초록 ▼

1. 신규 신호전달 유전자인 MPK38을 약 150개의 protein kinase cadidates로부터 분리 및 동정하였고, MPK38 cDNA는 SNF1 Ser/Thr kinase subfamily와 약 60% identity을 보여주었다. MPK38 유전자는 1932 bp의 coding 염기길이와 643 amino acids를 가진다.2. Northern blot analysis를 이용해 mouse의 조직 및 cell lines 을통한 MPK38 발현분석은 thymus 조직과 T-lineage cells, macrophage/

1. 신규 신호전달 유전자인 MPK38을 약 150개의 protein kinase cadidates로부터 분리 및 동정하였고, MPK38 cDNA는 SNF1 Ser/Thr kinase subfamily와 약 60% identity을 보여주었다. MPK38 유전자는 1932 bp의 coding 염기길이와 643 amino acids를 가진다.2. Northern blot analysis를 이용해 mouse의 조직 및 cell lines 을통한 MPK38 발현분석은 thymus 조직과 T-lineage cells, macrophage/monocyte cells에 특이적 발현을 보여주었다. 3. MPK38의 발현은 thymocytes의 in vitro culture 상태하에서 transcript가 거의 소실되고, Concanavalin A의 처리에 의해 다시 유도됨을 보여주었다. 이때 thymocyte의 활성화 정도는 marker인 CD25 발현을 측정하였으며, Con A처리에 의해 중가됨을 보여준다.4. MPK38 발현은 여러 mitogen 중 오직 T cell mitogen인 Con A 와 PHA에 의해 그 발현이 증가됨을 보여주었다. 또한 in vitro translation의 결과는 MPK38의 예상 분자량인 약 70 kDa과 더 불어 internal initiation로 부터 유래되는 약 50 kDa을 관측할 수 있었다.5. S. pombe에서의 Ser/Thr kinase candidate을 얻기위한 kinase-specific PCR은 southern blot analysis에의해 확인 하였고, 증폭된 PCR products들을 elution한 후 분석을 위한 kinase specific library을 만들 예정이다. 6. HBV-X 단백질의 인산화 작용연구를 위한 HBV-X 단백질은 pET3d vector system을 이용하였고 정제는 CM Sepharose을 이용하였다. 그 결과 16-17 kDa 크기의 순수한 단백질을 분리 하였다.7. HBV-X 단백질의 생리 활성도는 refolding된 단백질을 HeLa nuclear extract in vitro transcription system을 사용하여 측정 하였고, 첨가되는 단백질의 양에 비례하여 550 bp의 band 증가 현상을 측정할 수 있었다.8. HBV-X 단백질의 인산화 작용은 MAP kinase, PKC, PKA, casein kinase Ⅱ을 사용한 in vitro phosphorylation system을 이용하였고, 그 결과 HBV-X 단백질은 PKC에 의해 강하게 phosphorylation이 일어나고, PKA는 영향을 주지 못함을 보여주었다.

Abstract ▼

1. In an attempt to identify genes that are involved in the signal transduction for cell growth, proliferation, and differentiation, a PCR-based screen was employed and we finally selected one clone, named as MPK38. MPK38, a novel putative Ser/Thr kinase, showed about 60% identity to the kinase cata

1. In an attempt to identify genes that are involved in the signal transduction for cell growth, proliferation, and differentiation, a PCR-based screen was employed and we finally selected one clone, named as MPK38. MPK38, a novel putative Ser/Thr kinase, showed about 60% identity to the kinase catalytic domains of the protein SNF1 Ser/Thr kinase subfamily. There is a 1932 bp open reading frame encoding for a protein of 643 amino acids.2. The pattern of MPK38 expression among mouse tissues and cell lines was determined by Northern blot analysis using MPK38 cDNA probe. MPK38 was predominantly expressed in thymus and spleen, but was not detectable in other tissues. In addition, MPK38 was apparently expressed in T lineage cells and a macrophage/monocyte cell, but was not detectable in other cells tested.3. Incubation of thymocytes at 37 ℃ resulted in the loss of MPK38 transcript, however the transcript could be reinduced by treatment with ConA and PHA, but not other mitogens and growth factors. Consistent with the T cell activation by Con A, CD25, which are expressed in developing thymocytes and activated mature T cells, was proportionately increased in the thymocytes treated with ConA.4. To understand the coding capacity of the MPK38 cDNA, we transcribed and translated the cDNA in a rabbit reticulocyte lysate in the presence of 35S-methionine. The plasmid harboring the cDNA directed the synthesis of two protein species with relative mobilities corresponding to $^{\sim}70/; and/; ^{\sim}50$ kDa on SDS polyacrylamide gels.5. To identify Ser/Thr kinase candidates, kinase-specific PCR was employed using several oligonucleotide primers corresponding to the conserved catalytic domains of the protein kinases, and confirmed by Southern blot analysis. As a result, we obtained several positive kinase candidates, and will use them for construction of the kinase-specific library.6. To study the phosphorylation of HBV-X protein, we used the pET3d vector to make a recombinant HBV-X protein. The HBV-X protein was separated and purified using CM Sepharose. We can detect the pure HBV-X protein with 16-17 kDa on 12% SDS-PAGE.7. Physiological activity of the refolded HBV-X protein was determined by in vitro transcription assay. The results showed the increase of 550 bp band in proportion to the added amount of HBV-X protein.8. Phosphorylation of HBV-X protein was estimated by in vitro phosphorylation assay using MAP kinase, PKC, PKA, and casein kinase Ⅱ. The data showed that PKC could phosphorylate the HBV-X protein strongly, but PKA was not effective in phosphorylation of the HBV-X protein in vitro.

목차 Contents

• 제 1 부 신규 PROTEIN KINASE 의 분리 및 동정...15
• 1. 서론...15
• 2. 실험재료 및 방법...17
• 3. 결과 및 고찰...22
• 제 2 부 분열효모에서 PCR을 통한 새로운kinase cloning...31
• 1. 서론...31
• 2. 실험재료 및 방법...33
• 3. 결과 및 고찰...38
• 제 3 부 HBV-X 단백질의 인산화 작용...53
• 1. 서론...53
• 2. 실험재료 및 방법...54
• 3. 결과 및 고찰...57