• 미생물학회지
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• 제48권2호
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• pp.171-174
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• 2012

THOC1/Hpr1는 mRNA가 전사되는 동안 mRNP의 포장과 mRNA 방출에 관여하는 진화적으로 잘 보존된 THO 복합체의 한 소단위이다. 분열효모 Schizosaccharomyces pombe에서도 THOC1/Hpr1과 유사한 단백질을 암호화하는 유전자(sphpr1로 명명)를 찾아 그 특성을 조사하였다. 이배체 S. pombe 균주에 하나의 sphpr1 유전자만을 결실시킨 후 4분체 분석을 수행한 결과, 이 유전자는 생장에 필수적이었다. 티아민에 의해 발현이 조절되는 강력한 nmt1 프러모터를 이용하여 sphpr1를 과발현시키더라도 세포의 생장과 mRNA 방출에는 전혀 영향이 없었다. 하지만, sphpr1의 발현을 억제하면 생장이 억제되었으며 poly$(A)^+$ RNA가 핵 안에 축적되었다. 이와 같은 결과들은 sphpr1 유전자가 생장과 mRNA의 핵에서 세포질로의 방출에 관여하고 있음을 시사한다.

• Fisheries and Aquatic Sciences
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• 제17권3호
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• pp.377-380
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• 2014

RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectively downregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

• Animal cells and systems
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• 제5권3호
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• pp.217-224
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• 2001

The present study examines the expression and regulation of gonadotropin-releasing hormone (GnRH) and its receptor (GnRH-R) mRNA levels during mouse ovarian development. A fully processed, mature GnRH mRNA together with intron-containing primary transcripts was expressed in the immature mouse ovary as determined by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). The size of ovarian GnRH mRNA was similar to that of hypothalamus, but its amount was much lower than that in the hypothalamus. Quantitative RT-PCR procedure also revealed the expression of GnRH-R mRNA in the ovary, but the estimated amount was a thousand-fold lower than that in the pituitary gland. We also examined the regulation of ovarian GnRH and GnRH-R mRNA levels during the follicular development induced by pregnant mare's serum gonadotropin (PMSG) and/or human chorionic gonadotropin (hCG). Ovarian luteinizing hormone receptor (LH-R) mRNA was abruptly increased st 48 h after the PMSG administration and rapidly decreased to the basal level thereafter. Ovarian GnRH mRNA level was slightly decreased at 48 h after the PMSG administration, and then returned to the basal value. GnRH-R mRNA level began to increase at 24 h after the PMSG treatment, decreased below the uninduced basal level at 48 h, and gradually increased thereafter. HCG administration did not alter ovarian GnRH mRNA level, while it blocked the PMSG-induced increase in GnRH mRNA level. Taken together, the present study demonstrates that the expression of GnRH and GnRH-R mRNA are regulated by gonadotropin during follicular development, suggesting possible intragonadal paracrine roles of GnRH and GnRH-R in the mouse ovarian development.

• Animal cells and systems
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• 제1권1호
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• pp.115-121
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• 1997

To precisely analyze the role of ovarian steroids in the regulation of laminin chain gene expression in mouse uterine tissues, the ovariectomized mouse model was used. Ovariectomized mice received a single injection of steroid hormones and total RNA was isolated from whole uterine tissues. Messenger RNA levels of each laminin chain (A, 81, and 82) were determined by competitive RT-peR procedures. Estradiol decreased mRNA levels of laminin 81 chain about two-fold, and 82 chain rather moderately. Estradiol-induced inhibition of laminin 81 and 82 chain mRNA levels were completely blocked by pretreatment with estrogen receptor antagonist tamoxifen. Estriol, a short acting estrogen which cannot induce hyperplastic responses of rodent uterine tissues, also showed an inhibitory effect on 81 and 82 chain mRNA levels, while estrone, an inactive estrogen, failed to influence either 8 chain mRNA levels. Effects of steroids on A chain mRNA level were quite different from those on 8 chains. Laminin A chain mRNA level was slightly increased by estradiol treatment, but negatively affected by progesterone. Progesterone treatment greatly increased both 8 chain mRNA levels, but slightly decreased A chain mRNA level compared to the control. The effect of progesterone on laminin chain-specific mRNA levels was further increased by co-injection of estradiol in a time-dependent manner. Progesterone-induced 81 and 82 chain mRNA transcription was inhibited by RU486, a synthetic anti-progesterone /anti-glucocorticoid. The present study demonstrates for the first time that steroids are able to regulate laminin gene expression in mouse uterine tissues, indicating that steroid-regulated laminin gene expression is involved in uterine growth and probably differentiation.

• Asian-Australasian Journal of Animal Sciences
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• 제33권3호
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• pp.398-407
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• 2020

Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.339-347
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• 2012

동물세포배양 유래 생물의약품 생산 공정에서 다양한 외래성 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 보증을 위한 바이러스 검출시험이 필수적이다. Reovirus (Reo), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus (BPIV)는 동물 세포주와 동물 세포 배양 공정에 오염되는 대표적인 RNA 바이러스이다. 세포배양 유래 생물의약품의 안전성을 확보하기 위해, 세포주, 원료물질, 제조공정, 완제품에서 Reo, BVDV, BPIV를 동시에 검출할 수 있는 Multiplex Reverse Transcription (RT)-PCR 시험법을 확립하였다. Reo, BVDV, BPIV에 특이적인 primer를 선별하였으며, multiplex RT-PCR 시험법을 최적화하였다. Reo, BVDV, BPIV를 동시에 검출할 수 있는 multiplex RT-PCR 시험법의 민감도는 각각 $7.76{\times}10^2\;TCID_{50}/ml$, $7.44{\times}10^1\;TCID_{50}/ml$, $6.75{\times}10^1\;TCID_{50}/ml$이었다. 확립된 multiplex RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 각 바이러스를 오염시킨 CHO 세포에서 검출 시험을 실시한 결과 각 바이러스를 감염시킨 CHO 세포와 세포배양 상청액에서 각 바이러스를 검출할 수 있었다. 위와 같은 결과에서 확립된 multiplex RT-PCR시험법은 세포주, 원료물질, 제조공정, 완제품에서 Reo, BVDV, BPIV를 동시에 검출할 수 있는 특이성과 민감성이 우수한 시험법임을 확인하였다.

• 생명과학회지
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• 제21권1호
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• pp.141-145
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• 2011

본 연구에서는 전사억제제(transcriptional inhibitor)로 알려진 actinomycin D가 전사조절인자(transcription factor)인 STAT의 인산화를 유도한다는 것을 확인하였다. Actinomycin D 처리 시 STAT1의 Tyr701, Ser727 인산화는 유도되지 않았지만 STAT3의 Tyr705 잔기의 인산화를 특이적으로 유도하는 것을 확인하였다. Actinomycin D에 의한 STAT3의 Tyr705 인산화 유도가 어떠한 기전을 통한 것인지 확인하기 위해서 관련 인자의 단백질 및 mRNA 발현을 확인한 결과 SOCS3의 단백질 및 mRNA 발현의 감소를 확인하였다. STAT3의 탈인산화를 유도한다고 알려진 tyrosine phosphatase인 SHP-1와 STAT의 upstream kinase인 JAK2의 인산화는 변화가 없었다. 또한 actinomycin D 뿐 아니라 다른 전사억제제인 DRB를 처리 하였을 경우에도 STAT3의 Tyr705 인산화가 유도되는 것을 확인하였다. 이상의 결과는 전사억제제에 의하여 특이적인 SOCS3 단백질 발현감소는 SOCS3의 하류의 target인 STAT3 인산화를 유도하였다.

• Animal cells and systems
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• 제3권2호
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• pp.181-185
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• 1999

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetylcholine receptors (nAChRs) $\alpha$$_{6}$ subtype in oocytes was systematically increased by more than 100-fold, which was confirmed both by the $\alpha$-Bungarotoxin ($\alpha$,/TEX>Bgt) binding assay and the current measurement.t.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.421-426
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• 2005

In this paper, we investigate the nonlinear dynamic behavior of TPP (thiamine pyrophosphate) riboswitches in E. coli (Escherichia coli). TPP riboswitches are highly conserved RNA regulatory elements, embedded within the 5’'untranslated region of three TPP biosynthesis operons. The three operons thiCEFSGH, thiMD, and thiBPQ are involved in the biosynthesis, salvage, and transport of TPP, respectively. TPP riboswitches modulate their expressions in response to changing TPP concentration, without involving protein cofactors. Interestingly, the expression of thiMD is regulated at the translational level, while that of thiCEFSGH at both levels of transcription and translation. We develop a mathematical model of the TPP riboswitch’s regulatory system possessed by thiCEFSGH and thiMD, so as to simulate the time-course experiments of TPP biosynthesis in E. coli. The simulation results are validated against three sets of reported experimental data in order to gain insight into the nature of steady states and the stability of TPP riboswitches, and to explain the biological significance of regulating at level of transcription or translation, or even both. Our findings suggest that in the TPP biosynthesis pathway of E. coli, the biological effect of down-regulating thiCEFSGH operon at the translational level by TPP riboswitch is less prominent than that at the transcriptional level.

• Natural Product Sciences
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• 제17권1호
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• pp.26-32
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• 2011

Novel tyrosinase inhibitors are important for pigmentation in the skin. Following extraction of tyrosinase inhibitors from edible vegetables or fruits, we found that the Prunus persica flesh extract (PPFE) exhibited potential inhibitory activity for melanogenesis. PPFE showed tyrosinase inhibitory activity in an enzymatic assay and PPFE also significantly inhibited the melanin formation in cultured mouse melan-a cells. Moreover, real-time RT-PCR analysis revealed that the inhibition of melanin production by PPFE was closely related to marked suppression of mRNA expression of tyrosinase and tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) in melan-a cells. Further investigation found that the modulation of tyrosinase expression by PPFE was associated with the transcriptional regulation of the microphthalmia-associated transcription factor (MITF). PPFE inhibited the promoter activity of MITF and suppressed MITF mRNA expression in melan-a cells. These results indicate that PPFE down-regulates melanogenesis-associated gene expression through MITF-mediated transcriptional regulation and these events might be related to the hypopigmentary effects of PPFE.