완두 세포질 Fructose-1,6-Bisphosphatase cDNA의 PCR Cloning과 특성

손태종,한태룡 경희대학교 유전공학연구소 1996 遺傳工學論文集 Vol.8 No.-

Three positive phage clones were isolated from cDNA phage clones which had been first screened from a pea cDNA library with polyclonal antibodies for the purified pea cytosolic fructose-1 6-bisphosphatase (FBPase). Polymerase chain reaction was conducted with these phage clones using two primers synthesized from homology analysis of amino acid sequences for animal and plant cytosolic FBPases. A PCR product with 650 bp long was cloned into pGEM-T vector and sequenced The deduced amino acid sequence of the cDNA fragment was 98, 91, and 85% homologous with those of cytosolic FBPases from spinach, sugarbeet, and sugarcane, respectively. It was 86 and 51% homologous with amino acid sequences of FBPase from rat liver and pea chloroplasts, respectively. Northern blot analysis was proceeded with the cDNA clone resulting that 1.2 kb transcript was highly expressed in light grown pea leaves but almost not expressed in dark grown etiolated pea seedlings. Peas grown in the light for 10 days were transferred to darkness, showing that the transcript was gradually decreased during dark treatment, indicating that the expression of the enzyme was induced by continuous white light but suppressed by dark treatment. Pea cytosolic FBPase was highly expressed in leaves with trace amounts in stems, but almost not expressed in roots.

유화조건이 홍화적색소의 물성에 미치는 영향

이승룡,장규섭,이석건,윤혜현,한태룡 충남대학교 농업과학연구소 2000 농업과학연구 Vol.27 No.1

상기 실험으로부터 얻어진 결과를 요약하면 다음과 같다. 1. Hunter-value를 측정한 결과 hunter L-value 즉, 백색도에 있어서는 glycerin+carthamin+locithin의 경우가 실온저장 및, 열악조건 그리고 광조사시에 있어서 모두 8 시간이 경과하면서부터는 급격하게 상승하는 모습을 보였다. 2. 실온하에서 hunter a-value 즉, 적색도에 있어서는 carthamin 첨가구가, b-value 즉, 황색도에 있어서는 paprika 첨가구가 더 강하게 나타났다. 열악조건에서는 실온저장의 경우와 다소 다른 양상을 보여주었는데. D.W+carthamin+lecithin의 경우에 있어서도 hunter L-value가 낮은 값을 나타냈으며, hunter a-value의 경우 soybean oil+paprika에서도 높은값을 보여주었다. 3. 유변학적 특성에 대한 실험에서 비교적 고점도를 가지고 있는 glycerin을 base material로하였을 때는 이에 비해 저점도라고 할 수 있는 soybean oil이나 증류수가 쓰여졌을 때보다 spindle의 rpm에 따른 viscosity나 shear rate 및 shear stress등의 같이 월등히 높은 값을 나타냈다. 그리고, 일반적으로 spindle의 rpm에 대한 shear rate 및 shear stress의 값은 비례적으로 증가하는 경향을 나타냈다. 유화제의 사용여부에 있어서는 lecithin을 사용했을 경우. 사용하지 않았을 때보다 높은 점도를 나타냈고, 동일한 조건에서 carthamin보다는 paprika가 두 배 정도 더 높은 점도를 나타냈다. 4. soybean oil을 사용한 실험구에 있어서는lecithin의 첨가유무가 점도에 아무런 영향을 미치지 않는 것으로 나타났으나, glycerin을 사용한 실험구에 있어서는 유화제인 lecithin을 병행했을 경우, 특이하게도 오히려 paprika에서보다 carthamin에서 더 높은 점도를 나타냈다. 5. Shear stress의 경우 soybean oil 첨가구와 glycerin 첨가구가 현저한 차이를 보이며 증가한 반면, shear rate에 있어서는 두 가지 경우모두 거의 같은 경향을 나타낸 점이 특이했다. This study was peformed to evaluate the food technological properties according to different emulsion state of carthamus red pigment. For making emulsion, lecithin was used as an emulsifier and polyglycerol monooleate(PGMO) and polyoxyethylene sorbitan monooleate(Tween 80) were used as an assistant, and glycerin, distilled water and soybean oil were used as base materials respectively. Paprika stock solution was used for comparing carthamin on the rheological properties. The results were described as following; 1. Hunter L-value was not drastically increased until passed by 8 hours for glycerin, carthamin, and lecithin mixed sample. 2. Hunter a-value was higher at carthamin added sample than others, and b-value was higher to paprika added sample than others. 3. The viscosity, shear rate and shear stress levels in which glycerin was used as base material were higher than soybean oil or distilled water. 4. In which soybean oil was used as base material, lecithin was not affected on the rheological properties. But, in which glycerin was used, the lecithin was higher affected on carthamin than paprika. 5. The value of shear stress was increased both carthamin and soybean oil. However, that of shear rate was shown similar trends.

Site-Directed Mutagenesis법에 의한 RecA 단백질의 ATPase 활성 부위 검정 : Cysteinyl 129의 관련성 Involvement of Cysteinyl 129

배준성,한태룡 경희대학교 유전공학연구소 1992 遺傳工學論文集 Vol.4 No.-

ATPase activity of RecA protein is believed to be essential for the strand exchange and DNA repair reactions in E. coli. Chemical modification(Kuramitsu et al., Biochemistry. 23. 2363. 1984) and ATP photolabeling(Banks and Sedgwick. Biochemistry, 25, 5882, 1986) experiments suggest that cysteinyl-129 is one of the most probable residues for the binding and hydrolysis of ATP in RecA protein. Site-directed mutagenesis is employed to change cysteinyl-129 to serine and alanine to minimize any structural alterations. Wild type and mutant RecA proteins were purified and examined for ATPase activity. All the purified RecA proteins showed sigmoidal activity shapes as a function of substrate concentration, indicating that RecA catalyzed ATP hydrolysis is cooperative. Both mutant RecAs displayed almost same V_(max) with wild type RecA but much larger S_(1/2) values(l80 μM for Ser. 120 μM for ,A1a) than wild type(78 μM). These results suggest that cysteinyl-129 may involve in the ATP binding rather than ATP hydrolysis. Further experiments on direct ATP binding and strand exchange reactions are in progress.

완두 세포질 Fructose-1,6-bisphosphatase의 광의존적 발현

이상원,한태룡 경희대학교 유전공학연구소 1993 遺傳工學論文集 Vol.5 No.-

Cytoplasmic fructose-1,6-bisphosphatase from pea leaves was purified and characterized. Monomeric molecular weight of the purified protein was estimated as 37,000 by SDS polyacrylamide gel electrophoresis. The enzyme was active at neutral pH (pH 6.6). The chloroplasmic fructose-l,6-bisphosphatase, isoenzyme of this enzyme, is known to be active at alkaline pH (pH 8.8). The purified enzyme was relatively stable against heat. The activity of the enzyme was not affected by reducing agent such as dithiothreitol and the enzyme was inhibited by AMP and fructose-2,6-bisphosphate as all other gluconeogenic fructose-l,6-bisphosphatase from various species. The enzyme activity of cytoplasmic fructose-1,6-bisphosphatase was gradually increased by light illumination and decreased by dark treatment. Immunoblot analysis suggests that the expression of the enzyme is stimulated by light. Light pulse experiments with blue (420nm), and red (660 nm)/far-red (730 nm) light were performed to analyze the photoreceptors related to light signal transduction and photomorphogenesis. The cytoplasmic enzyme activity was almost insensitive to blue or red/far-red light pulse, suggesting that the cytoplasmic enzyme is not directly regulated by light signal. Thus the activity and/or expression of the cytoplasmic enzyme may be closely related to plant growth and development accompanied by continuous light illumination.

완두 엽록체 Fructose-1,6-Bisphosphatase 유전자의 특성에 관한 연구

남민엽,한태룡 경희대학교 생명자원과학연구원 1997 遺傳工學論文集 Vol.9 No.-

A gene encoding chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11, FBPase) was isolated from a pea genomic library with a pea chloroplast FBPase cDNA clone (1.2 kb) as a probe. Three positive clones were screened and analyzed by phage Southern blot method. A positive phage clone (pFHNl) was digested with restriction enzymes and subcloned. Nucleotide sequence analysis of the gene indicated that the pea chloroplast FBPase gene contained three introns and four exons. The structure of pea chloroplast FBPase gene was similar with that of wheat chloroplast FBPase gene, although the pea chloroplast FBPase gene has relatively longer introns. Light responsive cis-acting elements (GATA motif, AT-rich sequence, GT-1 site) were found in 5' upstream sequences of the pea chloroplast FBPase gene.

대두(Glycine max L.) phy A cDNA의 선별과 Cloning

이재호,한태룡 경희대학교 유전공학연구소 1993 遺傳工學論文集 Vol.5 No.-

cDNA for phytochrome A gene was screened with a cDNA library constructed from etiolated soybean seedlings. A genomic subclone (pB41, 1.5 kb) containing chromophore bearing region of phytochrome A was used as a probe. All the positive clones analyzed show identical restriction maps suggesting that phy A gene presents as a single copy. A clone with full coding region (3.6 kb) was subcloned and sequenced. The deduced amino acid sequence analysis of the coding regions suggest that pB44SC is categorized as phy A. The pB44SC is 62~63 % identical with monocot phy A and 74-84% identical with dicot phy A. It is only 49-51% identical with phy B and phy C.

무작위 염기서열법에 의한 미성숙 벼종자의 cDNA 분석

피봉관,한태룡 경희대학교 유전공학연구소 1995 遺傳工學論文集 Vol.7 No.-

Partial nucleotide sequences(150∼300 bp) of randomly selected cDNA clones from cDNA library constructed with immature rice seeds have been determined. The nucleotide sequences of 200 clones were analyzed with the databases of EMBL and Gen-Bank. The results showed that 83 cDNA clones were identical to known rice genes, 17 and 4 cDNA clones were homologous with other plant and nonplant genes, respectively. Among cDNAs identical with known rice genes, 35 clones were identified as glutelin, 26 clones as prolamin, and one clone as globulin genes, respectively. Northern blot analyses suggest that all the storage protein genes were mainly expressed in immature seeds, but not in leaves, stems, roots, or young seedlings. ADP-glucose pyrophosphorylase gene which is involved in starch synthesis was also expressed only at the early stage of rice seed formation.

생물변환 천연식용색소 생산공정기술 개발 : Ⅰ. 홍화 황색소의 안정성에 미치는 pH 및 온도의 영향 Ⅰ. Effect of pH and Temperature on the Stability of the Yellow Pigment from Carthamus tinctorious L.

홍성현,박성길,이종민,한태룡,백영숙,정인식 경희대학교 생명자원과학연구원 1999 硏究論文集 Vol.20 No.-

홍화로부터 열에 의한 안정성을 검토하였다. 황색소의 분해반응은 산성, 중성, 염기성 조건에서 UV/Vis spectral measurement에 의해 조사되었다. 시간변화에 다른 흡광도를 측정해 본 결과 온도가 상승함에 따라 황색소의 분해되는 정도가 점차적으로 증가하는 것을 확인 할 수 있었다. 40℃에서 황색소의 분해 반감기는 pH3.0, pH7.0, 그리고 pH12.0에서 각각 112.1, 39.3 과 25.3 시간이었다. 이것은 황색소가 pH3.0에서 비교적 더 안정함을 말한다. pH3.0, 7.0 그리고 12.0에서 황색소의 activation energy는 각각 519, 15.0 그리고 12.3 kJ/mol이었다 We investigated thermal stability of yellow pigment from Carthamus tinctorious L. Decomposition reactions of yellow pigment were monitored at acidic, neutral and alkaline conditions by UV/Vis spectral measurement. Decomposition of yellow pigment increased as temperature increased Decomposition half lives of yellow pigment at 40℃ were 112.1 39.3, and 25.3 hrs at pH 3.0 , pH 7.0, and pH 12.0, respectively, indicating that yellow pigment is more stable at pH 3.0. The activation energies of yellow pigment at pH 3.0, pH 7.0, and pH 12.0 were 51.9, 15.0, and 12. 3 kJ/mol, respectively.

Hydrophobic Properties of Phytochrome : Interactions of 8-anilinonaphthalene-1-sulfonate to the 59 kD and 124 kD Oat Phytochromes 59kD 및 124kD 귀리 phytochrome과 8-anilinonaphthalene-1-sulfonate의 상호작용

Hahn, Tae-Ryong 경희대학교 유전공학연구소 1989 遺傳工學論文集 Vol.1 No.-

귀리 59kD 및 124kD phytochrome의 소수성질을 8-anilinonaphthalene-1-sulfonate(ANS)를 써서 구명하였다. 59kD 및 124kD phytochrome의 흡광도와 광변환속도를 측정 분석한 결과 ANS는 Pr형보다 Pfr형에 대해 조금더 친화성이 있었다. 124kD phytochrome의 광변환에 따른 ANS의 형광을 측정한 결과 거의 변화가 없었다. 이러한 결과는 118kD phytochrome의 Pfr형에 노출된 소수 domain(Hahn and Song, 1981)이 59kD 및 124kD phytochrome에서는 숨겨져 있음을 의미한다. 이 소수 donain은 chromophore와 매우 근접하여 있을 것으로 생각되므로(Hershey et al., 1985; Hahn and Chae, 1986), 6kD 및 55kD proteolttic domain은 각각 Pfr형 phytochrome의 소수표면 노출에 부정적 및 긍정적으로 각각 작용할 것으로 믿어진다. 8-anilinonaphthalene-1-sulfonate (ANS) has been employed to probe the hydrophobic properties of 59 kD and 124 kD oat phytochromes. ANS interacts slightly more with the Pfr than the Pr for both of the 59 kD and 124 kD phytochromes in terms of spectral perturbation and phototransformation kinetics. Almost no changes in the ANS fluorescence emission upon phototransformation of 124 kD phytochrome have been observed. These results suggest that the hydrophobic domain which is exposed in the Pfr form of 118 kD phytochrome (Hahn and Song, 1981) is shielded for the 59 kD and 124 kD phytochromes. Since the hydrophobic domain is assumed to locate at/near the chromophore (Hershey et al., 1985; Hahn and Chae, 1986), the 6 kD and 55 kD proteolytic domains seem to negative and positive roles for the exposure of the hydrophobic site in the Pfr of phytochrome, respectively.

Comparative Proteomic Analysis of Blue Light Signaling Components in the Arabidopsis Cryptochrome 1 Mutant

Tae-Ryong Hahn,Bong-Kwan Phee,Sebyul Park,Jin-Hwan Cho,전종성,Seong Hee Bhoo 한국분자세포생물학회 2007 Molecules and cells Vol.23 No.2