Small GTP-binding Protein과 Vesicular Transport

천충일 한국생화학분자생물학회 1996 생화학분자생물학회 소식 Vol.16 No.2

Soybean의 Small GTP-binding Protein (sRab7)이 Endocytosis에 결함이 있는 Yeast ypt7 mutant 를 Complements

천충일,양효숙 숙명여자대학교 자연과학연구소 1997 자연과학논문집 Vol.- No.8

포유류의 Rab7에 대한 콩과식물의 homolog(sRab7)에 대한 cDNA가 soybean에서 분리된 바 있다. 이 단백질은 small GTP-binding protein에 특이한 공통 서열 motif(G1-G5)를 갖고 있으며, 세포내 수송신호로 알려진 C-terminal부위를 갖고 있다. sRab7이 정확한 Ypt7p(Rab7)의 homolog인지를 확인하기 위하여, srab7 cDNA가 YEp51의 GAL10 promoter의 downstream에 삽입되었다. 형질전환된 yeast mutant는 mutant와 대조적으로 하나의 큰 vacuole을 갖고 있음이 확인되었다. 이 발견은 sRab7이 yeast Ypt7p(Rab7)과 기능적으로 동일함을 보여준다. A cDNA encoding legume homolog of mammalian Rab7(sRab7) was isolated from soybean. sRab7 has the conserved wequence motifs (G1-G5) which are specific to small GTP-binding protein and C-terminal region known as intracellular targeting signal. In order to confirm that sRab7 is an authentic Ypt7p(Rab7) homolog, the srab7 cDNA was inserted downstream of the GAL10 promoter in YEp51(pHYE51). The transformed mutant showed a single large vacuole in contrast to mutant. Out finding suggests that sRab7 was confirmed to be a functional counterpart of yeast Ypt7p(Rab7).

Soybean의 Small GTP-binding Protein (sRab7)이 Endocytosis에 결함이 있는 Yeast ypt7 mutant 를 Complements

천충일 양효숙 숙명여자대학교 자연과학연구소 1997 자연과학논문집 Vol.- No.8

Small GTP-binding Protein, Rab7 Homologs의 Molecular Cloning

양효숙,천충일 숙명여자대학교 자연과학연구소 1996 자연과학논문집 Vol.- No.7

Soybean의 뿌리혹에서 두개의 small GTP-binding protein(sRab1, sRab7)이 질소고정시 유도됨이 밝혀졌고, cDNA가 분리된 바 있다. rab7의 유도되는 정도가 높은 점에 착안해 cDNA library screening을 실시한 결과, 추가로 3개의 sRab7과 유사한 단백질들이 cloning되었다(sRab71, -b, 그리고 -c). 이들은 DNA sequence 상에서 서로 높은 homology를 보이며, srab7 gene과도 높은 homology를 갖는다. 뿐만 아니라, small GTPase가 갖는 conserved sequence motifs(G1 to G5)를 보이고, intracellular targeting signal에 관련된 것으로 알려진 C-terminal region에는 다른 Rab proteins과 매우 다른 amino acid sequences를 갖는다. 반면에 sRab7과 또한 각 homologs간에는 C-terminal region까지도 높은 homology를 보였다. 이러한 결과는 soybean nodules의 발생시, endocytosis에 필요한 small GTP-binding protein이 여러 homology 의 형태로 존재하며, 이들의 세분된 각각의 기능에 대한 연구가 필요함을 보여주고 있다. cDNA's of two small GTP-binding proteins (sRab1, sRab7) were isolated and shown to be induced during nitrogen fixation. Since the rab7 induction was very high, additional screening cDNA library of soybean resulted in isolation of four more sRab7 homolotys (sRab7a, -b and -c). They have high homology to each other at DNA levels, and to srab7, too. They also show the conserved sequence motif (G1 to G5) which are specific to small GTPase. These clones have characteristic C-terminal region known as intracellular targeting signal which are highly homologous to sRab7 although sRab7d shows high homology to Rab7. We conclude that during soybean nodulation small GTP-binding proteins required for endocytosis may exist as many homologs which need to be studied in detail for the function of each homologs.

Small GTP-binding Protein, Rab7 Homologs의 Molecular Cloning

양효숙 천충일 숙명여자대학교 자연과학연구소 1996 자연과학논문집 Vol.- No.7

ABA에 유도되는 Athb-12유전자의 발현 패턴

오혜숙,천충일 숙명여자대학교 자연과학연구소 2000 자연과학논문집 Vol.- No.11

식물은 water stress를 Abscisic Acid (ABA)에 의한 신호전달 과정에 의해 반응하는 것으로 알려져 있는 데, homeobox gone인 Athb-12는 ABA에 의해 발현이 유도되는 것이 보고되어 water stress에 중요한 역할을 할 것으로 예상되었다. Athb-12유전자의 promoter지역 약 2.1 Kb를 β-glucuronidase (GUS)와 fusion하여 transgenic tobacco를 제작하였다. 이를 이용하여 ABA처리시 어떠한 양상으로 발현하는 지를 관찰하였다. 또한 다른 환경stress에 어떻게 반응하는지 조사하기 위해 UV처리와 wounding처리를 하여 GUS의 발현을 관찰하였다. 이 결과들은 Athb-12가 ABA에 의한 반응 뿐 아니라, 전반적인 스트레스들의 반응에 관여할 가능성을 보여 주었다. It has been known that plant responds to water stress by ABA-mediated signal transduction. Since Athb-12, a homeobox gene from Arabidopsis, was reported to be inducible to ABA, it was expected to be important to plant response to water stress. About 2.1 kb DNA of Athb-12 promoter region was fused to f-glucuronidase gene and the resulting construct was used in making transgenic tobacco plant. ABA-treatment to the transgenics was observed. Other environmental stresses such as UV and wounding were also tested. The results obtained showed a possibility that Athb-12 is involved in plant responses to most environmental stresses as well as ABA.

Screening for the interacting protein with Athb-12,an ABA-inducible homeodomain protein in Arabidopsis

Cho, Hee-Yeon,Cheon, Choong-Ill 숙명여자대학교 자연과학연구소 2002 자연과학논문집 Vol.- No.13

Athb-12는 homeobox gene이고 homeodomain 근처에 leucine zipper motif를 가지고 있다. 이 gene은 abscisic acid(ABA)와 가뭄(drought) 처리에 의해 발현이 유도된다. leucine zipper motif는 단백질간의 상호작용에서 역할을 하는 것으로 알려져 있기 때문에 Athb-12 단백질과 상호작용을 하는 단백질을 yeast two-hybrid library screening을 통하여 찾아보았다. Arabidopsis flower로부터 만들어진 cDNA library가 이 연구에 이용되었다. 1.1×10^6개의 clone이 screening 되었고, 그 결과 peroxisomal targeting signal typeⅠ receptor와 photosystemⅡ typeⅠ chlorophyll a/b binding protein같은 cytoplasmic protein이 Athb-12의 putative interacting partner로 찾아졌다. The Athb-12 gene is a homeobox gene and its amino acid sequence has a putative leucine zipper closely linked to the homeodomain. This gene was shown to be induced by exogenous abscisic acid (ABA) as well as drought. As the leucine zipper motif has been shown to be involved in protein-protein interaction, the interacting partner to Athb-12 was sought using a yeast two-hybrid library screening. The library made from Arabidopsis flower was used in this study. From 1.1×10^6 clones screened, the positives revealed some cytoplasmic proteins, such as peroxisomal targeting signal type receptor and photosystemⅡ typeⅠ chlorophyll a/b binding protein. The specific interaction of Athb-12 and these proteins will be tested in vitro and in vivo.

Stress-Inducible Gene Expression Pattern of the Athb-12, a Homeobox Gene in Arabidopsis

Son, Ora,Cheon, Choong-Ill 숙명여자대학교 자연과학연구소 2000 자연과학논문집 Vol.- No.11

Athb-12 gene은 homeobox gene이고, homeodomain의 가까이에 leucine aipper motif를 가지고 있다. 그 gene은 abscisic acid (ABA)의 처리에 의해 발현이 유도되고, 여러 개의 putative ABRES와 DREs가 5' flanking region에 존재한다. 다양한 길이의 Athb-12 promoter부위와 β-glucuronidase(GUS) reporter gene를 fusion시킨 여러 constructs의 ABA, drought, salt stresses에 의한 발현량이 분석되었다. Transgenic plants에서의 GUS acclivity의 정량적 분석을 통해 Athb-12 gene expression level이 promoter의 길이에 비례적으로 증가함을 알았다. Yeast two-hybrid test를 통해 Athb-12의 HD-Zip은 yeast내에서 homodimer를 형성하지 않음을 밝혔다. 이 연구에서 보여진 결과들로 Athb-12 gene이 ABA에 의해 중계되는 water stress의 signaling pathway에 관여한다는 것을 확인하였다. The Athb-12 gene is a homeobox gene, and has a putative leucine zipper motif closley linked to the homeodomain. It was shown to be induced by exogenous abscisic acid (ABA), and several putative ABA-responsive elements and dehydration-responsive elements were found in its 5'- flanking region. The expression of the Athb-12 gene fused with β-glucuronidase (GUS) reporter gene in response to ABA, drought, and salt stresses was analyzed. The quantitative analysis of GUS activity in transgenic plants showed that Athb-12 gene expression level is in proportion to the length of the promoter. A yeast two-hybrid test revealed that the HD-Zip of Athb-12 does not form a dimer with itself in yeast. The results shown in this study verified that the Athb-12 gene may participate signaling pathway of water stress mediated by ABA.

RNA Interference-Mediated Repression of S6 Kinase 1 Impairs Root Nodule Development in Soybean

엄지현,천충일,김성한,김윤경,송석보,이석하,Desh Pal S. Verma 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.3

Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified gluta-mine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.

Possible Dual Regulatory Circuits Involving AtS6K1 in the Regulation of Plant Cell Cycle and Growth

신윤정,천충일,김성한,Hui Du,최순영,Desh Pal S. Verma 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.5

The role of Arabidopsis S6 Kinase 1 (AtS6K1), a down-stream target of TOR kinase, in controlling plant growth and ribosome biogenesis was characterized after generating transgenic plants expressing AtS6K1 under auxin-inducible promoter. Down-regulation of selected cell cycle regulatory genes upon auxin treatment was observed in the transgenic plants, confirming the negative regulatory role of AtS6K1 in the plant cell cycle progression reported earlier. Callus tissues established from these transgenic plants grew to larger cell masses with more number of enlarged cells than untransformed control, demonstrating functional implication of AtS6K1 in the control of plant cell size. The observed negative correlation between the expression of AtS6K1 and the cell cycle regulatory genes, however, was completely reversed in protoplasts generated from the transgenic plants expressing AtS6K1, suggesting a possible existence of dual regulatory mechanism of the plant cell cycle regulation mediated by AtS6K1. An alternative method of kinase assay, termed “substrate-mediated kinase pull down”, was employed to examine the additional phosphorylation on other domains of AtS6K1 and verified the phosphorylation of both amino- and carboxy-terminal domains, which is a novel finding regarding the phosphorylation target sites on plant S6Ks by upstream regulatory kinases. In addition, this kinase assay under the stress conditions revealed the salt- and sugar-dependencies of AtS6K1 phosphorylations.