Agrobacterium tumefaciens의 Ti plasmid 재조합에 관한 연구

주충노,이희봉,김영명 연세대학교 자연과학연구소 1987 學術論文集 Vol.19 No.-

Crown gall의 병원체로 알려진 Agrobacterium tumefaciens가 함유하고 있는 Tumor inducing(Ti) plasmid가 식물 hormone과 opine의 합성원인이라는 것이 밝혀지고 식물 세포의 crown gall에는 Ti plasmid가 존재하며 Ti plasmid의 일부가 식물세포의 핵 genome에 삽입되어 형질 변화가 일어남이 보고 되었고 Ti plasmid의 식물체로의 gene도입 vector로서의 연구가 최근 활발히 진행되고 있으며 현재 PLGV vector와 PMON vector 등이 개발되고 있으나 조작이 복잡하고 아직은 초보적 단계인 것으로 알려져 있다. 본 연구에서는 원하는 식물 gene을 식물체에 도입하는 유용한 vector개발의 초보단계로서 Ti plasmid의 T-DNA의 일부인 nos gene을 분리하여 pBR322와의 재조합을 기도한 것이다. pBR 322를 포함하는 E. coli로 부터 Horwitz(1979)의 방법으로 pBR 322를 분리하였고 A. tumefaciens C58에서 Kado(1979)의 방법으로 Ti plasmid를 분리한 후 Ti plasmid 내의 nos gene을 분리하였다. 분리된 pBR 322를 Hind Ⅲ로 절단한 후 CIP로 처리하고 T_4 DNA ligase를 이용하여 Hind Ⅲ로 처리한 nos gene fragment와 재조합하였다. 이와같이 하여 얻은 recombinant DNA로 인한 E. coli HB101의 형질 변화를 Mandel과 Higa의 방법에 따라 조사한 결과 pBR 322의 tetracyclin gene 사이에 nos gene이 삽입된 것으로 확인되었다. 즉 ampicillin 배지에서는 92개의 colony가 형성되었는데 그중 59개는 tetracyclin 배지에서 colony를 형성하지 못하였다. 이러한 tetracyclin 감수성이면서 ampicillin 저항성인 colony를 배양하여 얻은 recombinant plasmid를 전기이동법으로 분리한 결과 예상대로 크기가 7.5kb 정도였고 Hind Ⅲ로 절단하였을 때 2개의 band가 확인되었다. 본 연구에서 얻은 nos gene-pBR 322 hybrid plasmid는 원하는 식물 gene을 이것에 삽입하여 A. tumefaciens 내에서 homologous recombination을 행한 후 식물체에 감염시켜 원하는 gene의 발현여부를 검출하는데 이용될 것으로 기대된다. It is mow realized that the tumor-inducing(Ti) plasmid in Agrobacterium tumefaciens is responsible for the induction of crown gall tumors in dicotyledonous plants and it has been demonstrated that a segment of the Ti plasmid, so called T-DNA, is stably integrated into and expressed in the genome of transformed plant cells. Recently, the use of A. tumefaciens Ti plasmid as a vector to introduce a foreign gene has been intensively studied and several vectors such as PLGV vector and PMON vector has been reported. However, their practical use is still at a primitive stage. It was attempted in the present study, therefore, to conduct the recombination of nos gene of T-DNA from A. tumefaciens Ti plasmid and E. coli pBR 322 as a vector it introduce foreign plant gene into dicotyledon plant cells. pBR 322 was isolated from E. coli containing pBR 322 accdrding to Horowitz(1979) and nos gene of T-DNA in Ti plasmid from A. tumefaciens C58 was obtained according to Kado(1979). The isolated pBR 322 was cut using Hind Ⅲ followed by the addition of CIP·pBR 322 fragments and Hind Ⅲ treated nos gene fragments were than recombined using T_4 DNA ligase and the E. coli cells were then transformed by recombinant DNA according to Mandel and Higa(1970). It was confirmed that nos gene was inserted into tetracyclin gene of pBR 322 by the finding that when the transformed cells were incubated in the ampicillin medium, 92 colonies were formed, in which 59 were tetracyclin sensitive. The above tetracyclin sensitive but ampicillin resistant cells were then cultured and the recombinant pBR-nos gene was isolated and subjected to electrophoresis. It was found that size of the recombinant DNA was approximately 7.5 Kb as expected and two bands were appeared on electrophoretogram when the recombinant DNA was treated with Hind Ⅲ. It might be possible to insert the foreign plant genes to the pBR-nos gene hybrid plasmid obtained in the present study and the resultant DNA could be then homolgously recombined with Ti plasmid of A. tumefaciens and the final recombined Ti plasmid could be used for the introduction of the foreign plant genes into dicotyledon plant cells.

균 spheroplast 및 large unilamellar vesicle의 식물 protoplast로의 도입

주충노,강철호,이희봉 연세대학교 자연과학연구소 1987 學術論文集 Vol.19 No.-

A. tumefaciens의 식물 protoplast로의 도입을 확인하는 방법으로 방사성 인산(Na_2H^32PO_4)을 함유한 0.8% nutrient broth에서 증식한 Agrobacterium tumefaciens C58를 사용하여 방사성 spheroplast와 방사성 Ti plasmid encapsulated liposome을 만들어 Danus carota protoplast로의 도입을 시도한 결과 다음과 같은 실험 성적을 얻었다. 1. A. tumefaciens에서 추출한 Ti plasmid를 Reverse Phase Evaporation 법으로 phosphatidyl-choline, stearylamine과 함께 Ti plasmid encapsulated liposome을 제조한 결과 17%의 Ti plasmid가 liposome안으로 편입되었다. 2. 방사성 Ti plasmid encapsulated liposome과 Dacus carota root protoplast를 polyethyleneglycol-Ca^2+법으로 처리한 결과 introduction frequency는 38%이었다. 3. A. tumefaciens C58의 spheroplast와 Dacus carota root protoplast를 polyethyleneglycol Ca^2+ 법으로 처리한 결과 introduction frequency가 47.6%였다. 4. 본 연구에서 얻은 실험결과로 부터 liposome법, spheroplast법 모두 Ti plasmid의 식물 protoplast로의 도입은 용이하게 일어나는 것으로 생각된다. It was attempted in the present study to prepare radioactive Ti plasmid to identify the introduction frequency of Ti plasmid encapsulated liposome into Dacus carota root protoplast. Agrobcterium tumefaciens C58 was cultured in 0.8% nutrient broth containing radioactive phosphoric acid(H_3^32PO_4) and the radioactive Ti plasmid wad isolated. Radioactive Ti plasmid was encapsulated into phosphatidyl choline-stearylamine liposomes by reverse phase evaporation technique and the encapsulation frequency was found to be about 17%. Radioactive Ti plasmid encapsulated liposome and Dacus carota root protoplast was treated by polyethyleneglycol Ca^2+ procedure. The introduction frequency was 38%. The spheroplast of A. tumefaciens C38 was prepared from the bacteria cultured in 0.8% nutrient broth containing radioactive phosphoric acid using lysozyme. As much as 47% of the spheroplast were found to be introduced into Dacus carota protoplast.

인삼 사포닌이 지용성 비타민(A와 E)의 흡수에 미치는 영향

朱忠魯,金斗植,全英運 연세대학교 자연과학연구소 1978 學術論文集 Vol.1 No.-

본 연구에서는 人蔘 Saonin 이 不水溶性 Vitamin인 Vitamin A와 Vitamin E의 吸收에 미치는 영향을 쥐(albino rat, 100~120g ♂)를 使用하여 觀察하고 다음과 같은 結果를 얻었다. 1) Vit. A 를 人蔘 Saponin 과 함께 Stomach tubing法으로 投與한후 쥐의 血淸을 조사한 結果 試驗群의 Vit.A의 含量은 對照群보다 2倍나 된다. 그러나 3時間後부터는 過量의 Vitamin含量이 減少되기 시작하였다. 試驗群의 肝의 Vitamin A含量도 對照群의 1.5倍였으며 3時間後에는 正常 Vitamin量으로 回復되었다. 2) Vit.E의 경우도 Vitamin E를 投與하고 40分後에 血淸과 肝에서의 Vit. E含量을 調査한 結果 試驗群이 對照群에 比하여 1.4~1.5倍였다. 3) 위와같은 實驗結果는 人蔘 Saponin이 Vit. A와 Vit. E의 吸收를 促進한다는 것을 意味하는 것이다. 4) 한편 試驗群의 腎藏에서의 Vitamin含量이 對照群에 比하여 훨씬 높은 것은 人蔘 Saponin이 不水溶性 Vitamin의 吸收뿐 아니라, 移動 및 排泄에도 크게 영향을 미칠것으로 예상된다. It was attempted in this experiment to observe the effect of ginseng saponin on the absorption of water insoluble Vitamins such as Vitamin A and Vitamin E using albino rat (150-200g,♂) and the following result were obtained. 1) When the Vitamin A and ginseng saponin were fed by stomach tubing, Vit. A concentration of blood serum of test group was found as much as twice that of control, but the Vitamin A content started to decrease 3 hours after. The Vitamin A content of the liver of test animal was also found about 1.5 times that of control and its value returned to normal value three hours later. 2) The Vitamin E contents of the blood and lover of test animals were found as much as 1.4-1.5 times that of control 40 minutes after feeding. 3)The above results suggested tha saponin stimulated the absortion of water insolubl Vitamin A and E 4) On the other hand, the contents of the above Vitamins in the kidney of test animals were significantly higher suggesting that the ginseng saponin also affects the transport of the above water insoluble Vitamins.

現代 産業技術의 特質과 科學敎育

朱忠魯 연세대학교 교육대학원 1977 연세교육과학 Vol.11 No.-

We are all living in the age of science and it is now well recognized that politics, economic prosperity and civilization of our society anti even philosophical problems cannot be dealt with without considering science and technology today. It is also true that science and technology became an indispensale strategic tool for the rapid development of society, particularly for the developing country like Korea. Therefore, science education and its development became one of the serious facing issues of this country. Science of today is growing rapidly and it is one of the characteristics of modern science that it is finely dividing into a number of specialized fields but on the other hand they are going to group into several catagories. Furthermore, industrial technology is so intimately interlinked to science that no industrial development can be made without the background of science and scientific research. Actually science today acts a leading role in industry and few people would venture to assert that the prosperity and industrial development of any society can expect otherwise the rational science education and scientific research being carried out steadily. Science today occupies the heart of modern industry and it is rapidly spreading over industry through scientific production and administration. This rejects the social significance of modern Science. Historical events tell us that well organized and planned policy of science and technology did catalytic role unexceptionally for the prosperity of society in the past. Furthermore, it should he reminded that new technologies were brought by scientific findings and it is extremly dangerous to consider science and technology separately. It cannot he overestimated to emphasize the signifance of fundamental science for industrial development and prosperity of society. We know that science and technology have been developed before industrial prosperities were made, and well organized science education and policies were prepared before the development of science and technology. It is clear therefore that improvement of science education, both teaching contents and methods, must be urgently required to meet the development of industry and prosperity of thin society. There is no doubt that the purpose of science education today is not any more to teach systematic scientific knowledge as before. Rather I think it is necessary to teach fundumental concepts governing today's science and technology and scientific methods so that they may handle their own problems in the future and enjoy their lives rationally in the highly developed scientific society. Accordingly the content of the text of science education must be carefully selected to meet their lives in the future. It is realized that modern science governing today's industry would be consisted of nuclear physics, electronics, polymer chemistry and molecular biology. Baaed on the above fields of science, what it is called universe development has been initiated. From the above considerations, it is suggested that we all, educators, scientists as well as industrial people and government officials have to pay their extreme attention to set up rational programs for science education and scientific research in the very near future, and hoped the programs being carried out properly and steadily for the prosperity of this country.

科學 Curriculum 改善의 動向

朱忠魯 연세대학교 교육대학원 1979 연세교육과학 Vol.14 No.-

Curriculum reform movement of Science education was initiated in 1976 and the Curriculum study has been made towards formulating "Concept and process centered Science Curriculum", what is called "investigative curriculum. " Recently developed integrated and/or individualized Science curricula are also based on the idea of the above concept and process centered curriculum but extended the area that the fundamental concept fevers and/or considered not only concepts and process of the Science but also the feeling side of the students. It is quite true that the purpose of science educntion cannot be accomplished successfully even with a perfect curriculum when the ability and feeling of the students are ignored. Therefore the individualized science curriculum will be ideal one toward which the currumt study is approching. For the successful achievement of the above developed curriculum, the role of teachers are of great significance. As pointed out through the experimental teaching of PSSC course at school, it is necessary to retrain school teachers to meet the new science curriculum. Particularly in a developing country like ours, we are suffering from the lack of qualified teachers. Of course, there are a number of well trained teachers, but most school teachers in this country have received old fashioned education at home and they have not enough experience of scientific experiments to meet the new curriculum requirements. It is urgent, therefore, to establish a sound and practical retraining courses for school science teachers to carry out the new science curriculum successfully. I suggest that it is possible to retrain the school teachers at academic institution such as graduate school of education. It is also realized that the number of experiments required in the current curriculum is far beyond the practically limited available teading time at middle school. This signified that a new experimental program must be organized under the full cooperation of scientist and school teachers. Finally it must be pointed out that the supply of laboratory equipments, reagents, materials and assistants should be satisfied and the teacher's over-load should be avoided. It is quite natural that the satisfactory science education cannot be expected without a sufficient educational investigation as the good products are not produced without properly organized instruments and machines. It is clear that improvement of science education, both teaching contents and methods, must be urgently required to meet the developing and properity of this society. There is no doubt that the purpose of science education today is not any more to teach systemtic scientific knowledge as before but to teach fundamental concepts grovering today's science and scientific methods so that they may handle their own problems in the future and enjoy their lives rationally in the highly developed scientific society. Accordingly the contents of the text of science education must be carefully reselected to meet the above requirment. From the above consideration, the government officials, industrial people and school authorities have to pay their atention to organized rational programs for science education.

中心壓軸力을 받는 高强度 鐵筋콘크리트 기둥의 耐力과 延性에 關한 실험적 硏究

곽노현,이영호,김남주,이경종,정헌수 중앙대학교 기술과학연구소 1997 기술과학연구소 논문집 Vol.27 No.-

A study on the behavior of short reinforced concrete columns was experimently described. Twenty-four concrete columns, with 20cm square and 60cm height, containing 8 longitudinal steel bars and different the amount of ties, spacing and concrete strengths, were subjected to concentric load by failure. Test results indicated that 1) It is incompatible in high-strength concrete to arrange stirrups to the AIK code of D/2 throughout the example of 10cm stirrup spacing following AIK codes. 2) As the amount of stirrup increased, the strength and ductily of reinforced concrete column improved. 3) As the concrete strength is high, the effect of the was reduced. 4) If strength enhancement factors increased, strain at maximum load also does and ductility ratios does almost linearliary. 5) The rate of strength enhancement factor does well with the modified kent & Park's theory. 6) As to the result of calculating the effective confined area which is suggested in Sheikh & Uzumeri's theory, the middle vertical steel is effective, only if the stirrups are arranged with spacing of 3cm.

Some Physiological and Biochemical Aspects of Saponin Fraction of Panax Ginseng C. A. Meyer

(Chung No Joo) 고려인삼학회 1990 Proceedings of International Symposium on Korean G Vol.- No.-

Effect of Ginseng Saponin on Alcohol Metabolism in the Animal Body

Joo, Chung-No The Korean Society of Ginseng 1992 Journal of Ginseng Research Vol.16 No.3

Unlike carbohydrats and fats, alcohol is essentially foreign to the body and it is known that the body get rid of it by oxidizing alcohol maily in the liver. Acetaldehyde is produced during ethanol metabolism and is known to be oxidized mainly by aldehyde dehydrogenase (ALDH). ALDH activity was found mainly in the mitochondrial fraction but a significant ALDH activity was also present in microsomal and cytosol fraction. Wistar rats (150~200 g, male) were given freely with 12% ethanol (Control) and/or 12% ethanol containing 0.1% ginseng saponins (Test) instead of water for 6 days and the liver was analyzed. ALDH activities of both control and test group were lower than that of normal group but test AkDH was less inhibited than control. ADH activies of both control and test were slightly higher than that of normal group but our previous data showed that it became gradually steady after prolonged ethanol feeding. MEOS activities of both control and test group were much higher than that of normal group. MEOS enzymes are inducible but the activity of test group was greatly higher than that of control. Ethanol containing [1-i4C] ethanol (5 $\mu$Ci) was injected to the above three groups and 30 min later, the distribution of radioactivity of hepatic lipids was investigated. Radioactivities of hepatic lipids of both control and test group were higher than that of normal group, however, that of test group was much lower than that of control. Analysis of individual lipids showed that phospholipid biosynthesis was significantly impaired and fatty acid and triglycerides biosynthesis were greatly stimulated. However, it was realized that the saponin prevented phospholipid biosynthesis depression and the increase of triglyceride biosynthesis considerably. It seemed that the saponin might stimulate ADH, ALDH and MEOS and the acetaldehyde formed would be removed faster. The excess hydrogen can be shunt more quickly into lipid biosynthesis. Electron microscopic observation showed that the hepatic cell of control group was si gnificantly damaged. Mitochondria were swollen and rough endoplasmic reticulum were dilated, however, hepatocytes of test group were not damaged.

심포지움(고려인삼 국제학술대회 1990년 7월 20일-21일, 서울) 고려인삼사포닌 분획의 생리, 생화학적 작용

Joo, Chung-No 고려인삼학회 1990 Journal of Ginseng Research Vol.14 No.2

Purification and Reaction Mechanism of Rat Brain Succinic Semialdehyde Dehydrogenase

Joo, Chung No,Kim, Kyu Tae 생화학분자생물학회 1977 BMB Reports Vol.28 No.2

Rat brain succinic semialdehyde dehydrogenase (EC 1.2.1.24 SSADH) activity was detected in mitochondrial, cytosolic and microsomal fractions. Brain mitochondrial soluble SSADH was purified by ammonium sulfate precipitation, DEAF Sephacel, and 5'-AMP Sepharose 4B affinity chromatography. The purified enzyme was shown to consist of four identical subunits, and the molecular weight of a subunit was 55 kD. The Km for short chain aliphatic aldehydes and aromatic aldehydes were at the 10^(-3) M level but that for succinic semialdehyde was 2.2 1iM. Either NAD^+ or NADP^+ can be used as a cofactor but the affinity for NAD^+ was 10 times higher than that for NADP^+. The brain cytosolic SSADH was also purified by ammonium sulfate precipitation, DEAF Sephacel, Blue Sepharose CL-6B and 5'-AMP Sepharose 4B affinity chromatography and its Km for short chain aliphatic aldehydes was at the 10^(-3) level but that for succinic semialdehyde was 3.3 μM. NAD^+ can be used as a cofactor for this enzyme. We suppose that both enzyme might participate in the oxidation of succinic semialdehyde, which is produced during GABA metabolism. The activity of both cytosolic and mitochondrial SSADH was markedly inhibited when the concentration of succinic semialdehyde was high. The reciprocal plot pattern of product inhibition and initial velocity indicated a sequential ordered mechanism for mitochondrial matrix SSADH. Chemical modification data suggested that amino acid residues such as cysteine, serine and lysine might participate in the SSADH reaction.