• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.362-368
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• 1997

Five formae speciales of Fusarium oxysporum in Korea were examined using RFLP analysis to find the possibility for classification and analyze genetic variations. DNAs from F. oxysporum f. sp. lycopersici, cucumerinum, fragariae, garlic and sesami were used with three recombinant probes such as pFC46, pFC52 and pFC57. Distinct differences among five formae speciales of this fungus were detected in RFLP band patterns based on southern hybridization of genomic DNA using each recombinant clone, which was a repetitive copy probe. Strains belong to four formae speciales could be very stable in genetic variation except f. sp. sesami which has more variation than the others based on the RFLP analysis. They formed their own cluster which has high similarity within the same formae specialis resulted from the UPGMA analysis for genetic relationship analysis and each cluster represented its own formae specialis. The method using three recombinant DNA probes could be a good tool for classification of formae speciales in F. oxysporum.

• YAKHAK HOEJI
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• v.38 no.3
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• pp.293-299
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• 1994

Forty nine clinical isolates of S. aureus showing resistance to erythromycin(EM) were selected from 83 strains isolated recently in Korea. Fourteen strains of S. aureus showing inducible resistance to MLS antibiotics were selected by disc agar diffusion method. Colony hydridization was executed using two MLS inducible resistance genes, ermA and ermC, identified previously from S. aureus as probes. S. aureus 375 and S. aureus 507 whose genes were not homologous to those probes were finally selected. It was confirmed that the resistance genes of S. aureus 375 and S. aureus 507 had no homology with those probes in southern hybridization test using ermA, ermC and ermAM as probes. It was determined that S. aureus 375 had a plasmid whose size was about 35 kb. To know if the plasmid may have the genes related to inducible resistance to MLS antibiotics, it was attempted to transform Bacillus subtillis BR151 and S. aureus RN4220 with the plasmid isolated from S. aureus 375. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. aureus showing inducible resistance to MLS antibiotics have novel genes that have no homology with MLS resistance genes identified so far. It is assumed that these genes may exist in chromosomal DNA.

• Journal of Plant Biotechnology
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• v.43 no.3
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• pp.341-346
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• 2016

The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hybridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.

• Journal of Life Science
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• v.10 no.4
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• pp.397-402
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• 2000

Polyphosphate kinase catalyzes the formation of polyphosphate from ATP. To understand the mechanism of phosphate accumulation, the Serratia marcescens gene encoding ppk was cloned from the genomic library by the method of Southern hybridization. The hybridization positive DNA fragment region from pDH3 was subcloned into the expression vector. The ppk gene product, a polypeptide of 75 kDa, was confirmed by SDS-PAGE. Expression of the Serratia marcescens ppk is regulated by the catabolite repression system. The enzyme activity polyphosphate kinase was increased in the E. coli strain harboring plasmid pMH4 with ppk gene.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.215-221
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• 1991

An autonomous replication sequence (ARS) derived from Cephalosporium acremonium ATCC 20339 was cloned in Sarchuromyces cerevisiae SHY 3 using YIp5 as a cloning vector. A new recombinant plasmid, designated pCY-2, which contained a 3.7 kb BamHI fragment of C. acrenzonium DNA showed the highest stability among the 40 recombinant plasmids composed of the YIp5 2nd ARS of C. ucremoniztm. Also, Southern hybridization and transformation of E, cull with DNA purified from yeast transformants verified that pCY-2 autonomously replicates in yeasts. Transformation efficiency and plasmid stability of pCY-2 in yeast were higher than those ol YRp 7 containing ARS which originated from yeast. Detailed studies by subcloning revealed that two ARSs existed within 2.6 kb of the insert, which is a novel discovery. However, it was concluded that these two ARSs were ligated during the gene manipulation in vitro.

• Journal of Life Science
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• v.13 no.4
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• pp.522-528
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• 2003

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the RAD4 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. In order to investigation whether the increase of transcripts by DNA damaging agent, transcripts levels were examined after treating the cells. The level of transcript did not increase by untraviolet light (UV). This result indicated that the RAD4 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the RAD4 homologous gene is essential for cell viability.

• Horticultural Science & Technology
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• v.32 no.1
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• pp.91-99
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• 2014

The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line 'Chiifu' under cold stress condition ($4^{\circ}C$). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. $T_1$ transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under $4^{\circ}C$ treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.

• Journal of Life Science
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• v.12 no.4
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• pp.496-503
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• 2002

To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor $\alpha$1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ $m\ell$.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.286-290
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• 1992

The present study was performed to isolate the fluorescent pseudomonads from Kwang-Ju soil and to construct a mutant strain defective in siderophore biosynthesis. The siderophore-secreting pseudomonads were screened on Blue agar (Chrome Azuol S agar) plates and one strain of them was designated to Pseudominas fluorescens (P. fluorescens) PY002. To construct a mutant defective in siderophore biosynthesis, P. fluorescens PY002 was randomly mutagenized with a transposon Tn5. The location of Tn5 integrated into chromosomal of the mutants strain was determined by Southern blot analysis. The mutagenized strain showed non-fluorescent on a King's B agar plate and were defective in iron (III) acquisition ability.

• The Korean Journal of Zoology
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• v.38 no.2
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• pp.177-185
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• 1995

두 지역에서 채집한 초파리 자연집단에 대하여 난소발생이상 실험에 의한 P 인자 활성과 세포질형을 분석하여 P 전이인자의 계통형을 조사하였다 전체 238 isofemale line을 조사한 결과 strong P와 true M은 존재하지 않았고, 0(weak P)와 M'(pseudo M) strain이 전체의 98.74%를 차지하여 가장 우세하게 분포하고 있었다. P$\pi$25.1 probe를 이용한 in situ hybridization을 행하여 P 전이인자의 copy수를 조사한 결과 평균 42.12개로 나타났으며, 0와 M'의 계통형 간에 유의적인 차이는 없었다 그러나 염색체 firm당 COPy수는 X염색체가 상염색체의 좌 우 각 arm보다 다소 높게 분포하고 있었고. 염색체상 P 전이인자의 삽입부위에 대한 특이적 좌위는 존재하지 않았다 P 전이인자의 분자구조에 대한 변이형을 조사하기 위하여 southern blot hybridization을 행한 결과 2.9kb의 완전한 크기의 분자를 포함하여 여러종류의 단편들이 확인되었다 조사한 모든 isofemale line에서 KP(1. 15kb)인자를 포함하고 있었으며 이들 KP인자가 P-M System의 난소발생이상을 표현하는데 있어 억제적 작용을 하는 것으로 판단되었다.