Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Though the effectiveness of ceramide is not understood fully, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries....
Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Though the effectiveness of ceramide is not understood fully, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. However, ceramide production from Saccharomyces cerevisiae has not been widely studied and the quantity are very low. Gene deletion in the cell is used frequently to investigate the function of gene and verification research of drug target. Specially, deletion mutant library is useful for a large amount functional analysis of gene. In this study, deletion mutants of genes on the metabolic pathway of ceramide synthesis in S. cerevisiae were grown in a batch culture and the cellular content of ceramide was measured. The ceramide content was highest in ${\triangle}$ydc1 mutant and 6 mg ceramide/g cell was obtained.
Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Though the effectiveness of ceramide is not understood fully, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. However, ceramide production from Saccharomyces cerevisiae has not been widely studied and the quantity are very low. Gene deletion in the cell is used frequently to investigate the function of gene and verification research of drug target. Specially, deletion mutant library is useful for a large amount functional analysis of gene. In this study, deletion mutants of genes on the metabolic pathway of ceramide synthesis in S. cerevisiae were grown in a batch culture and the cellular content of ceramide was measured. The ceramide content was highest in ${\triangle}$ydc1 mutant and 6 mg ceramide/g cell was obtained.
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문제 정의
있다(1). 본 연구에서는 sphingolipid를 생산하는 균주인 BY4742 와 deletion mutant 9종류의 배양에서 각각의 균주에 따라 세라마이드의 생산에 미치는 영향을 알아보는데 그목적이 있다.
제안 방법
배양액은 0.2 丿加 cellulose acetate filter (Satorius Co., Gemany)로 filtering 하여 RI detector LC-10AD (Shimadzu, Japan)을 장착한 HPLC 10A system (Shimadzu, Japan)으로 분석하였다. Glucose 분석에 사용된 column은 Aminex HPX-87H (Bio-Rad Laboratories, U.
세라마이드 분석은 ceramide standard (Avanti U.S.A.) 를 기준물질로 하여 ELSD를 장착한 HPLC로 측정하였다([Fig. 2]). 9종류의 deletion mutant의 세포내 세라마이드양을 [Fig.
추출된 total lipid에서 sphingolipid 부분을 얻기 위해 mild 이kaline hydrolysis를 수행하였다(8). 클로로포름을첨가한 샘플에 methanol carbon tetrachloride (5 : 1, v/v) 30 ml오} 0.
대상 데이터
Saccharomyces cerevisiae 배양을 위한 배지로는 YPD 를 사용하였으며 배지의 조성은 glucose (Samchun pure chemical Co., Korea) 20 g/1, bacto pepton (BD, U.S.A.) 20 g/1, bacto yea마 extract (BD, U.S.A.) 10 g/1 이다.
1](11)과 같다. 본 연구에서 사용된 균주는각각火化七 ypcl, scs7, surl, csg2, iptl, lcb4, lcb5, c#, 〃이 deletion 된 균주이며 control로는 BY4742를 사용하였다.
2 伽 RC filter (Satorius, Germany)로 필터링하여 분석에 사용하였다. 사용된 HPLC는 Acme 9000 HPLC system (Younglin Instrument, Korea)이며 data system은 Autochro 3000 (Younglin Instrument, Korea), detection system은 ELSD Sedex 75 (Sedere, France) 이다. ELSD 조건은 drift temperature 55°C, 분사 가스는질소 3.
성능/효과
ydcl gene의 deletion은 세포를 heat stress에민감하게 하여, 세포내에서 YDClp과 YPClp이 각각 dihydroceramide와 phytoceramide를 기질로서 사용하게된耳. \ydcl mutant 균주의 세라마이드 생산량은 6 mg ceramide/g cell로 BY4742 보다 1.57배 많은 생산량을나타내었으며, △ypcl mutant 균주의 세라마이드 생산량은 BY4742 보다 오히려 낮은 1.93 mg ceramide/g cell 을 나타내었다. 이는 ph>toceiamide에서 phytosphingosine 으로 가는 대사회로에서 ydcl gene이 ypcl gene 보다더 많은 영향을 미친다는 다른 연구 결과들과 경향성이일치한다(13, 14).
후속연구
즉면에서 쉽지 않다. 따라서, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabdits elegans 등의 모델 세포 또는 모델 생물체를 활용한 미지의 유전자나 또는 목적 유전자의 생물학적기능분석, pathway 규명, 질병기전과정의 연구가 가능하다(2, 3). 특히 S.
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