• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.544-549
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• 1995

The alkaline elastase is an extracellular serine protease of the alkalophilic Bacillus strain Ya-B. To increase the gene copy number and the production level of the alkaline elastase Ya-B, we designed, on the B. subtilis chromosome, a gene amplification of the 10.6 kb repeating unit containing amyE, aleE (alkaline elastase Ya-B gene) and tmrB. The aleE was inserted between amyE and tmrB, and B. subtilis APT119 strain was transformed with this amyE-aleE-tmrB-junction region fragment. As a result, we succeeded in obtaining tunicamycin-resistant (Tm$^{r}$) transformants (Tf-1, Tf-2) in which the designed gene amplification of 10.6 kb occurred in chromosome. The transformants showed high productivity of $\alpha $-amylase and alkaline elastase Ya-B. The copy number of the repeating unit (amyE-aleE-tmrB) was estimated to be 25, but plasmid vector (pUC19) was not integrated. The amplified aleE of chromosome was more stable than that of plasmid in absence of antibiotics.

• KSBB Journal
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• v.5 no.3
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• pp.299-306
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• 1990

Escherkchla coli/Cownebacterium glutamicum shuttle vectors, pECCGl and pECCG2 were constructed by joining a 3.0 kb C. glutamicum cryptic plasmid pCBl and a 3.94 kb E. coli plasmid pACYC177. Using the plasmid pECCGl, various parameters involved in electroporation system including electric field strength, resistance, DNA concentration, cell concentration and growth stage were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Transformation efficiencies of 106 transformants/$\mu\textrm{g}$ of plasmid DNA were achieved with Corynebacterium glutamicum.

• Mycobiology
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• v.29 no.3
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.

• Journal of the Korean Society of Tobacco Science
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• v.15 no.2
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• pp.161-166
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• 1993

A double - stranded cDNA fragment (436bp) encoding coat protein of tobacco mosaic virus(TMV) was derived from the total 480nucleotides gene after reverse transcription of TMV RNA, and subclorled into a plant expression vector pBl 121, resulting in pBL 430. The plasmid DNA containing this chimeric gene was moved from E. cofi to Agrobacterium tumefaciens strain A28l, and was introduced in시 the tobacco plant by the Agrobocterium Ti - mediated transformtion system. The transformants were selected on a selection media containing kanamycin. The shoots add roots could be differentiated from the explants and whole plants were obtained. From Southern blot hybridization analysis, DNA extracted from transformants, it could be conformed that the chimeric gene fragment was inserted into the genomic DNA of tobacco plant.

• Animal cells and systems
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• v.2 no.2
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• pp.269-275
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• 1998

The reporter plasmid pMT-lacZ containing the metallothionein (MT) promoter region (-320∼+58 with respect to the transcription initiation site) fused to the lacZ gene in a P-element vector was constructed. Transgenic Drosophila bearing the MT-lacZ fusion gene were established by P-element mediated transformation. Expression of the MT-lacZ fusion gene in transformants was examined during development. By treatment with low concentration of cadmium (>1O uM) or paraquat (>50 uM), increased expression of B-galactosidase was shown in fat body, brain lobe, and ganglion transgenic larval tissues. The results show that transformants bearing the MT-lacZ fusion gene are useful for further studies on the mechanism of regulation of MT gene expression and for monitoring toxic metals.

• Journal of Microbiology
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• v.35 no.3
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• pp.181-187
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• 1997

Filamentous cyanobacteria are an ecologically important group of bacteria because they are able to provide both organic carbon fixed nitrogen that can support the nutritional requirements for other microorganisms. Because of their prokaryotic nature, they can also be used as potentially powerful model systems for the analysis of oxygenic photosynthesis and nitrogen fixation. Gene transfer is an indispensable procedure for genetic analysis of filamentous cyanobacteria. Electroporation was used to introduce foreign DNA into cyanobacterial cells. In experiments designed to optimize the electroporation technique, the effects of the field strength (amplitude of pulse) and time constant (duration of pulse), DNA concentration and host restriction/modification of DNA on the efficiency of electro-transformation were investigated. The results of this research revelaed that a high voltage pulse of short duration was effective for the electro-transformation of Anabaene sp. M131. The maximal number of transformants was obtained at 6 kV/cm with a pulse duration of 5 msec. The efficiency of electro-transformation was also sensitive to concenetration of DNA; even small amounts of DNA (0.01 .mu.g/ml) were able to gie a large number of transformants (1.0 * 10$\^$3/ cfu/ml).

• Plant Resources
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• v.4 no.1
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• pp.26-30
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• 2001

To improve the iron content of red pepper, we have transferred the entire coding sequence of the ferritin gene(Fpl) into Capsicum annuum (L. cv. Chungyang and Bukang) by Agrobacterium mediated transformation. Transformants were found to contain the Fp1 gene at up to three loci, increased distinct iron content changes. In transgenic plants, iron content was as much as 7-fold to 8-folds greater than that of their untransformed counterparts. Furthermore, the Rl progenies from transformant(A7, A8) co-segregated into a 15:1 ratio for both Kanamycin resistance and genotype of high iron.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• Applied Biological Chemistry
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• v.30 no.4
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• pp.293-299
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• 1987

Bradyrhizobium japonicum spheroplasts were prepared by culturing cells in the presence of glycine, follwed by treatment with lysozyme. The cells were examined by electron microscopy during the formation of spheroplast. Then Ti plasmid from Agrobacterium tumefaciens 15955 was introduced into Bradyrhizobium japonicum by glycine-lysozyme induced spheroplast transformation. After cell wall regeneration, transformants were selected by the ability of utilization of octopine. Transformation were received at a frequency of $1{\times}10^{-7}$. The transformants obtained from spheroplast transformation harbored the introduced Ti plasmid, which was identified by agarose gel electrophoresis. Furthermore, the differences in their gene products were observed between the transformant and the recipient cell by two-dimensional polyacrylamide gel electrophoresis. The transformants which still possessed the same ability nodulate soybean (Glycine max.) as that of the original host strain, acquired the ability to induce tumor on Petunia hybrida like Agrobacterium, but formed the small crown galls in size compared to those of Agrobacterium tumefaciens.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.450-455
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• 1998

Aspergillus oryzae M-2-3 strain (argB$\^$-/) was transformed with pTAalp plasmid which was constructed for expression of the alkaline pretense gene, alpA, and 16 transformants were selected on arginine minus medium. When these transformants were tested for productivity of alkaline proteases using agar plate containing skim milk, the halo was observed around each colony of transformants, but not observed around the host strain in this condition. Southern analysis showed that the pTAalp plasmid having alpA gene was integrated into the chromosome of the host strain. The highest level of alkaline protease production was obtained in the culture filtrate of the transformant No. 14, which was estimated to 80-90% of total secreted proteins, and the enzyme activity was 64-450 times higher than those of host strain and industrial strain. Total nitrogen content and the digestion rate in soybean Koji extracts were also increased to 1.5 times in Aspergillus oryzae transformant No. 14.