• KSBB Journal
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• v.25 no.2
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• pp.167-172
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• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.111-118
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• 1997

Four separate pairs of oligonucleotide primers within the coding region in a T sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme 3nd Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. morginnle and 3. ouata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive. PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.103-108
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• 2003

LinA gene involving in the ${\gamma}$-benzenehexachloride degradation have been cloned from Sphingmonas paucimobilis UT26. This linA gene which catalyzes the first dechlorination step of ${\gamma}$-benzenehexachloride is known to play a key role in the ${\gamma}$-benzenehexachloride degradation pathway in UT26. In this study, the linA gene was designed to clean-up the ${\gamma}$-benzenehexachloride and its derivatives contaminated in soil, water and air using transgenic tobacco plants. The linA transgene was introduced into the chromosome of tobacco using leaf-disk transformation approach as revealed by Southern blot analysis. In addition, mRNA and protein produced by linA gene was expressed at a high level in the leaf tissue as demonstrated by both northern blot analysis and Western bolt analysis with polyclonal antibody against S. paucimobilis UT26. in vitro analysis using GC-MS showed that transgenic tobacco plant produced the linA protein which effectively degraded ${\gamma}$-benzenehexachloride into ${\gamma}$- pentachlorocyclohexene and 1,2,4-trichlobenzene compounds which are less toxic.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.115-125
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• 1994

In this study, we try to establish the rapid and specific detection system for Salmonella species. The PhoE gene of Salmonella species was amplified with two specific primers, ST5 and ST8c, using PCR. The probe prepared from the amplified PhoE gene was sequenced and applied for Southern blot analysis. After PCR with ST5 and ST8c primers for PhoE gene, DNA bands of expected size(365bp) from 7 different Salmonella species were detected, but not from 12 enterobacteriaceae and 3 gram positive bacteria. PCR was highly sensitive to detect up to 10fg of purified DNA template and to identify Salmonella species with only 320 heat-lysed bacterial cells. The inhibition of PCR amplification from stool specimen was occurred with 50-fold dilution but disappeared over 100 fold dilution of samples. It was confirmed that the PhoE genes were amplified and cloned with over 97% nacleotide sequence homology of PCR products compared with that of S. typhfmurium LT2. The DNA probe derived from S. typhimurium TA 3,000 showed highly specific and sensitive reaction with PCR products of all tested Salmonella species. These results indicate that PCR was rapid and sensitive detection method for Salmonella species and DNA probe prepared from S. typhimurium TA 3,000 was specific to identify PCR products of different Salmonella species.

• The Korean Journal of Mycology
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• v.25 no.3 s.82
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• pp.167-175
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• 1997

The PCR fragments of two distinct chitin synthase genes, PdCHSl and PdCHS2, were cloned from Penicillium diversum KCTC 6786. The nucleotide sequences of PdCHSl and PdCHS2 contained uninterrupted open reading frames (ORFs) of 570 bp excluding the primer sequence. The similarity analysis of the deduced amino acid sequences using BLASTP indicated that the possible evolutionary relationship between P. diversum and ascomycetous fungi. Multialignment of the deduced amino acid sequences of PdCHSs using CLASTAL W revealed that the PdCHSs fell into two different classes: PdCHSl into Class I and PdCHS2 into Class II of chitin synthase defined by Bowen et al. (1992). By Southern blot analysis, it was shown that each of the two genes is present as a single copy in the genome of P. diversum KCTC 6786.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.191-197
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• 1994

It is generally known that mutations in any of several genes encoding photoreceptor-specific proteins have resulted in retinitis pigmentosa (RP), a disease characterized by losing photoreceptor function with progressive degeneration of photoreceptor cells and eventually leading to blindness. To study the procure and cure of photoreceptor degeneration, we produced transgenic mice. Transgene consisted of a 12.5kb genomic DNA fragment that contains mutated pig rhodopsin gene (Pro-347-Ser) including both the 5'-franking (4.0 kb) and the 3'-franking (2.9 kb) sequences. This gene was used for the production of transgenic mouse. The mutated rhodopsin DNA was microinjected into male pronuclei of fertilized mouse (C57BL /6]) embryos. We detected transgenic animals harboring mutated rhodopsin gene by PCR and Southern blot analysis. These transgenic mice showed stable transmission of microinjected rhodopsin gene into their offspring. Therefore these animals will provide a novel approach to study the mechanism of the photoreceptor degeneration and be provided as a disease model for the treatment of the blind in human.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.502-509
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• 2005

Sexual reproduction plays an important role in the biology and epidemiology of oomycete plant pathogens such as the heterothallic species Phytophthora infestans. Recent worldwide dispersal of A2 mating type strains of P. infestans resulted in increased virulence, gene transfer, and genetic variation, creating new challenges for disease management. To develop a genetic assay for mating type identification in P. infestans, we used the Amplified Fragment Length Polymorphism (AFLP) technique. The primer combination E+AT/M+CTA detected a fragment specific to A1 mating type (Mat-A1) of P. infestans. This fragment was cloned and sequenced, and a pair of primers (INF-1, INF-2) were designed and used to differentiate P. infestans Mat-A1 from Mat-A2 strains. The Mat A1-specific fragment was detected using Southern blot analysis of PCR products amplified with primers INF-1 and INF-2 from genomic DNA of 14 P. infestans Mat-A1 strains, but not 13 P. infestans Mat-A2 strains or 8 other isolates representing several Phytophthora spp. Southern blot analysis of genomic DNAs of P. infestans isolates revealed a 1.6 kb restriction enzyme (EcoRI, BamHI, AvaI)-fragment only in Mat-A1 strains. The A1 mating type-specific primers amplified a unique band under stringent annealing temperatures of $63^{\circ}C-64^{\circ}C$, suggesting that this PCR assay could be developed into a useful method for mating type determination of P. infestans in field material.

• Journal of Life Science
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• v.14 no.2
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• pp.368-373
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• 2004

We have obtained fertile transgenic rice plants resistant to the broad spectrum herbicide Bast $a^{(R)}$ (active ingredient phosphinothricin, PPT) by PEG-mediated transformation procedure. The plasmid pCaMV35S::Bar was used to deliver the bar gene into embryogenic suspension culture-derived protoplasts of rice (Oryza sativa L.). Transformed plants were regenerated and selected on medium containing 15 mg/l of phosphinothricin. Stable integration and expression of the bar gene in transgenic rice plants was confirmed by Southern and Northern blot analysis. Transgenic $R_1$ plants were also confirmed by assays for phosphinothricin acetyltransferase (PAT) activity. The bar gene was expressed in the primary transgenic rice plants and in the next generation progeny, in which it showed a 3 : 1 Mendelian inheritance pattern. Transgenic $R_1$ and $R_2$ plants were resistant to the herbicide Bast $a^{(R)}$ when sprayed at rates used in field practice.ice.

• Animal cells and systems
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• v.12 no.3
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• pp.137-141
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• 2008

Molecular cloning revealed the three isoforms($Ca_v3.1,\;Ca_v3.2,\;and\;Ca_v3.3$) of the T-type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T-type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat $Ca_v3.1$ T-type channel by reverse-transcription-polymerase chain reaction(RT-PCR) to further explore diversity of $Ca_v3.1$. Interestingly, analyses of cloned RT-PCR products displayed that there were at least four splicing variants of rat $Ca_v3.1$ in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was $Ca_v3.1a$(492 bp), which were rarely detected in most of peripheral tissues. Other two variants($Ca_v3.1c$, 546 bp; $Ca_v3.1d$, 525 bp) were detected in most of the tissues examined. The smallest isoform($Ca_v3.1b$, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.

• Korean Journal of Plant Resources
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• v.27 no.4
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• pp.392-399
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• 2014

Plant senescence is one of the survival strategies to use limited nutrients efficiently during growth, development and adaptation. In this study, we isolated a gene (PagSEN1) homologous to SENESCENCE 1 from Populus alba ${\times}$ P. glandulosa. The PagSEN1 gene encodes a putative protein consisting of 243 amino acids containing a rhodanese domain. Southern blot analysis suggested that two copies of the PagSEN1 gene are present in the poplar genome. We characterized its transcriptional expression under various conditions mimicking senescence and environmental stresses. The PagSEN1 was expressed most strongly in mature leaves but most weakly in roots. The gene was significantly up-regulated by treatments with mannitol, NaCl, ABA and JA, but not by cold, SA and GA3. These results indicate that PagSEN1 is involved in senescence response induced by environmental stresses.