• 원예과학기술지
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• 제33권2호
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• pp.242-250
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• 2015

기능 유전체 연구에서 이동유전자 또는 T-DNA 도입을 이용한 삽입돌연변이체 분석은 미지의 유전자 기능을 분석할 수 있게 한다. 이에 따라 본 연구에서는 배추(Brassica rapa ssp. pekinensis)와 같은 고등 식물에서 genomic DNA조각을 분리할 수 있는 효과적인 PCR 방법을 개발하였다. 본 연구에서 개발한 variable argument thermal asymmetric interlaced PCR(VA-TAIL PCR)은 single-step annealin-gextension PCR 방법과 길게 구성된 유전자 특이적 primer 및 touch-up PCR 방법이 적용되었으며, 증폭 효율을 증가시키기 위하여 autosegment extension 증폭 방법이 적용되었다. 또한 개발된 VA-TAIL PCR 방법은 배추에 특화된 변성 primer를 Integr8 단백질체 데이터베이스를 분석하여 작성하였으며 대량의 배추 T-DNA 삽입 돌연변이 집단에서 각각의 T-DNA 인접 염기 서열을 분석하는 데 매우 정확하고 효과적인 것으로 분석되었다. 따라서 본 연구에서 개발된 VA-TAIL PCR 방법은 기존에 확인된 염기 서열을 이용하여 양쪽 방향을 모두 분석할 수 있기 때문에, 유전체 연구에서 T-DNA 또는 기 밝혀진 염기 서열에 인접한 미지의 염기서열을 확인하는데 매우 효과적일 것으로 기대된다.

• 한국버섯학회지
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• 제22권2호
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• pp.48-52
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• 2024

This study investigated the microscopic characteristics and genetic relationships of Ganoderma applanatum fruiting bodies. Basidiospores were brown, ellipsoid, and had one or two large vacuoles and a double wall. The surface of basidiospores was smooth or wrinkled and most had numerous small and shallow holes. The length and width of basidiospores of Ganoderma applanatum isolates GBGA-01, GBGA-02, ASI 50167, ASI 52821, ASI 52822, ASI 52823, and ASI 53399 were on average 7.6×4.8 ㎛, 7.9×4.6 ㎛, 7.7×4.9 ㎛, 8.2×5.3 ㎛, 7.7×5.0 ㎛, 8.0×4.9 ㎛, and 7.9×4.9 mm, respectively. In contrast, the basidiospores of Ganoderma lucidum isolate ASI 7125 were 7.7×5.2 ㎛. Using the universal ITS1/ITS4 primer set, the ITS region of the isolates were amplified and sequenced. The ITS sequences were very closely related to G. applantum isolate GBGA-01, GBGA-02, ASI 50167, ASI 52821, ASI 52822, ASI 52823 and ASI 53399, but were not the same species. Whereas, G. lucidum isolate ASI 7125 belongs to different group.

• BMB Reports
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• 제30권2호
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• pp.162-165
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• 1997

RNA I. a negative controller of ColE1-type plasmid replication, is metabolized by several RNases in Escherichia coli. Two small derivatives of RNA I are accumulated at nonpermissive temperatures in an E. coli strain carrying the rnpA49 mutation, a thermosensitive mutation in the rnpA gene encoding the protein component of RNase P. A primer extension analysis was carried out to compare 5' processing of RNA I in the E. coli rnpA49 cells at both permissive and nonpermissive temperatures. Derivatives of RNA I having different 5' ends were observed in the cells grown at permissive and nonpermissive temperatures. Some of the derivatives may be generated by the cleavage of RNase P.

• BMB Reports
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• 제36권3호
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• pp.305-311
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• 2003

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities ($K_a\;=\;5-10{\times}10^6\;M^{-1}$ oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.131-137
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• 1995

The alkaline phosphatase (K-ALPase) gene of Kluyveromyces fragilis has been cloned (1) and determined its base sequences (2) previously in our laboratory. When the K-ALPase gene was expressed in Escherichia coli and Saccharomyces cerevisiae, it showed a constitutive activity in E. coli, and a derepressed activity in S. cerevisiae in phosphate-limited medium. Northem hybridization experiment was performed to elucidate the transcription level of the K-ALPase gene. Northern experiment showed that transcription level of K-ALPase gene in S. cerevisiae was higher in phosphate depletion, but it was higher in high phosphate medium than in phosphate limited medium in K. fragilis. The transcription initiation site of the K-ALPase gene was determined by primer extension analysis. It matched nucleotide position - 169 in relation to the putative trnslational start site.

• BMB Reports
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• 제36권6호
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• pp.529-532
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• 2003

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.

• 미생물학회지
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• 제40권3호
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• pp.254-256
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• 2004

Polymerase chain reaction (PCR) is a powerful tool for precisely amplifying selected DNA sequences that have had a broad impact on genomic studies. When examining human $\alpha$- and $\beta$- tryptase genes which have 95% DNA homology, inconsistent PCR amplification of genomic sequences hampered our progress. This study suggests that long PCR technique on the original DNA digested with restriction enzymes improves both efficiency and sensitivity of PCR. These improved results seem to derived from the effective denaturation of the original genomic DNA template or reduction of formation of secondary structures that block either primer annealing or extension in PCR. Elimination of homo- or hetero-duplex products derived from highly homologous genes provides an additional advantage in this study. This communication describes how the use of restriction enzymes improved these efficiencies, and also facilitated studies of highly homologous genes including tryptase genes.

• Journal of Microbiology
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• 제34권2호
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• pp.137-143
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• 1996

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

• Bulletin of the Korean Chemical Society
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• 제30권4호
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• pp.791-793
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• 2009

Escherichia coli RNase P is composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). Since both subunits are assembled in a 1:1 ratio, expression of M1 RNA and C5 protein should be coordinately regulated for RNase P to be efficiently synthesized in the cell. However, it is not known yet how the coordination occurs. In this study, we investigated how overexpression of C5 protein affects expression of the rnpB gene encoding M1 RNA, using a lysogenic strain, which carries an rnpB-lacZ transcription fusion. Primer extension analysis of rnpB-lacZ fusion transcripts showed that the overexpression of C5 protein increased the amount of the fusion transcripts, suggesting that rnpB expression increases with the increase of intracellular level of C5 protein.

• 식물병연구
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• 제25권1호
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• pp.8-15
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• 2019

전 세계적으로 자두곰보바이러스는 핵과류에 발생하는 중요한 바이러스이다. 2015년 국내 복숭아에서 자두곰보바이러스 감염이 확인된 이후 국내 핵과류 과원에 대한 자두곰보바이러스 발생 조사가 시작되었다. 2016-2017년 국내 핵과류 1,985과원의 30,333주 나무에서 시료를 채집하여 특이 프라이머를 이용한 RT-PCR 검정 결과, 10과원의 21주 나무에서 자두곰보바이러스가 확인되었고, 박멸조치에 따라 감염 주에 대한 폐기 등 방제가 실시되었다. 2016년 자두곰보바이러스에 감염된 복숭아 나무 7주의 total RNA에서 PCR 산물 약 547 bp을 얻은 후 direct sequencing 하여 염기서열을 결정하였다. BLAST 검색 결과 Genbank에 등록된 자두곰보바이러스 D 계통 분리 주들(LC331298, LT600782)과 99% 높은 염기서열 상동성을 보였으며, 국내 복숭아 나무 7개 분리 주 간에 98-100% 염기서열 상동성을 보였다. Phylogenetic tree 분석 결과 한국, 일본, 미국, 캐나다에서 보고된 D 계통과 높은 유연관계를 가지는 것을 확인 할 수 있었다. 이 보고는 2016-2017년 국내 핵과류 과원의 자두곰보바이러스 발생 현황에 대한 첫 보고이며, 이를 바탕으로 발생 주 제거 등 국내 금지급 자두곰보병의 예방 및 방제 대책 수립을 위한 기초자료로 활용하고자 하였다.