• The Plant Pathology Journal
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• v.23 no.2
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• pp.51-56
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• 2007

A plant pathogenic fungus, Gibberella zeae (anamorph: Fusarium graminearum), not only generates economic losses by causing disease on cereal grains, but also leads to severe toxicosis in human and animals through the production of mycotoxins in infected plants. Here, we characterized a histidine auxotrophic mutant of G. zeae, designated Z43R1092, which was generated using a restriction enzyme-mediated integration (REMI) procedure. The mutant exhibited pleiotropic phenotypic changes, including a reduction in mycelial growth and virulence and loss of sexual reproduction. Outcrossing analysis confirmed that the histidine auxotrophy is linked to the insertional vector in Z43R1092. Molecular analysis showed that the histidine requirement of Z43R1092 is caused by a disruption of an open reading frame, designated GzHIS7. The deduced product of GzHIS7 encodes a putative enzyme with an N-terminal glutamine amidotransferase and a C-terminal cyclase domain, similar to the Saccharomyces cerevisiae HIS7 required for histidine biosynthesis. The subsequent gene deletion and complementation analyses confirmed the functions of GzHIS7 in G. zeae. This is the first report of the molecular characterization of histidine auxotrophy in G. zeae, and our results demonstrate that correct histidine biosynthesis is essential for virulence, as well as sexual development, in G. zeae. In addition, our results could provide a G. zeae histidine auxotroph as a recipient strain for genetic transformation using this new selectable marker.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.255-259
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• 2004

Agrobacterium tumefaciens-mediated cotyledonary node transformation was used to produce transgenic soybean. Cotyledonary node explants of three cultivars and one genotype were co-cultivated with strains Agrobacterium (LBA4404, GV3101, EHA101, C58) containing the binary vectors (pCAMBIA3301 and pPTN289) carrying with CaMV 35S promoter-GUS gene as reporter gene and NOS promoter-bar gene conferring resistance to glufosinate (herbicide Basta) as selectable marker. There was a significant difference in the transformation frequency depend on bacteria strain. The EHA101 strain of the bacterial strains employed gave the maximum efficiency (3.6%). One hundred-six lines transformed showed the resistance in glufosinate. Histochemical GUS assay showed that at least 11 plants transformed with the GUS gene were positive response. The soybean transformants were obtained from the Thorne (5 plants), 1049 (5 plants) and Bakun (1 plant), respectively. Southern blot analysis and leaf painting assay revealed that the GUS and bar gene segregated and expressed in their progeny.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.1
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• pp.19-26
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• 2001

Transformed Spodoptera frugiperda (Sf9) cells expressing baculovirus very late factor (VLF-1) were constructed by using Autograha nuclear polyhedrosis virus (AcNPV) immediate earthy gene (ie1). Neomycin-resistance gene as a selectable marker was introduced under the control of AcNPV ie1 promoter, and Bombyx mori nuclear polyhedrosis (BmNPV-K1) vlf-1 gene was introduced under the control of the Drosophila heat shock protein gene (hspr70) promoter to yield dual expression plasmid with two independent transcription units. It was transfected into Sf9 cells and cell clones expressing vlf-1 were selected by G4l8 treatment. Genomic DNA from transformed cells was isolated and integration of AcNPV iel harboring vlf-1 was confirmed by PCR using AcNPV iel-specific primers and Southern blot analysis. The transformed cells expressing VLF-1 in an infection-independent manner expressed foreign gene product of recombinant baculovirus in the earlier stage of infection compared with control Sf9 cells. These results suggest the possible to develop highly efficient transformed insect cells for baculovirus expression vector system.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.243-247
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• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.162-167
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• 2007

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-hostrange IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-hostrange IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The $Km^r\;and\;Sm^r$ selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.45-53
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• 2010

We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of a gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic resistance gene to create a "mazF-cassette". A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Journal of Korea Multimedia Society
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• v.6 no.3
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• pp.419-428
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• 2003

Several methods have been studied to prune the parasitic branches that cause unfortunately from thinning a shape to get its skeleton. We found that the symmetric path finding method was most efficient because it followed the boundary pixels of the shape just once. In this paper, its extended method is proposed to apply to removing the noise branches that protrude out of the boundary of a segmented or extracted shape in a given image. The proposed method can remove a noise branch with one-pixel width and also remove the noise branch that includes a round shape called a noise blob. The method uses a 4-8-directional boundary-following technique to determine symmetric paths and finds noise branches with noise blobs by detecting quasi-symmetric paths. Its time complexity is a linear function of the number of boundary pixels. Interactively selectable parameters are used to define various types of noise branches flexibly, which are the branch - size parameter and the blob-size parameter. Experimental results for a practical shape and various artificial shapes showed that the proposed method was very useful for simplifying the shapes.

• Journal of Plant Biotechnology
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• v.5 no.4
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• pp.215-219
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• 2003

Optimal shoot regeneration and transformation conditions of root type chicory (Cichorium intybus L. var. sativus cv Cesare) were studied. Leaf explants were co-cultured with Agrobacterium tumefaciens, which contained NPTII as a selectable marker and a rice homeotic gene, OsMADS1, that encodes a MADS-domain-containing transcription factor. After one day of co-cultivation, explants were transferred to selection media consisting of MS basal medium supplemented with 0.5 mg/L BAP, 0.1 mg/L IAA, 70 mg/L kanamycin, and 250 mg/L cefotaxime. PCR and Southern blot analyses revealed stable integration of the OsMADS1 gene in the chicory genome. Four-teen original transgenic plants ($T_o$ plants) were acclimatized in the greenhouse and examined for their morphological characters. Most of the transgenic plants showed altered morphologies, such as short, bushy, and early-flowering phenotypes with reduced apical dominance. Additionally, half of the transgenic plants exhibited altered leaf shapes, and 4 out of 14 plants were sterile. These phenotypes were inherited by the next generation. Northern blot analysis confirmed expression of the OsMADS1 gene in both floral and vegetative organs.