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Target DNA 염기서열 내에 존재하는 비상동성 간격이 상동성재조합을 이용한 클로닝 빈도에 미치는 영향
김재우,도은주,윤세련,정윤희,윤영호,임선희,선우양일,박인호,Kim Jae-Woo,Do Eun-Ju,Yoon Se-Lyun,Jeong Yun-Hee,Yoon Young-Ho,Leem Sun-Hee,Sunwoo Yangil,Park In-Ho 한국미생물학회 2005 미생물학회지 Vol.41 No.4
Transformation-associated recombination (TAR) 클로닝 법은 복잡한 게놈으로부터 염색체 내의 특정부위나 유전자를 선택적으로 분리할 수 있다. 이 방법은 목적 유전자에 근접한 작은 게놈DNA 염기서열 정보를 필요로 한다. 이 기술은 효모의 spheroplast transformation을 시키는 동안 목적으로 하는 유전자의 5' 또는 3' 서열을 포함하고 있는 TAR vector와 게놈DNA사이에서 일어나는 상동성재조합에 의해 이루어진다. 본 연구에서는 plasmid 모델시스템을 이용하여 target hooks 내에 존재하는 비상동성 염기서 열이 상동성재조합에 미치는 영향을 조사하였다. plasmid에 존재하는HIS3유전자와 변형시킨 his3-TRP1-his3 단편 사이의 상동성재조합의 효율은 $Ura^+$ 형질전환체의 형질분석에 의해 이루어졌다. $Ura^+$ 형질전환체의 수는 7종류의 서로 달리 변형된 his3-TRP1-his3 단편들을 사용하였을 매 거의 동일하게 나타났다. 그러나 $Trp^+His^+$ positive recombinants의 빈도는 변형된 his3-TRP1-his3 단편 내에 비상동성 영역에 부정확한 간격을 지닐 때 현저한 감소를 나타내었다. 이러한 결과로서, 부정확한 간격이 target hook과 substrate DNA 사이에 일어나는 상동성재조합을 방해하는 것으로 사료된다. 그러므로 이종간의 상동유전자를 클로닝 할 때에는 target hook내의 비상동성 염기서열이 존재한다면 이것이 정확한 간격을 지니는지 여부를 중요란 요인으로 고려해야 한다. Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences. In this study, we examined the effect of non-homologous spacing sequence in target hooks on homologous recombination using a plasmid model system. The efficiency of homologous recombination between the modified his3-TRP1-his3 fragments and HlS3 gene on plasmid were analyzed by the characterization of $Ura^+$ transformants. The numbers of $Ura^+$ transformant showed same level when seven different modified his3-TRP1-his3 fragments were used. But the percentage of positive recombinants. $Trp^+His^-$, dramatically decreased when used the modified his3-TRP1-his3 fragments contained incorrect spacing of nonhomologous region. As a result, we suggest that incorrect spacing inhibits the homologous recombination between target hook and substrate DNA. Therefore, we should consider the correct spacing in target hook when the target hook are used for cloning of orthologue gene.
Anabaena 의 광합성 전자전달 활성에 미치는 Simazine 의 억제효과
권혁동(Hyuk Dong Kwon),이혜주(Hae Joo Lee),선우양일(Yangil Sunwoo),이진범(Chin Bum Lee),박인호(In Ho Park) 한국식물학회 1988 Journal of Plant Biology Vol.31 No.3
Effects of simazine [2-chloro-4,6-bis(methylamino)-s-triazine] on the photochemical reactions of isolated spinach chloroplasts and crude thylakoids of Anabaena inequalis UTEX B-381 were compared. Simazine inhibited photosynthetic O_2 evolution and increased the chlorophyll fluorescence in whole cells of Anabaena. The electron transfer from diphenylcarbazide to 2,6-dichlorophenolindophenol was inhibited by simazine treatment in spinach chloroplasts, but not in crude thylakoids of Anabaena. In spinach chloroplasts, the chlorophyll fluorescence was increased by simazine treatment in the presence of diphenylcarbazide and ferricyanide, but not in the presence of diphenylcarbazide and silicomolybdate. In crude thylakoids of Anabaena, simazine treatment did not increase the chlorophyll fluorescence in the presence of either diphenylcarbazide and silicomolybdate, or diphenylcarbazide and ferricyanide. These results suggest that the inhibitory site of simazine on photosynthetic electron transport chain of Anabaena is different from that of spinach chloroplasts. And there may be a possibility that the inhibition site of simazine in Anabaena lies on the donor side of photosystem Ⅱ, before the site of electron donation by diphenylcarbazide.
엽록체의 전자전달과 광음향 신호에 미치는 Simetryne 의 영향
전현식(Hyun Sik Chun),이혜주(Hae Joo Lee),선우양일(Yangil Sunwoo),박인호(In Ho Park),이진범(Chin Bum Lee) 한국식물학회 1988 Journal of Plant Biology Vol.31 No.3
The effects of simetryne on light induced electron transport and phosphorylation in isolated spinach (Spinacia oleracea L.) chloroplasts were investigated in comparison with sencor and DCMU. Simetryne, like sencor and DCMU, completely, inhibited PSⅡ electron transport and phosphorylation with 10^-6 M treatment but did not inhibit PSⅠ electron transport. Interference with the electron transport pathway was evidenced by the greater sensitivity of oxygen evolution and uptake than phosphorylation. The following order of decreasing inhibitory effectiveness was exhibited; DCMU>simetryne>sencor. The photoacoustic technique was also used to monitor the relative photosynthetic activity in the leaves treated with the herbicides (simetryne, sencor or DCMU) in viva and in vitro. Photoacoustic measurements on intact leaves provide quantitative information on two related aspects of the photosynthetic process, namely, photochemical energy storage and oxygen evolution. The relative photoacoustic signal of leaves treated with the herbicides showed low level in 21 Hz, but high level in 380 Hz and on isolated chloroplasts (both 21 Hz and 380 Hz) in comparison with that of the untreated leaves. These results suggest that some of photochemical energy is converted into the heat owing to the inhibition of electron transport pathway by the herbicides.
Polyvinylalcohol 에 고정화한 시금치 엽록체의 광합성특성에 대한 연구
박인호(In Ho Park),서상희(Sang Hee Seo),이혜주(Hae Joo Lee),이진범(Chin Bum Lee),선우양일(Yangil Sunwoo) 한국식물학회 1991 Journal of Plant Biology Vol.34 No.3
Photosynthetic properties of polyvinylalcohol (PVA)-immobilized chloroplast especially regarded to stability of photosynthetic electron transport and the fluorescence induction pattern were studied. When isolated spinach chloroplasts were immobilized with PVA, it showed good preservation of photosynthetic electron transport activity, especially PS Ⅱ activity, during storage at -15℃, 4℃ and 20℃. And immobilized chloroplasts revealed similar thermostability of whole chain electron transport to free chloroplasts. And the absorption peak of red band of chloroplasts showed the blue-shift of 2-4 ㎚ after immobilization. F_v/F_m ratio of chlorophyll fluorescence slightly decreased after immobilization. While light pulse after continuous light do not induced the additional fluorescence rise. This means chlorophyll fluorescence at room temperature reached to F_max under continuous light in the immobilized chloroplasts. It seems that PVA may be a good candidate for immobilization matrix for the preservation of photosynthetic function of thylakoids and for the continuous use of chloroplast membranes of higher plants for solar energy storage and conversion.
SLC6A19 Minisatellites 7(SLC6A19-MS7)의 심근경색과의 관련성과 진화적 분석
설소영(So-Young Seol),이상엽(Sang-Yeop Lee),염지훈(Ji-Hoon Yum),윤해순(Hae-Soon Yoon),선우양일(Yangil Sunwoo) 대한약학회 2010 약학회지 Vol.54 No.1
SLC6A19 which reported as a neurotransmitter was composed of seven minisatellites. In previous our study, the minisatellites variants of SLC6A19-MS7 showed the susceptibility for hypertension. When this minisatellte sequences were analyzed using the bioinformatic tool, USF1 (upstream transcription factor 1) was found in this region as a putative transcription factor binding site. USF1 is binding with E-boxes which has a consensus sequence of CACGTG. USF1 is a ubiquitously expressed transcription factor and involved in the transcriptional control of many genes including the molecular pathogenesis of cardiovascular disease. Thus, we investigated that the putative functional relationship between the minisatellites variants and susceptibility for myocardial infarction. A case-control study was performed that compared genomic DNA from 400 controls and 225 cases with myocardial infarction. There were no significant differences observed in the overall allelic distribution of minisatellites between controls and cases, which indicates that this polymorphism is not responsible for myocardial infarction susceptibility. Hence, we analyzed the five different minisatellites alleles from this study and characterized 14 different repeats units (Unit1~Unit14). Then, we evaluated the DNA composition, phylogenic tree, and pairwise distances of its repeats. The variability of each repeats differed from 2.33% to 16%. The phylogenic trees for the four SLC6A19-MS7 minisatellites exhibited very different shapes in their braches and distances, and present most common 8 repeats allele was the longest 14 repeats allele. Therefore, this result may help to understand for the evolutional level of the length of minisatellites.