Diptera produced bipyramidal inclusions consisting of two major polypeptides of approximately 130- and 70-kDa. Proteolytic processing by trypsin and gut juice of Bombyx mori yielded predominant proteins with molecular masses of about 66-kDa. The whole crystal protein of B. thuringiensis NT0423 immunologically was related to that of B. thuringiensis subsp. aizawai. PCR analysis showed that B. thuringiensis NT0423 has at least five crystal protein genes including cryIA(a), cryIA(b), cryIC, cryID and cryIIA, and southern blot was determined the location of each gene on intact and enzyme-digested plasmid DNA fragments. Except for cryIA(a) gene on the high molecular weight plasmid of 165-kb, all of four genes were located on the plasmid of 66-kb. The production of $\beta$-exotoxin from B. thuringiensis NT0423 was identified by the HPLC analysis. In addition, the $\beta$-exotoxin showed its ability to prevent pupation of treated larvae of house flies (Musca domestica) from developing into normal adults.

• Korean journal of applied entomology
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• v.32 no.3
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• pp.300-306
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• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.

• Journal of Marine Life Science
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• v.9 no.1
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• pp.47-52
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• 2024

Light is a major external environmental factor that influences the circadian rhythm of photosynthetic organisms and various physiological phenomena, such as growth, maturation, and behavior. The number of light-reaching organisms changes depending on the season and atmospheric conditions, and the intensity and wavelength of light differ depending on the organisms inhabiting the environment. Altered light changes the circadian rhythm of fish, which is controlled by clock genes, such as period 2 (Per2), cryptochrome 1 (Cry1), and melatonin. In this study, we set the zeitgeber time (ZT; 14 light-10 dark, LD) based on the actual sunrise and sunset times and examined Per2 and Cry1 activities, levels of aralkylamine N-acetyltransferase (AANAT), and melatonin in Pholis nebulosa, a drifting seaweed species exposed to irregular light. Per2 and Cry1 levels increased during the daytime and decreased after sunset. The AANAT levels decreased during the daytime and increased during the night. Melatonin concentration was highest around midnight (ZT21, 23:30), but exhibited similar concentrations during the daytime. While the activity of Per2, Cry1, and AANAT levels exhibited a typical circadian rhythm observed in most vertebrates, melatonin concentrations did not show a significant difference between the daytime and nighttime. These findings provide insights into the circadian rhythm patterns of organisms exposed to irregular light environments, such as P. nebulosa, which differ from those of typical fish species.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.142-147
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• 1989

Six plasmids of B. thuringiensis serovar. kurstaki HD73 were detected, with approximate sizes of 7.4, 7.8, 8.1, 11.3, and 75 Kb, as well as a low copied plasmid of similar length to 75 Kb. Partially cured mutants from B. thuringiensis HD73 were obtained either by the treatment of the curing agent, ethidium bromide(0.02 $\mu\textrm{g}$/$m\ell$) or by spontaneous curing, Acrystalliferous mutants(Cry$^-$) were identified by microscopic observation and immunoblotting with polyclonal antibody against 133 KD deltaendotoxin of HD73. Ten Cry$^-$ mutants were found to be lack of 75 Kb plasmid. These results implicated that this plasmid was associated with delta-endotoxin production, After isolating the mutants, we streaked them on potato dextrose agar, spizizen casamino acid glucose, starch agar, and nutrient agar. Only on starch agar medium did morphologies of Cry$^-$ appear translucent and light greyish. On the other hand, the mutants of B. thuringiensis isolated from Korean soil, designated KBS722, were obtained by the treatment of novobiocin (3 $\mu\textrm{g}$/$m\ell$). Acrystalliferous mutants of KBS722 were less translucent than HD73 mutants' only on nutrient agar medium. Compared the plasmid profile of the mutants with delta-endotoxin production, the results seemed to indicate that the insecticidal protein gene of B. thuringiensis isolates KBS722 located on about 225 Kb plasmid DNA.