Trilinear Analysis for the Resolution of Overlapping Fluorescence Sepctra

이춘환,김경숙,Lee, Choon-Hwan,Kim, Kyung-Sook,Ross, Robert T. Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.4

중첩된 형광 스펙트럼의 삼중 선형 분석

이춘환,김경숙,Robert t . Ross ( Choon Hwan Lee,Kyung Sook Kim,Robert T . Ross ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.4

The information contained in vivo fluorescence spectra of biological specimens is often obscured by severe overlapping of component spectra. Fluorescence intensity is separately linear in functions of excitation wavelength, emission wavelength, and any chemical treatment which alters overall fluorescence yield. When data are collected for several values of each of these three independent variables, strongly overlapping spectra can be resolved without the use of any a priori information about their shapes. Using concentration of a fluorescence quencher, potassium iodide, as a third variable, a mixture of rhodamine 6G, rhodamine B, and sulforhodamine 101 was used as a test system; the spectra resolved agreed closely with the spectra of the pure dyes. Previously published applications of trilinear analysis to luminescence spectroscopy have required either an instrument capable of time resolution or multiple specimens with varying relative concentrations of fluorophores; use of quencher concentration as a variable permits use of the method with a steady-state fluorometer and without changing fluorophore concentrations. Requiring that the spectra deduced be non-negative can increase the accuracy of the results.

수은 처리에 의한 보리 잎 엽록소 형광의 광의존적 변화

이춘환(Choon Hwan Lee),장호식(Ho Sik Chang),하석봉(Suk Bong Ha),이진범(Chin Bum Lee) 한국식물학회 1995 Journal of Plant Biology Vol.38 No.1

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형질전환된 담배 어린 싹의 녹화과정 중 Arabidopsis Cab 프로모터의 활성 차이

이춘환(Choon Hwan Lee),고예정(Ye Jeong Ko),최홍근(Hong Keun Choi),유태형(Tae Hyong Rhew),이진범(Chin Bum Lee) 한국식물학회 1993 Journal of Plant Biology Vol.36 No.3

Promoters of the chlorophyll a/b binding protein genes, cab1, and cab2, of Arabidopsis thaliana were studied for their functions in differential expression during greening of etiolated shoots. The etiolated shoots were derived from leaves of transgenic tobacco plants with the cab-CAT (chloramphenicol acetyltransferase) translational fusions, and CAT activity was measured to monitor the activities of the cab promoters. Cab1 promoter activity increased rapidly and showed saturation after about 24 hours of greening, but that of cab2 increased with about 2 day-lag period and showed saturation after 6 days, Cab1 promoter activity was more sensitive to levulinic acit (LA) compared with cab2 activity. Cab2 promoter activity was inhibited more sensitively by chloramphenicol (CAP) than by inhibitors of Chl formation. Cab1 promoter activity was, however, inhibited less sensitively by CAP than by LA. The treatment of abscisic acid (ABA) did not block Chl synthesis so significantly as LA treatment did, and cab2 promoter activity was much less sensitive to ABA compared with that of cab1. These results suggest that cab1 expression is strongly related with Chl formation, possibly with δ-aminolevulinic acid accumulation, and cab2 expression is suppressed more by the blockage of translation of Chl a-aporproteins than by the blockage of Chl a accumulation.

온도가 저서규조류 광합성 반응에 미치는 영향: 형광을 이용한 추정

윤미선,이춘환,정익교,Yun, Mi-Sun,Lee, Choon-Hwan,Chung, Ik-Kyo 한국해양학회 2009 바다 Vol.14 No.2

Benthic diatoms are very important primary producers in understanding estuary ecosystems and their productions are largely varied by their photo-physiological characteristics. The short-term effects of increased temperature on the photosynthetic and photo-physiological characteristics of cultured different species of benthic diatoms (Navicula sp., Nitzschia sp., Cylindrotheca closterium, and Pleurosigma elongatum) were investigated by measuring their PSII-fluorescence kinetics using a Diving-PAM. Photosynthesis versus irradiance curves were measured every two hours at six different temperatures (10, 15, 20, 25, 30, and $35^{\circ}C$) for twenty-four hour. The effective quantum yield of PSII ($\Phi_{PSII}$) for most of the species showed a decreasing trend with increased temperature. The relative maximum electron transport rate (rETRmax) was significantly increased up to the optimum temperature level and then sharply decreased. Relative to the values of other parameters, the maximum light use coefficient ($\alpha$) was not substantially changed at lower temperature levels (<$30^{\circ}C$) but significantly decreased only at higher temperatures (30 and $35^{\circ}C$). The light saturation coefficient ($E_K$) mirrored the rETRmax temperature response. In regards to the temperature acclimation abilities of the four species with time, Navicula sp. and C. closterium acclimated to short-term changes in temperature through their photo-physiological adjustments. 저서규조류는 하구역 먹이망을 이해하는 데 중요한 생물로서 그들의 광생리 특성에 따라 일차생산력이 크게 변화한다. 본 연구에서는 단기간 온도 변화가 저서규조류 4종(Navicula sp., Nitzschia sp., Cylindrotheca closterium, Pleurosigma elongatum)의 광합성 반응에 미치는 영향을 Diving PAM을 이용한 엽록소 형광 분석법으로 측정하여 광생리 특성을 분석하였다. 6개의 온도 조건(10, 15, 20, 25, 30, $35^{\circ}C$)에서 2시간 간격으로 24시간 동안 엽록소 형광을 측정하여 P-I 곡선을 도출하였다. 제2광계의 유효양자수율($\Phi_{PSII}$)은 대부분의 종에 있어서 온도가 증가함에 따라 감소하였으며, 상대 최대 전자전달율(rETRmax)은 최적 온도까지 증가한 후 급격하게 감소하였다. 최대 빛이용 효율($\alpha$)은 다른 광합성 매개변수에 비해 온도에 덜 민감하였으나, 높은 온도에서는 감소하였으며, 광포화 계수($E_K$)는 상대 최대 전자전달율의 반응과 매우 유사하였다. 종별 광생리 특성을 분석한 결과, Navicula sp.와 Cylindeotheca closterium가 광생리적 조절을 통하여 단시간의 온도 변화에 광순응하는 것을 확인할 수 있었다.

Generation and Selection of Promoter Trap Lines for the Investigation of Shoot Development in Arabidopsis

이화목,박희연,이춘환,문용환,Lee Hwa-Mok,Park Hee-Yeon,Zulfugarov Ismayil S.,Lee Choon-Hwan,Moon Yong-Hwan Korean Society of Life Science 2006 생명과학회지 Vol.16 No.3

T-DNA 매개 형질전환은 삽입 돌연변이를 가지는 형질전환 식물체를 만들기 위한 유용한 방법이다. 애기장대의 shoot 발달 과정에서 중요한 역할을 하는 유전자를 확인하기 위해, 프로모터 trap 식물체 라인을 제작하고 분석하였다. 이를 위해 효율적인 프로모터 trap 백터인 pFGL561을 제작하였다. pFGL561은 basta 저항성 유전자, multiple splicing donor acceptor 서열들, 그리고 프로모터가 없는 GFP 리포터 유전자를 포함하고 있다. Agrobacterium 균주인 GV3101에 pFGL561을 도입하고, 이를 매개로 하여 300개의 $T_1$ 프로모터 trap 식물체를 제작하였고, 이들 식물체에서 GFP 발현을 조사하였다. $T_1$ 식물체에서 GFP 발현 비율은 26.7%로 매우 높았고, 이러한 발현은 $T_2$ 식물체에서도 재확인되었다. 한편, 식물의 shoot 발달에 관여하는 주요 유전자를 동정하기 위해서, shoot에서 GFP발현을 보인 19개 $T_1$ 식물체에서 유래한 $T_2$ 식물체를 대상으로 표현형을 조사한 결과, 비정상적인 shoot발달을 보이는 6개 $T_1$ 라인을 확인하였다. 이들 식물체는 shoot발달의 조절 기작 분석에 매우 유용하게 사용될 수 있을 것이다. T-DNA-mediated transformation is a common method for generating transgenic plants with insertional mutagenesis. In order to identify important genes involved in shoot development, a system of promoter trap insertional mutagenesis was employed in Arabidopsis thaliana. For this system, an efficient promoter trap vector, pFGL561 was developed. The pFGL561 includes a basta-resistant gene, an intron with multiple splicing donor and acceptor sites, and a promoter-less GFP reporter gene. Using floral-dipping method, we made total 300 $T_1$ promoter-trap lines which were screened for GFP expression. GFP signals in the $T_1$ plants were detected with high frequency, 26.7%, and the signals were reconfirmed in $T_2$ plants. To isolate the genes that are involved in shoot development, phenotypes were analyzed in $T_2$ plants of the 19 $T_1$ lines that had GFP signals in shoot apex, and 6 $T_1$ lines were selected that had abnormal shoot development. These lines will be very useful for the investigation of shoot development.

엽록소 농도 결정을 위하여 측정한 흡광도 값의 신뢰도 검정 지표로서 엽록소 a/b 비례치

오광석(Guangxi Wu),이춘환(Choon-Hwan Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.5

비어·람버트 법칙에 의하면 충분히 묽은 용액에서 주어진 파장에서 측정한 흡광도는 용질의 농도에 비례한다. 반대로 충분히 묽은 용액이란 측정한 흡광도 값이 용질의 농도에 비례관계를 가지는 범위에 있는 용액을 말하지만 실제 확신하기가 어렵다. 본 연구에서는 벼 잎에서 추출한 엽록소 80% 아세톤 용액을 사용하여, 측정한 흡광도 값의 신뢰도를 판단할 수 있는 기준으로서 엽록소 a/b 비례치의 사용 가능성을 4가지 다른 흡광분광분석기(Cary4E, UV-1650PC, Versamax, NanoDrop 100)을 사용하여 조사하였다: 우선 0.2 ㎍/ml의 매우 저농도에서 200 ㎍/ml까지의 다양하게 희석한 엽록소 용액을 사용하여, 645 ㎚와 663 ㎚에서 측정한 흡광도 값을 측정한 후, 흡광도 값과 엽록소 농도의 비례관계를 조사하였다. 그 결과, 이 비례관계로 판단한 측정치의 신뢰 범위에서 흡광도로부터 계산한 용액에서의 엽록소 농도와 이미 알고 있는 엽록소 농도 비교적 일치하였다. 그러나, 비례관계의 한계치 안에 있어 신뢰도가 인정된 일부 고농도와 저농도에서의 일부 값이 엽록소 a/b 비례치가 용액의 희석에 변화지 않을 것이라는 판단 기준에 있어 신뢰도가 떨어짐을 알 수 있었다. 그러므로, 본 연구결과는 엽록소 농도 결정을 위하여 측정한 흡광도 값의 신뢰 여부는 대상 용액을 희석하여 측정한 흡광도의 비례관계로 본 비어-램버트 법칙의 기준 보다는 엽록소 a/b 비례치가 변하지 않는다는 기준으로 측정치의 신뢰도를 판단하는 것이 더 좋다는 것을 확인할 수 있었다. The Beer-Lambert law states that absorbance is proportional to the concentration of a solute in a solution at a given wavelength. This linearity works for an ideal or a ‘sufficiently diluted’ solution, so this linearity is often used as a criterion for the fidelity of the absorbance value measured. In this study, we used a chlorophyll (Chl) solution, isolated from rice leaves with 80% acetone to test the use of the Chl a/b ratio as an additional criterion for checking the fidelity of measured values using four different absorption spectrophotometers: Cary4E, UV-1650PC, Versamax (a microplate reader), and NanoDrop 1,000(which can handle a 4 μl aliquot). We used Chl solutions of varying concentrations from 0.2 ㎍/ml to 200 ㎍/ml to measure absorbance values at 645 ㎚ and 663 ㎚ and checked the linearity first. The results indicated that the range of Chl concentrations that we can rely on based on the linearity was similar to the range in which the calculated Chl concentrations based on the measured absorbance values agreed with the known concentrations. However, some border cases or cases with very low Chl concentrations inside the fidelity range of Chl concentrations did not agree with the criterion that the Chl a/b ratio should not change after dilution of the Chl in the solution. These results suggest that the Chl a/b ratio is a better criterion for the reliability of the absorbance values measured for the determination of chlorophyll concentration than the criterion based on the linearity suggested by the Beer-Lambert law.

수종 담수적조 원인종들의 형광특성과 적용연구

손문호,권오섭,문병용,정익교,이춘환,이진애,Son, Moon-Ho,Zulfugarov, Ismayil S.,Kwon, O-Seob,Moon, Byoung-Young,Chung, Ik-Kyo,Lee, Choon-Hwan,Lee, Jin-Ae 한국조류학회(藻類) 2005 ALGAE Vol.20 No.2

The freshwater blooms mainly blue-green algal blooms occur frequently in the lower Naktong River in summer, which provoke many socio-economical problems; therefore, the early detection of bloom events are demanding through the quantitative and qualitative analyses of blue green algal species. The in vivo fluorescence properties of cultured strains of Microcystis aeruginosa, M. viridis, M. wesenbergii, M. ichthyoblabe, Anabaena cylindrica, A. flos-aquae, and Synedra sp. were investigated. Wild phytoplankton communities of the lower Naktong River were also monitored at four stations in terms of their standing stocks, biomass and fluorescence properties compared with its absorption spectram. The 77K fluorescence emission spectra of each cultured strains normalized at 620 nm was very specific and enabled to detect of blue green algal biomass qualitatively and quantitatively. The relative chlorophyll a concentration determined by chlorophyll fluorescence analysis method showed significant relationship with chlorophyll a concentration determined by solvent extraction method ($R^2$ = 0.906), and the blue-green algal cell number determined by microscopic observation ($R^2$ = 0.588), which gives insight into applications to early detection of blue green algal bloom.

Chilling Tolerance of Photosynthesis in Plants is Dependent on the Capacity to Enhance the Levels of the Xanthophyll Cycle Pigments in Response to Cold Stress

김현주,강인순,이신범,이춘환,조성호,문병용,Kim, Hyun-Ju,Kang, In-Soon,Lee, Chin-Bum,Lee, Choon-Hwan,Cho, Sung-Ho,Moon, Byoung-Yong 한국광과학회 2005 Journal of Photosciences Vol.12 No.1

Plants possess the ability to dissipate the excitation energy for the protection of photosynthetic apparatus from absorbed excess light. Heat dissipation is regulated by xanthophyll cycle in thylakoid membranes of chloroplasts. We investigated the mechanistic aspects of xanthophyll cycle-dependent photoprotection against low-temperature photoinhibition in plants. Using barley and rice as chilling-resistant species and sensitive ones, respectively, chilling-induced chlorophyll fluorescence quenching, composition of xanthophyll cycle pigments and mRNA expression of the zeaxanthin epoxidase were examined. Chilled barley plants exhibited little changes in chlorophyll fluorescence quenching either of photochemical or non-photochemical nature and in the photosynthetic electron transport, indicating low reduction state of PS II primary electron acceptor. In contrast to the barley, chilled rice showed a marked decline in those parameters mentioned above, indicating the increased reduction state of PS II primary electron acceptor. In addition, barley plants were shown to have a higher capacity to elevate the pool size of xanthophyll cycle pigments in response to cold stress compared to rice plants. Such species-dependent regulation of xanthophyll cycle activity was correlated with the gene expression level of cold-induced zeaxanthin epoxidase. Chilled rice plants depressed the gene expression of zeaxanthin epoxidase, whereas barley increased its expression in response to cold stress. We suggest that chilling-induced alterations in the pool size of xanthophyll cycle pigments related to its capacity would play an important role in regulating plant's sensitivity to chilling stress.

A Simple Method for Extraction of High Molecular Weight DNA fromPorphyra Tenera (Rhodophyta) Using Diatomaceous Earth

김태훈,황미숙,송주동,오민혁,문용환,정익교,류태형,이춘환,Kim, Tae-Hoon,Hwang, Mi-Sook,Song, Ju-Dong,Oh, Min-Hyuk,Moon, Yong-Hwan,Chung, Ik-Kyo,Rhew, Tae-Hyoung,Lee, Choon-Hwan The Korean Society of Phycology 2006 ALGAE Vol.21 No.2

The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.