• Genomics & Informatics
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• 제5권4호
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• pp.174-178
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• 2007

The earliest stages of mammalian embryogenesis are governed by the activity of maternally inherited transcripts and proteins. Cytoplasmic polyadenylation of selected maternal mRNA has been reported to be a major control mechanism of delayed translation during preimplantation embryogenesis in mice. The presence of cis-elements required for cytoplasmic polyadenylation (e.g., CPE) can serve as a useful tag in the screening of maternal genes partaking in key functions in the transcriptionally dormant egg and early embryo. However, due to its relative simplicity, UA-rich sequences satisfying the canonical rule of known CPE consensus sequences are often found in the 3'-UTR of maternal transcripts that do not actually undergo cytoplasmic polyadenylation. In this study, we developed a method to confirm the validity of candidate CPE sequences in a given gene by a multiplex comparison of 3'-UTR sequences between mammalian homologs. We found that genes undergoing cytoplasmic polyadenylation tend to create a conserved block around the CPE, while CPE-like sequences in the 3'-UTR of genes lacking cytoplasmic polyadenylation do not exhibit such conservation between species. Through this cross-species comparison, we also identified an alternative CPE in the 3'-UTR of tissue-type plasminogen activator (tPA), which is more likely to serve as a functional element. We suggest that verification of CPEs based on sequence conservation can provide a convenient tool for mass screening of factors governing the earliest processes of mammalian embryogenesis.

• 미생물학회지
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• 제46권1호
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• pp.104-108
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• 2010

Leucine-responsive regulatory protein (Lrp)는 대장균 (Escherichia coli)에서 발견된 '글로벌 조절자(global regulator)'로서 Lrp-regulon이 leucine에 의하여 상이한 형태의 조절 양상을 나타낸다. 6XHis-tag 시스템으로 제조한 야생형 Lrp (Lrp Wt)와 돌연변이 Lrp (Lrp R145W) 단백질을 정제하여 그들의 생화학적 성질을 조사하였다. 이들은 gel retardation assay를 통하여 ilv 오페론의 프로모터 영역 consensus 염기서열인 21bp의 이중가닥 DNA와 결합하여 복합체를 형성하는 것을 확인 하였다. 형광성 아미노산인 tryptophan을 지닌 Lrp R145W은 단백질의 농도가 증가함에 따라 형광이 커졌으며, 아미노산 leucine에 의하여 형광성의 변화가 관찰되었다. 즉 1 ${\mu}M$의 Lrp R145W 단백질에 leucine을 첨가하여 결합시키면 약 20 ${\mu}M$까지는 형광이 증가하다가 그 이상의 농도에서는 감소하는 양상을 얻었다. 이들 실험 결과는 leucine과 Lrp의 결합 양상 및 구조변이에 관한 심층연구에 있어서 Lrp의 고유 형광성이 요긴하게 쓰일 수 있음을 시사한다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• 식물조직배양학회지
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• 제25권4호
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• pp.237-243
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• 1998

야생종 토마토의 자가불화합성에 관여하는 S RNase유전자의 조직특이적 발현 양상을 조사하기 위하여 $\textrm{S}_{11}$ 및 $\textrm{S}_{12}$ allele에 속하는 RNase 유전자의 Promoter영역에 대한 염기 서열을 비교 분석한 결과, 전사개시점에서 상류측으로 350-500bp사이에서 양쪽 allele의 Promoter간에 상동성을 나타내는 3부분과 direct repeat sequence를 발견하였다. Promoter영역에서 이러한 부분이 S RNase 유전자가 화주특이적으로 발현하는데 영향을 줄 것으로 예상하고, 이들 영역을 중심으로 6종류의 deletion fragment를 만들어 GUS 유전자에 연결하여, 토마토의 생식조직에 microprojectile bombardment를 수행하였다. 그 결과 토마토 자가불화합성에 관여하는 S RNase 유전자의 promoter는 TATA box를 포함한 127 bP만으로도 화주조직 특이적 발현을 조절할 수 있었다. 또한 S RNase 유전자의 promoter영역내에는 토마토 화변, 자방과 심피조직들에서 negative 혹은 positive로 유전자의 발현을 유도하는 부분이 발견되었다.

• Molecules and Cells
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• 제26권5호
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• pp.474-480
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• 2008

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.63-68
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• 2005

Transcription factors regulate gene expression by binding to gene upstream region. Each transcription factor has the specific binding site in promoter region. So the analysis of gene upstream sequence is necessary for understanding regulatory mechanism of genes, under a plausible idea that assumption that DNA sequence motif profiles are closely related to gene expression behaviors of the corresponding genes. Here, we present an effective approach to the analysis of the relation between gene expression profiles and gene upstream sequences on the basis of kernel canonical correlation analysis (kernel CCA). Kernel CCA is a useful method for finding relationships underlying between two different data sets. In the application to a yeast cell cycle data set, it is shown that gene upstream sequence profile is closely related to gene expression patterns in terms of canonical correlation scores. By the further analysis of the contributing values or weights of sequence motifs in the construction of a pair of sequence motif profiles and expression profiles, we show that the proposed method can identify significant DNA sequence motifs involved with some specific gene expression patterns, including some well known motifs and those putative, in the process of the yeast cell cycle.

• Fisheries and Aquatic Sciences
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• 제15권4호
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• pp.317-324
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• 2012

We characterized the cytoskeletal beta-actin (${\beta}$-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.

• 약학회지
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• 제50권2호
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• pp.124-128
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• 2006

Astrocyte has emerged as an active regulator of brain function, which connects between blood vessels and neurons as well as is a structural component of the blood-brain barrier, From its structural characteristics, astrocyte seems to sensitively respond to oxygen tension, and, in turn, generate diverse cellular cascades. Therefore, to reveal astrocytlc events by oxygen change, we screened genes whose expressions are upregulated under reoxygenation after hypoxic stress using cDNA representational difference analysis (RDA) technique. Meteorin that regulates glial differentiation was isolated from primary cultured rat astrocytes as a hypoxia/reoxygenation regulatory factor. We cloned rat version of Meteorin (rMe-teorin) and determined full-size sequences of rMeteorin. In addition, RT-PCR analysis revealed that Meteorin was increased under reoxygenation in astrocytes and highly expressed in the developing brain. Collectively, these results suggest that Meteorin may regulate astrocyte-mediated effects in response to the change of oxygen tension in the pathophysiological states.

• 한국가금학회지
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• 제50권4호
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• pp.261-266
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• 2023

가축의 골격근은 동물성 단백질 식품으로서 중요한 역할을 하며, 가금육의 소비는 전세계적으로 꾸준히 증가하고 있다. 근육의 형성과 발달에는 근형성조절인자를 포함한 많은 유전자들이 관여하며, 발달 단계에 따라 유전자 발현의 정확한 조절이 필요하다. 본 연구에서는 근육에서 특이적으로 발현하는 유전자를 선발하고, 해당 유전자의 프로모터를 클로닝하고 기능을 분석하였다. 동물의 조직별 유전자 발현을 분석한 결과, 다수의 유전자들이 골격근 특이적인 발현양상을 보였는데, 특히 TNNT3와 TNNC2, MYF6 유전자들은 가금에서도 유사한 양상을 나타냈다. TNNT3, TNNC2, MYF6 유전자의 프로모터 부위를 중합효소연쇄반응을 통해 각각 1.2 kb, 1.03 kb, 1.43 kb씩 증폭하여, 녹색형광단백질 유전자를 포함한 벡터의 앞부분에 삽입하였다. 염기서열 분석 결과, 세 프로모터는 기존에 밝혀진 유전체 서열과 거의 일치함을 확인하였다. QM7 메추리 근육세포주에서 각각의 프로모터를 포함한 벡터를 도입한 결과, 세 프로모터 모두녹색형광단백질을 성공적으로 발현시켰다. 녹색 형광의 밝기는 대조군으로 사용한 CMV-IE 프로모터와 비교 시, 약 7배 정도 어두웠다. 클로닝한 프로모터들에는 230개 이상의 전사인자들이 결합할 수 있을 것으로 예측되었으며, 특히 MYF5나 MYOD, MYOG와 같은 근형성조절인자를 포함한 근육에서 발현하는 다양한 전사인자들이 결합할 수 있을 것으로 예측되었다. 이 프로모터들은 가금의 근육세포에서 유전자 발현을 유도하는 연구에 활용이 가능할 것이며, 추후연구를 통해 프로모터 부위별 발현 조절 기능 연구가 필요할 것으로 사료된다.