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담배 Callus 와 Shoots 에서 Isoperoxidase 와 Translatable Isoperoxidase mRNAs 의 비교 연구
김은성,김승수 ( Eun Sung Kim,Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3
Tobacco callus grown on 5×10^(-7) M kinetin/10^(-8) M IAA or 10^(-5) M kinetin/10^(-5)IAA formed shoots after 15-20 days of culture. The total peroxidase activities in shoots were lower than those of non-shoot-forming callus. However, cathodic isoperoxidases were increased significantly in shoots formed on 10^(-8) M IAA media while anodic isoperoxidases were increased in shoots formed on 10^(-5) M kinetin media as is the case of shoot initiating callus. The gene expression patterns of isoperoxidases in shoot and non-shoot-forming tissue were also examined by rabbit reticulocyte lysate in vitro translation system. These studies indicate that fraction of translatable poly(A)isoperoxidase mRNA was increased considerably in shoots formed on 10^(-5) M kinetin, whereas poly(A)^+ isoperoxidase mRNA contents were twice higher than poly(A)isoperoxidase mRNAs in non-shoot-forming callus. At the present time, at least five isoperoxidases could be detected from the translation mixture of total cellular RNA. Preliminary translation data indicate that less cathodic isoperoxidases and more anodic isoperoxidases were expressed from poly(A)^- RNAs than from poly(A)^+ RNAs.
Shoot 을 형성시키는 두 호르몬 levels 에 있어서 peroxidase 의 활성과 isoenzyme pattern 변화에 관한 연구
김은성,김승수 ( Eun Sung Kim,Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2
The effects of various ratios of kinetin and IAA in media on shoot initiation, peroxidase activity and isoperoxidase pattern changes of tobacco callus were examined. Tissues grown on 5×10^(-7) M kinetin/10^(-8) M IAA or 10^(-5) M kinetin/10^(-5) M IAA began to form masses of cells in nodular form for shoot initiation after 15 days. The peroxidase activities of shoot intiating calli were higher than the normal non-shoot forming calli at the initial stage of shoot formation. It was also noted that the activity of peroxidase in 10^(-8) M IAA cultures was about twice higher than that of 10^(-5)M kinetin cultures. The increased enzyme activity in 10^(-8) M IAA cultures was due to the increased amount of cathodic isoperoxidases while anodic isoperoxidases were responsible for the increase of peroxidase activity in 10^(-5)M kinetin cultures. The results indicate that kinds or groups of isoperoxidases not over-all concentration of enzymes are important in shoot initiating process.
Isoperoxidases and Translatable Isoperoxidase mRNAs in Shoots and Non-shoot-forming Tobacco Callus
김은성,김승수,Kim, Eun-Sung,Kim, Soung-Soo 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3
담배 callus를 $5{\times}10^{-7}\;M$ kinetin/$10^{-8}\;M$ IAA 배지 또는 $10^{-5}\;M$ kinetin/$10^{-5}\;M$ IAA 배지에서 15~20일 배양했을 때 shoot이 형성되었다. 두 조건에서 형성된 shoot에 있어서 peroxidase의 활성도는 shoot을 형성하지 않은 callus 보다 낮았다. 또한 $10^{-8}\;M$ kinetin 배지에서 형성된 shoot에서는 cathodic isoperoxidases의 양이 증가한 반면 $10^{-5}M$ Kinetin 배지에서 형성된 shoot에서는 anodic isoperoxidases가 증가하였다. 담배 callus의 mRNA를 분리하여 in vitro translation을 행한 결과 최소한 5개의 isoperoxidases mRNA가 존재함을 확인하였으며, $poly(A)^-RNA$에서도 peroxidase가 합성되었다. 정상배지에서 4주 배양한 callus에서는 $poly(A)^+$ RNA와 $poly(A)^-$ $RNA{\mu}g$ 당 거의 같은 양의 peroxidase가 합성되었으나, $10^{-5}M\;M$ kinetin 배지에서 형성된 shoot에서는 $poly(A)^+$ RNA에서보다 $poly(A)^-$ RNA에서 더많은 peroxidase가 합성되었다. 또한 $poly(A)^-$ RNA에서는 $poly(A)^+$ RNA보다 cathodic isoperoxidases가 더 적게 translation되는 사실도 확인하였다. Tobacco callus grown on $5{\times}10^{-7}\;M$ kinetin/$10^{-8}\;M$ IAA or $10^{-5}\;M$ kinetin/$10^{-5}$ IAA formed shoots after 15-20 days of culture. The total peroxidase activities in shoots were lower than those of non-shoot-forming callus. However, cathodic isoperoxidases were increased significantly in shoots formed on $10^{-8}\;M$ IAA media while anodic isoperoxidases were increased in shoots formed on $10^{-5}\;M$ kinetin media as is the case of shoot initiating callus. The gene expression patterns of isoperoxidases in shoot and non-shoot-forming tissue were also examined by rabbit reticulocyte lysate in vitro translation system. These studies indicate that fraction of translatable $poly(A)^-$ isoperoxidase mRNA was increased considerably in shoots formed on $10^{-5}\;M$ kinetin, whereas $poly(A)^+$ isoperoxidase mRNA contents were twice higher than $poly(A)^-$ isoperoxidase mRNAs in non-shoot-forming callus. At the present time, at least five isoperoxidases could be detected from the translation mixture of total cellular RNA. Preliminary translation data indicate that less cathodic isoperoxidases and more anodic isoperoxidases were expressed from $poly(A)^-$ RNAs than from $poly(A)^+$ RNAs.
Peroxidase Activity and Isoenzyme Pattern Change in Two Shoot Initiating Hormonal Levels
김은성,김승수,Kim, Eun-Sung,Kim, Soung-Sao 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2
담배 callus 배양시 kinetin과 IAA가 shoot형성과 peroxidase 활성 및 isoenzyme pattern 변화에 미치는 영향을 조사하였다. 담배 callus를 $5{\times}10^{-7}$ Kinetin/$10^{-8}\;M$ IAA 또는 $10^{-5}\;M$ kinetin/$10^{-5}M$ IAA 배지에서 15일 정도 배양하였을 때 shoot형성이 시작되었다. Shoot 형성 초기의 peroxidase 활성은 위의 두 경우 모두 대조군 ($5{\times}10^{-7}\;M$ kinetin/$10^{-5}\;M$ IAA) 보다 증가하였으 며 $10^{-8}\;M$ IAA 배지에서 배양한 callus 에서는 $10^{-5}\;M$ kinetin 배지에서 배양한 callus 보다 shoot 형성시 약 두배가량 높은 peroxidase 활성도를 나타냈다. 또한 shoot형성시 $10^{-8}\;M$ IAA callus에서 peroxidase의 활성도 증가는 cathodic isoperoxidase의 증가와 일치 하였으며 $10^{-5}\;M$ kinetin callus에서는 anodic isoperoxidase의 증가와 일치 하였다. 이와 같은 사실로 보아 shoot 형성과정에 있어서 total peroxidase의 양보다는 isoperoxidase의 종류가 중요한 역할을 하는 것으로 사료된다. The effects of various ratios of kinetin and IAA in media on shoot initiation, peroxidase activity and isoperoxidase pattern changes of tobacco callus were examined. Tissues grown on $5{\times}10^{-7}\;M$ kinetin/$10^{-8}\;M$ IAA or $10^{-5}\;M$ kinetin/$10^{-5}\;M$ IAA began to form masses of cells in nodular form for shoot initiation after 15 days. The peroxidase activities of shoot intiating calli were higher than the normal non-shoot forming calli at the initial stage of shoot formation. It was also noted that the activity of peroxidase in $10^{-8}\;M$ IAA cultures was about twice higher than that of $10^{-5}M$ kinetin cultures. The increased enzyme activity in $10^{-8}\;M$ IAA cultures was due to the increased amount of cathodic isoperoxidases while anodic isoperoxidases were responsible for the increase of peroxidase activity in $10^{-5}M$ kinetin cultures. The results indicate that kinds or groups of isoperoxidases not over-all concentration of enzymes are important in shoot initiating process.
담배 6 - Phosphogluconate Dehydrogenase Band Il 의 동일 Subunits 에 관한 화학적 증거
손혜정,김은성,김승수 ( Hye Jeong Sohn,Eun Sung Kim,Soung Soo Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.4
6-Phosphogluconate dehydrogenase band II isoenzyme was isolated from a tobacco callus culture line, Nicotiana tabacum L., var. Virginia 115, utilizing ammonium sulfate precipitation, DEAF-cellulose chromatography, Red Sepharose CL-6B affinity chromatography followed by Sephadex G-150 gei filtration. N-terminal amino acid of the purified enzyme by dansylation method was lysine. Amino acid analysis indicated that the enzyme had 11 lysine and 17 arginine per 45,000 molecular weight subunit. Approximately 30 ninhydrin-positive spots were observed from tryptic peptide mapping study. Therefore, the results of N-terminal amino acid analysis, amino acid composition, and tryptic petide mapping suggest that the dimeric 6PG DH is composed of chemically identical subunits.