• 식물병연구
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• 제13권2호
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• pp.88-92
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• 2007

우리나라 참다래 과수원에서 참다래 꽃썩음병을 일으키는 원인 세균인 Pseudomonas syringae pv. syringae를 분리하였다. 총 41개 균주 중 스트렙토마이신 저항성을 보이는 2개의 균주를 대상으로 PCR과 염기서열 결정을 통해 저항성 유전자 구조를 조사하였다. PCR결과 스트렙토마이신 저항성유전자는 Tn5359a에 들어 있는 strA-strB 구조인 것으로 밝혀졌다. Xanthomonas campestris와 Erwinia amylovora에서 알려진 IS6100과 IS1133은 발견되지 않았다. strA-strB의 염기서열은 이미 밝혀진 Tn5393a와 동일하였다. 두 스트렙토마이신 저항성 균주는 각각 3개의 플라스미드를 가지고 있었으며 저항성 유전자는 그 중 100kb의 플라스미드에 들어 있었다.

• 한국연초학회지
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• 제23권2호
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• pp.123-132
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• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• 대한수의학회지
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• 제39권2호
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• pp.327-337
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• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.

• 대한수의학회지
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• 제40권1호
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• pp.42-48
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• 2000

Akabane disease is transmitted through mosquitoes in cattle, sheep and goats. It shows congenital abnormalities including encephalomyetitis, hydranencephaly, neurogenic arthrogryposis, and deformed neonatal calves. Akabane viruses, 93FMX and K-9 strain, were isolated from fetal matrix of aborted cow and blood of healthy cow, respectively. S gene sequences of 93FMX and K-9 showed 100% homology with that of OBE-1 strain isolated in Japan. Based upon our sequencing data, we synthesized specific primers for PCR diagnosis. Using these primers, we were able to amplify the S gene of Akabane virus not only from the culture fluid of Vero cells but also from the brain tissue of suckling mouse inoculated with, Akabane virus. These PCR products were confirmed by Southern blot hybridization. Not only the sensitivity of PCR test was high enough to detect the viruses of $10^{1.0}TCID_{50}/ml$, but also the time for diagnosis was significantly shorter than that of the virus isolation by tissue culture method. This method was also effective for the detection of Akabane virus in the cerebrum of fetus. RT-PCR method may be used for a useful diagnostic test of the clinical cases of Akabane disease.

• Journal of Microbiology
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• 제34권2호
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• pp.144-150
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• 1996

During growth of the genetically engineered E. coli CU103 in different media, extracellular DNAs released from the cells were studied. The extracellular DNAs released in the medium were concentrated by an thanol precipitation method and then quantified by a fluorescence method using Hoechst 33258. The released extracellular DNAs were also examined by gel electrophoresis and identified by Southern hybridization for the cloned pcbCD genes. The chromosomal DNAs and recombinant plasmid containing the cloned genes were observed to be released in an exponential growth phase. In Luria-Bertani (LB) broth and MM2-GLUCOSE, 210 and 69 ng/ml of DNAs were detected, respectively, after 3-4 days incubation at $30^{\circ}C$ and at pH 7.0. But the released DNAs were measured to be about 10-15 ng/ml in filtered river water (FW) and Tris-EDTA (TE). The at both $15^{\circ}C$ and $4^{\circ}C$, but the released DNAs were more easily degraded at the higher temperature. The extracellular DNAs were produced about 2 times more at pH 7.0 than at both pH 5.0 and pH 9.0 in MM2-glucose medium at $30^{\circ}C$. Therefore, the extracellular DNAs were found to be released actively from the cells during growth in liquid media.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.17-20
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• 1999

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.222-227
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• 1991

Rhizobium fredii USDA193은 대두(Peking)의 근모 세포벽에 침투하여 근류를 형성한다. 본 실험에서는 전보 (1)에서 분리 보고한 R.fredii의 pectate lyase 유전자 clone(SY1)으로부터 $\Omega$변이를 시켰다. 이것을 R.fredii USDA193에 다시 marker-exchange시켜 얻은 변이주(R.fredii USDA193$\Omega$와 R.fredii USDA193omega1)의 pectate lyase 활성이 완전히 block되지 못하였다. R.fredii 내에서는 다른 종류의 pel 유전자가 존재하 것으로 생각되며 pelB 및 pelE의 Rhizobium mutants들은 근류형성 초기단계에서 직접적으로 영향을 미치지 못하였다.

• 한국양식학회지
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• 제22권1호
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• pp.74-78
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• 2009

동자개(PF) 암컷과 대농갱이(LU) 수컷에 의해 유도된 잡종(FU)을 대상으로 동자개 및 대농갱이 암컷과 역교배체(PFU, LFU)를 유도하여 수정률, 부화율, 초기생존을, 염색체 수 및 핵형분석을 수행하였다. 유도된 PFU ($PF{\times}FU$)의 수정률은 86.8%, 부화율은 70.7% 및 생존율은 76.4%로 양친으로 사용된 PF 및 LU보다 유의적으로 높게 나타났다. 그러나 FU를 사용한 LFU ($PF{\times}FU$)는 모두 사망하였다. PF (2n=52)와 FU(2n=52)간의 PFU의 염색체 수는 2n = 52로 양친과 동일하게 나타났으며 핵형분석 결과 22 metacentrics + 20 submetacentric + 10 acrocentric으로 구성되어 있었다.

• Journal of Plant Biology
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• 제36권2호
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• pp.127-132
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• 1993

Self-incompatibility in Brassica campestris is controlled by multi-allele system in a single genetic locus, the S locus, and it is elucidated that S-glycoproteins are S gene products. In this experiments, we examined the genetic mode(pollen tube behavior and segregation of S-glycoprotein), characteristic of S-glycoproteins and DNA constitution within nuclear genome on S gene family that unexplained by single locus model, and investigated the segregation pattern of S-glycoproteins in bred F1 generation. By diallel cross among the 15 plants within one family the existence of three types of homozygotes and three types of heterozygotes were observed, and segregation of S-allele could not explained by single locus model. From the results of IEF-immunoblot analysis for non-segregated individual plant, the segregation pattern of S specific bands was corresponded with results of diallel cross except with one case(SaSa genotype). The molecular weight of 6 different S-genotype varied in near by 50 kD, and each genotype expressed with 2 or 3 bands. Specific bands in SaSa, SbSb, ScSc has almost similar molecular weight between them. Southern analysis of genomic DNA probed with S-glycoprotein cDNA for 6 different genotypes revealed that there are clear difference in polymorphism, multiple bands of hybridization, when restriction enzymes of EcoR I were used. It could be assumed that there are several sequences related to the S-glycoprotein structural genes within their nuclear genome. Therefore, we suggested the possibilities that S-allele system could be controlled by multi-locus, that dominance-recessive interactions could be explained by modifier gene or supressor gene based on the results of abnormal segregation of S-glycoprotein in bred F1. The F2 analyses are progressing in now.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.