• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• International Journal of Industrial Entomology and Biomaterials
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• 제19권1호
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.15-20
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• 2007

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.517-522
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• 1998

The cry1 gene content of a collection of Bacillus thuringiensis strains, which include new isolates from Malaysia and Indonesia, was determined by a multiplex PCR using a set of eight oligonucleotide forward primers specific to cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1Da, cry1Ea, and cry1Fa genes, and two reverse primers, one specific to cry1Ab and the other common to the remaining cry1 genes. Two-thirds of the 59 strains screened were cry1 positive and contained one to four different genes. The cry gene profiles correlated well with toxicities of the strains to lepidopteran insects. The method can be used for rapid screening of a large number of new isolates as the total DNA extracted by boiling cells from single colonies can be used directly in the PCR. However, it is not suitable for follow-up monitoring of specific commercial strains after application in the field as the PCR product profiles of these strains could not be differentiated from those of new isolates.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• Plant Biotechnology Reports
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• 제4권4호
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• pp.293-301
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• 2010

The feasibility of two strategies for transgene pyramiding using Agrobacterium-mediated transformation was investigated to develop a transgenic potato (Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) (Phthorimaea operculella (Zeller)). In the first approach, cry1Ac9 and cry9Aa2 genes were introduced simultaneously using a kanamycin (nptII) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry1Ac9 gene, using a hygromycin resistance (hpt) selectable marker gene, into an existing line transgenic for a cry9Aa2 gene and a kanamycin resistance (nptII) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.

• 한국미생물·생명공학회지
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• 제33권2호
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• pp.154-158
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• 2005

Twenty-three Bacillus thuringiensis strains isolated from Korea were screened to detect the cry-type genes using PCR with 21 specific oligonucleotide primers. Eight strains contained distinct multiple crystal genes; cry1Aa2, cry1Ab1, cry1Ac1 and cry2Aa1. These results indicate that the strains coincided with the B. thuringiensis subsp. kurstaki strain. The other 15 strains were not recognised to the 21 specific primers.

• Journal of Microbiology and Biotechnology
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• 제4권3호
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• pp.171-175
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• 1994

To find new crystal protein genes, we screened 42 Bacillus thuringiensis strains of serovar standards by Southern hybridization with a cryI-specific probe which was amplified from B. thuringiensis subsp. kurstaki HDl by polymerase chain reaction (PCR). Two strains, B. thuringiensis subsp. entomocidus HD9 and subsp. subtoxicus HD109, generated weak signals under the low-stringency hybridization conditions. Further analysis with Southern hybridization revealed that the two strains contained cryIB genes which are slightly different from those of B. thuringiensis subsp. thuringiensis HD2. These results were confirmed by PCR with cryIB-specific primers followed by the restriction analysis of PCR products.

• Asian Pacific Journal of Cancer Prevention
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• 제13권11호
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• pp.5725-5728
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• 2012

Growing evidence shows that deregulation of the circadian clock plays an important role in the development of malignant tumors, including gliomas. However, the molecular mechanisms of gene chnages controlling circadian rhythm in glioma cells have not been explored. Using real time polymerase chain reaction and immunohistochemistry techniques, we examined the expression of two important clock genes, cry1 and cry2, in 69 gliomas. In this study, out of 69 gliomas, 38 were cry1-positive, and 51 were cry2-positive. The expression levels of cry1 and cry2 in glioma cells were significantly different from the surrounding non-glioma cells (P<0.01). The difference in the expression rate of cry1 and cry 2 in high-grade (grade III and IV) and low-grade (grade 1 and II) gliomas was non-significant (P>0.05) but there was a difference in the intensity of immunoactivity for cry 2 between high-grade gliomas and low-grade gliomas (r=-0.384, P=0.021). In this study, we found that the expression of cry1 and cry2 in glioma cells was much lower than in the surrounding non-glioma cells. Therefore, we suggest that disturbances in cry1 and cry2 expression may result in the disruption of the control of normal circadian rhythm, thus benefiting the survival of glioma cells. Differential expression of circadian clock genes in glioma and non-glioma cells may provide a molecular basis for the chemotherapy of gliomas.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1057-1062
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• 2004

The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. Insecticidal proteins, coded by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. We isolated a Bacillus thuringiensis strain having insecticidal activity against larvae of the house fly (M. domestica) from the soils at a pig farm in Korea, and named it Bacillus thuringiensis SM. The culture filtrate from Bacillus thuringiensis SM showed strong lethality (83.3%) against M. domestica larvae. The parasporal crystal is enclosed within the spores' outermost envelope, as determined by transmission electron microscopy, and exhibited a bipyramidal form. The crystal proteins of strain SM consisted of five proteins with molecular weights of approximately ~130, ~80, ~68, ~42, and ~27 kDa on a 10% SDS-PAGE (major band, a size characteristic of Cry protein). Examination of antibiotic resistance revealed that the strain SM showed multiple resistant. The strain SM had at least three different plasmids with sizes of 6.6, 9.3, and 54 kb. Polymerase chain reactions (PCRs) revealed the presence of cry1, cry4A2, and cry11A1 genes in the strain SM. The cry1 gene profile of the strain SM appeared in the three respective products of 487 bp [cry1A(c)], 414 bp [cry1D], and 238 bp [cry1A(b)]. However, the strain SM has not shown the cry4A2 md cry11A1 genes. In in vivo toxicity assays, the strain SM showed high toxicity on fly larvae (M. domestic) [with $LC_{50}$ of 4.2 mg/ml, $LC_{90}$ of 8.2 mg/ml].