• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.791-797
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• 2018

Objective: Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Methods: Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions ($Fold-change{\geq}2$, $FDR{\leq}0.01$) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 (CRABP1), growth differentiation factor 10 (GDF10), and gremlin 1 (GREM1) gene explorations. Results: The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes-CRABP1, GDF10, and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. Conclusion: According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

• Fisheries and Aquatic Sciences
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• 제13권1호
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• pp.56-62
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• 2010

To determine whether long double-stranded RNA (dsRNA) induces RNA interference and type I interferon (IFN) responses in fish, long dsRNAs encoding enhanced green fluorescent protein (EGFP), GFPuv, and polyinosinic-polycytidylic acid sequences were co-injected with an EGFP expressing plasmid, into rock bream (Oplegnathus fasciatus). We investigated the EGFP mRNA and protein levels, and the transcriptional responses of dsRNA-dependent protein kinase and Mx1 genes. Long dsRNAs were strong inducers of a type I IFN response in rock bream, resulting in nonspecific suppression of exogenous gene expression. Furthermore, sequence-specific knockdown of exogenous gene expression at the mRNA level was detected at an early phase (24 h). These results suggested that long dsRNA may inhibit exogenous gene expression through an early mRNA interference response and a later type I IFN response in fish.

• Radiation Oncology Journal
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• 제19권4호
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• pp.381-388
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• 2001

목적 : 사람의 유방암 세포인 MCF-7 세포주를 대상으로 gultathione S-transferase K1 (hGSTK1) 유전자의 발현 정도 및 방사선 조사에 의한 hGSTK1 유전자의 발현 변화를 관찰하고 hGSTK1 유전자를 과발현시킴으로써 hGSTK1이 방사선 감수성에 어떤 영향을 미치는 지를 관찰하였다. 재료 및 방법 : 사람의 모유두 세포 pBluescript phagemid cDNA library로부터 선별한 hGSTK1 cDNA를 pcDNA3.1/Myc-His (+) vector에 결합시킨 후, 사람의 유방암 세포인 MCF극 세포에 이입시켰다. hGSTK1 유전자를 이입시키지 않은 MCF극 세포와 hGSTK1을 이입시킨 MCF-7 세포에 $2\~12\;Gy$의 엑스선을 조사하여 생존 분획을 비교하였다. hGSTK1 유전자를 이입시키지 않은 MCF-7 세포와 hGSTK1을 이입시킨 MCF-7 세포에서 방사선량, 분할조사 여부, 방사선 조사 후 경과 시간에 따른 hGSTK1 mRNA 발현의 차이를 보기 위하여 RT-PCR 분석을 시행하였다. 결과 : hGSTK1 유전자를 이입시키기 전의 MCF-7 세포보다 hGSTK1 유전자를 이입시킨 MCF-7 세포에서 생존 분획이 유의하게 높은 것으로 나타났다. hGSTK1 유전자를 이입시키지 않은 MCF-7 세포에서 2 Gy 생존 분획은 $0.3250{\pm}0.0319$였고, hGSTK1 유전자를 이입시킨 MCF-7 세포에서 2 Gy 생존 분획은 $0.4125{\pm}0.0325$였다 (p<0.05). 그러나 RT-PCR에 의한 hGSTK1 mRNA 분석에서는 방사선량, 분할조사 여부, 방사선 조사 후 경과 시간에 따른 발현의 차이를 볼 수 없었다. 결론 : MCF-7 세포주에서 hGSTK1의 과발현은 방사선 감수성에 영향을 미쳐서 MCF-7 세포의 생존 분획을 증가 시켰다고 볼 수 있으나 이에 대한 정확한 기전을 알기 위해서는 더 많은 연구가 필요할 것이다.

• 한국환경성돌연변이발암원학회지
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• 제23권2호
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• pp.45-50
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• 2003

Biological activities of PAHs are not known although PAHs are considered as carcinogens. Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Our laboratory have been studied the effect of PAHs in the human breast cancer MCF-7 cells. In this study, we examined the human breast cancer MCF-7 cells as a new system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using CYP1A1-luciferase reporter gene expression system where CYP1A1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into MCF-7 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter, CYP1A1 mRNA and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. None of PAHs that we have tested showed stronger stimulatory effect on CYP1 gene expression than TCDD. Benz(a)anthracene and benzo(b)fluoranthene were weak responders to CYP1A1 promoter activity stimulation, CYP1A1 mRNA and EROD induction in MCF-7 cells and these chemicals seemed to respond less either CYP1A1 mRNA or EROD than CYP1A1 promoter activity. Benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene showed strong response to CYP1A1 promoter activity stimulation, CYP1A1 mRNA increase and also EROD induction in MCF-7 cells. Results of dose response study suggested that two strong responding PAHs, such as benzo(k)fluoranthene and dibenzo(a, h)anthracene might be mediated through Aryl hydrocarbon receptors system in MCF-7 cells.

• Journal of Oral Medicine and Pain
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• 제19권2호
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• pp.57-71
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• 1994

Gingival fibroblasts were cultured and subjected to the test of Northern blot analysis for the demonstration of various mRNA expression in response to the low level laser treatment. For duplication of in vivo. Wound healing process, fibroblasts were pretreated with proinflammatory cytokine interleukin-1$\beta$(IL-1$\beta$) or mitogenic substance phorbol 12-myristate 13-acetate(PMA) prior to laser irradiation. The results were as follows : 1. By the laser irradiation, the gene expression of collagen type I was markedly increased I n gingival fibroblasts, especially in the case of PMA pretreatment. The gene expression of collagen type IV, however, was not only affected by laser irradiation but also by chemical cell stimulation. 2. Oncogene v-myc expression was affected by both laser irradiation and IL-1$\beta$ or PMA stimulation, But v-fos gene expression was not detected in any case of this experimental system. 3. Heat shock gene(Hsp 70)was expressed constiutively, but slightly increased by laser irradiation. 4. mRNA of fibroblast growth factor(FGF) was induced by both laser irradiation and IL-1$\beta$ or PMA treatment.

• The Korean Journal of Physiology and Pharmacology
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• 제1권1호
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• pp.45-53
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• 1997

In humans and many animal models with chronic progressive renal diseases, angiotensin-converting enzyme (ACE) inhibitor markedly attenuates the progression of nephropathy. Several studies have reported augmented gene expression and redistribution of renal renin in partial nephrectomized rats. Although precise mechanism(s) is not known, the renin-angiotensin system (RAS) may play an important role in the progression of renal diseases. Thus, this study was undertaken to examine the gene expression of renal renin, angiotensinogen, and $AT_1$ subtypes ($AT_{1A}$ and $AT_{1B}$) in rats with diabetic nephropathy, and the influences of lipopolysaccharide (LPS)-induced septicemia on the gene expression. Four weeks after streptozotocin (STZ) treatment (55 mg/kg, i.p.), rats were randomly divided into LPS-treated (1.6 mg/kg, i.p.) and control rats. At 6 hours after LPS treatment, the rats were killed and the kidney was removed from each rat. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR)techniques were used to detect mRNA expression. STZ treatment markedly attenuated body weight gain and significantly increased blood glucose level. Renal renin content (RRC) was significantly decreased in the STZ-treated rats compared to that in control rats. The renal ACE activity between STZ-treated and control rats was not significantly different. Renal renin mRNA level was prominently increased, while angiotensinogen and $AT_{1A}$ mRNA levels were slightly decreased in STZ-treated rats compared to those in controls. $AT_1$B mRNA level did not differ in both groups. Acute LPS treatment did not show any significant changes of mRNA levels of intrarenal RAS components in both groups. These results suggest that intrarenal RAS components were differentially regulated in STZ-treated diabetic rats. Further studies are required to evaluate the relationship between intrarenal RAS and other vasomodulatory systems.

• 한국동물생명공학회지
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• 제36권3호
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• pp.121-128
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• 2021

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl₂. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 ㎛ of CdCl₂, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl₂ untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl₂ in HC11 cells decreased the mRNA expression of Rad51.

• Journal of Applied Biological Chemistry
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• 제48권1호
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• pp.1-6
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• 2005

Analysis of chlorophyll (Chl) fluorescence implicated treatment of 40 mM NaCl decreased maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm), actual quantum yield of PSII (${\Phi}_{PSII}$), and photochemical quenching (qP) in rice, but increased non-photochemical quenching (NPQ). Decreases in Fv/Fm, ${\Phi}_{PSII}$, and qP were significantly alleviated by $30\;{\mu}M$ jasmonic acid (JA), while NPQ increase was enhanced. Transcription levels of antioxidant isoenzyme genes were differentially modulated by NaCl treatment. Expression of cCuZn-SOD2 gene increased, while those of cAPXb, CATb, and CATc genes decreased. JA prevented salt-induced decrease of pCuZn-SOD gene expression, but caused greater decrease in mRNA levels of cAPXa and Chl_tAPX genes. Investigation of vacuolar $Na^+/H^+$ exchanger (NHX2) and 1-pyrroline-5-carboxylate synthetase (P5CS) gene expressions revealed transcription level of NHX2 gene was increased by JA, regardless of NaCl presence, while that of P5CS gene slightly increased only in co-presence of JA and NaCl. Unlike JA, ${\gamma}$-radiation rarely affected expressions of antioxidant isoenzyme, NHX2, and P5CS genes, except for increase in mRNA level of Chl_tAPX and decrease in that of pCuZn-SOD. These results demonstrate enhanced salt-tolerance in JA-treated rice seedlings may be partly due to high transcription levels of pCuZn-SOD, NHX2, and P5CS genes under salt stress.

• 한국어병학회지
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• 제6권2호
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• pp.93-101
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• 1993

효모의 RNAI유전자는 RNA processing에 관여 하는지 혹은 RNA transport에 관여 하는지 아직까지 유전자의 기능이 정확히 알려져 있지 않은 실정이다. 효모의 RNAI 유전자의 기능을 파악하기 위한 방법으로 본 연구에서는 rna1-1 mutant gene을 cloning하여 이에 대한 DNA sequence를 조사함으로써 RNAI 유전자와 rna1-1 유전자의 차이점을 이해하고자 하였다. rna1-1 marker를 갖는 yeast strain(R49)로 부터 genomic DNA를 추출하여 이를 BglII로 절단하여 genomic southern blotting을 행한 결과 wild type의 경우와 동일하게 3.4 kb에서 hybridization되는 signal을 얻었으며, RNAl 및 rna1-1이 yeast genome내에 single site로 존재함을 보여 주는 결과를 얻었다. mutant strain으로 부터 얻은 3.4 kb의 BglI fragment를 pUC19의 BamHI site에 subcloning하여 transformant들을 얻었고, wild type RNAl 유전자를 probe로 하여 rna1-1 mutant 유전자를 cloning할 수 있었다. pUC19에 cloning된 RNA1유전자 및 rna1-1유전자로부터 다양한 Ba131유도체를 얻어 이들에 대한 염기 서열을 비교한 결과 transcription initation site에서부터 down stream쪽으로 17 아미노산위치에 TCC가 TTC로 대치되어 있었으며 그 결과 serine이 phenylalanine으로 변환되는 결과를 얻었다. Wild type RNAI gene의 5'-region에는 3군데의 TATA-like sequence가 true TATA box인지 확인하기 위하여 Bal3I deletion에 의해 -103nt까지 deletion된 유도체를 얻었으며 ${\Delta}RNAI$, rna1-1, 81-2-6 clone이 rna1-1 allele와 complementation한지 확인하였으나 ${\Delta}RNAI$은 TS-complementation을 하지 못하였다. 따라서 현재까지 TATA-box라고 알려진 부분은 promoter로 작용하지 못함을 확인하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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• pp.1651-1654
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• 2007

The mouse myxovirus resistance protein 1 (Mx1) is known to be sufficient to confer resistance to influenza viruses, and the gene encoding Mx1 is, therefore, an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; the full-length coding region of the pig Mx1 gene spans 2,545 bp (M65087) and is organized into 17 exons compared with the human ortholog mRNA. In this study, the exons 9, 10 and 11 and introns 6 and 9 of the porcine Mx1 gene were cloned and sequenced. Two SNPs were identified in exons 9, 10 and 11 but none of the SNPs led to an amino acid exchange, and the other eleven variants were detected in introns 6 and 9, respectively. Differences in allele frequency between Meishan and other pig breeds were observed within intron 6, of which an $A{\rightarrow}G$ substitution at position 371 was detected as an SnaBI PCR-RFLP. The association analysis using the Large White${\times}$Meishan $F_2$ offspring suggested that the Mx1 genotype was associated with variation in several immunity traits that are of interest in pig breeding. However, further investigations in more populations are needed to confirm the above result.