• Animal cells and systems
• /
• 제4권4호
• /
• pp.389-394
• /
• 2000

The Tcf-1 (1-cell factor-1) protein binds to the T-cell specific enhancer sequences and plays an architectural role in the assembly of transcriptional machinery. One of the Tcf family proteins, Tcf-4, was found to be an important regulator for colon cancer development where it activates specific genes upon binding to $\beta$-catenin following Wnt signaling. We were interested in the transcriptional regulatory activities of Tcf-1 and Tcf-4 proteins in T-cells and colon cancer cells. Transactivation assay was developed using a reporter plasmid containing luciferase gene under the control of Tcf responsive elements. Luciferase activity was determined following co-transfection of the reporter along with Tcf-1 and/or $\beta$-catenin expressing plasmids. Transcription was significantly induced by $\beta$-catenin expression in all cells. Tcf-1 by itself did not induce transcription in the mature T-cell lines, but overexpressed Tcf-1 greatly activated transcription in the immature T-cell line. In addition, transfected $\beta$-catenin induced apoptosis, but co-transfected Tcf-1 suppressed apoptosis in HEK293 cells. These results suggest that Tcf-1 and $\beta$-catenin differently regulate transcription and apoptosis.

• Asian-Australasian Journal of Animal Sciences
• /
• 제25권2호
• /
• pp.163-169
• /
• 2012

Interferon regulatory factor 6 (IRF6) gene is a member of the IRF-family, and plays functionally diverse roles in the regulation of the immune system. In this report, the 13,720 bp porcine IRF6 genomic DNA structure was firstly identified with a putative IRF6 protein of 467 amino acids. Alignment and phylogenetic analysis of the porcine IRF6 amino acid sequences with their homologies to other species showed high identity (over 96%). Tissues expression of IRF6 mRNA was observed by RT-PCR, the results revealed IRF6 expressed widely in eight tissues. One SNP (HQ026023:1383 G>C) in exon7 and two SNPs (HQ026023:130 G>A; 232 C>T) in the 5′ promoter region of porcine IRF6 gene were demonstrated by DNA sequencing analysis. A further analysis of SNP genotypes associated with immune traits including IFN-${\gamma}$ and IL10 concentrations in serum was carried out in three pig populations including Large White, Landraces and Songliao Black pig (a Chinese indigenous breed). The results showed that the SNP (HQ026023:1383 G>C) was significantly associated with the level of IFN-${\gamma}$ (d 20) in serum (p = 0.038) and the ratio of IFN-${\gamma}$ to IL10 (d 20) in serum (p = 0.041); The other two SNPs (HQ026023:130 G>A; 232 C>T) were highly significantly associated with IL10 level in serum both at the day 20 (p = 0.005; p = 0.001) and the day 35 (p = 0.004; p = 0.006). Identification of the porcine IRF6 gene will help our further understanding of the molecular basis of the IFN regulation pathway in the porcine immune response. All these results should indicate that the IRF6 gene can be regarded as a molecular marker associated with the IL10 level in serum and used for genetic selection in the pig breeding.

• 미생물학회지
• /
• 제41권1호
• /
• pp.1-7
• /
• 2005

Methylophaga aminosulfidovorans SK1 (KCTC 10323 BP)은 단일 탄소원, 질소원 그리고 에너지원으로 난분해성 화합물인 트리메틸아민을 이용할 수 있다. M. aminosulfidovorans SK1는 진핵세포의 flavin-containing monooxygenase와 유사한 유전자(bFMO)를 지니고 있으며 대장균에서 발현된 재조합 단백질은 강력한 트리메틸아민 산화활성을 보인다. 본 연구에서는 bEMO의 기능과 조절 메커니즘을 연구하기 위하여 bfmo의 상단부 및 하단부 유전자의 염기서열을 결정하였다. bfmo 상단부의 세 개의 열린해독틀은 잘 보존된 nitrate/nitrite response regulators와 methyl accepting protein 유사단백질을 암호화하였다. 하단부의 두 개의 작은 열린해독틀은 기능은 알려져 있지 않지만 진정세균계에서 잘 보존된 단백질의 일종으로 나타났다. 역전사효소 중합효소증폭반응을 통하여 여섯 개의 유전자는 세 개의 독립된 오페론으로 구성되어 있음을 확인하였다. bfmo의 상단부에 위치하는 세 개의 조절유전자는 두 개의 프로모터에서 전사되었다. 그리고 이와 독립적으로 bfmo와 두 개의 하단부 유전자가 하나의 전사단위를 이루고 있다.

• Applied Biological Chemistry
• /
• 제37권5호
• /
• pp.361-369
• /
• 1994

Ribulose-1,5-bisphosphate carboxylase small subunit(rbcS)의 광유도 발현과 엽록체로의 단백질 이동 메카니즘을 연구하기 위해 벼의 게놈으로부터 rbcS 유전자를 분리하여(GrbcS) 그의 염기서열을 결정하였다. GrbcS의 유전자 염기서열 결정 결과, 단백질 암호부위는 한 개의 intron과 두 개의 exon으로 이루어져 있고 이들은 47개의 transit peptide를 포함하는 175개의 아미노산을 암호화하는 것으로 밝혀졌다. GrbcS의 이러한 구조적인 성질은 다른 단자엽 식물의 그것과 비교적 일치하고 genomic Southern blot analysis 결과 rbcS 유전자는 벼의 게놈상에 상대적으로 적은 규모의 multigene family로 존재한다는 것이 밝혀졌다. GrbcS의 유전자 염기서열과 그로부터 유추된 아미노산의 염기서열은 벼로부터 분리된 다른 rbcS와 매우 유사함을 보였고 다른 식물체로부터 분리된 그것과도 높은 유사성을 보였다. GrbcS의 5’ 앞쪽 부분에는 G-box, 3AF1-binding site, GATA site와 같은 광유도 발현 유전자에 공통적으로 존재하는 염기서열을 지니고 있었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제32권8호
• /
• pp.1095-1103
• /
• 2019

Objective: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme $1{\alpha}$ ($IRE1{\alpha}$)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates $IRE1{\alpha}$ signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. Methods: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. Results: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. Conclusion: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

• 한국육종학회지
• /
• 제50권4호
• /
• pp.434-441
• /
• 2018

본 연구에서는 배 '원황'(Pyrus pyrifolia)의 유전체 정보를 바탕으로, 유용 유전자 관련 SSR 마커를 선발하였고, 선발된 SSR과 SNP 마커를 이용하여 '원황' ${\times}$ 'Bartlett' $F_1$ 교배집단에 대한 유전자 지도를 작성하였다. '원황'의 scaffold에서 제작된 SSR 마커 유래 염기서열들과 NCBI nucleotide DB와 BLASTn 분석하여, 유용한 유전자들과 높은 상동성을 보이는 510개 SSR 마커를 선발하였다. 이들 마커를 사용하여 양친과 F1 집단 94개체의 대립 단편의 증폭 양상을 확인한 결과, 88개 마커들이 헤테로 집단에 맞는 분리비를 보였다. 선발된 88개의 SSR 마커는 GBS 분석을 통해 획득한 579개 SNP 마커와 함께 '원황'의 유전자지도를 작성하였다. 70개의SSR 마커들은 배 염색체 수와 같은 17개의 염색체에 잘 위치하였고, 모든 염색체에 한 개 이상의 마커로 위치하였다. 유전자지도의 총 유전거리는 3784.2cM이고 마커간 평균거리는 5.8cM이었다. 본 연구에서 개발된 SSR 분자마커 및 이를 기반으로 만들어진 유전자지도는 배의 육종 및 유전 연구에 유용한 정보를 제공할 것으로 기대한다.

• Molecules and Cells
• /
• 제37권10호
• /
• pp.734-741
• /
• 2014

Histone modifications on major transcription factor target genes are one of the major regulatory mechanisms controlling adipogenesis. Plant homeodomain finger 2 (PHF2) is a Jumonji domain-containing protein and is known to demethylate the histone H3K9, a repressive gene marker. To better understand the function of PHF2 in adipocyte differentiation, we constructed stable PHF2 knock-down cells by using the mouse pre-adipocyte cell line 3T3-L1. When induced with adipogenic media, PHF2 knock-down cells showed reduced lipid accumulation compared to control cells. Differential expression using a cDNA microarray revealed significant reduction of metabolic pathway genes in the PHF2 knock-down cell line after differentiation. The reduced expression of major transcription factors and adipokines was confirmed with reverse transcription- quantitative polymerase chain reaction and Western blotting. We further performed co-immunoprecipitation analysis of PHF2 with four major adipogenic transcription factors, and we found that CCATT/enhancer binding protein (C/EBP)${\alpha}$ and C/EBP${\delta}$ physically interact with PHF2. In addition, PHF2 binding to target gene promoters was confirmed with a chromatin immunoprecipitation experiment. Finally, histone H3K9 methylation markers on the PHF2-binding sequences were increased in PHF2 knock-down cells after differentiation. Together, these results demonstrate that PHF2 histone demethylase controls adipogenic gene expression during differentiation.

• Interdisciplinary Bio Central
• /
• 제4권1호
• /
• pp.1.1-1.7
• /
• 2012

Background: The F-box proteins represent one of the largest families of proteins in eukaryotes. Apart from being a component of the ubiquitin (Ub)/26 S proteasome pathways, their regulatory roles in other cellular and developmental pathways have also been reported. One interesting feature of the genes encoding the proteins of this particular family is their variable selection patterns across different lineages. This resulted in the presence of lineage specific F-box proteins across different species. Findings: In this study, 48 non-redundant F-box proteins in E. siliculosus have been identified by a homology based approach and classified into three classes based on their variable C-terminal domains. A greater number of the F-box proteins have domains similar to the ones identified in other species. On the other hand, when the proteins having unknown or no C-terminal domain (as predicted by InterProScan) were analyzed, it was found that some of them have the polyglutamine repeats. To gain evolutionary insights on the genes encoding the F-box proteins, their selection patterns were analyzed and a strong positive selection was observed which indicated the adaptation potential of the members of this family. Moreover, four lineage specific F-box genes were found in E. siliculosus with no identified homolog in any other species. Conclusions: This study describes a genome wide in silico analysis of the F-box proteins in E. siliculosus which sheds light on their evolutionary patterns. The results presented in this study provide a strong foundation to select candidate sequences for future functional analysis.

• Reproductive and Developmental Biology
• /
• 제29권1호
• /
• pp.49-55
• /
• 2005

본 연구에서는 돼지의 체지방을 감소시키고 성장을 촉진시키는 인자인 PGH를 cloning하여 이 유전자를 외래 유전자의 발현이 유도적으로 조절되는 Tet system에 도입하고자 하였다. 또한 유전자의 발현이 turn on되었을 때 그 발현 정도를 최대화하기 위하여 WPRE 서열을 도입하였다. 구축된 각각의 vector는 retrovirus 생산 세포주에 도입하여 virus를 생산하였으며 이를 여러 종류의 표적세포에 감염시켜서 PGH 유전자의 발현을 확인한 결과, 1×10/sup 6/ 세포에서 350∼2,100 ng의 PGH가 분비되었으며 특히 PFF 세포에서 가장 높은 발현을 나타내었다. Tet system에 도입된 PGH의 발현이 유도적으로 조절되는지를 PFF 세포에서 확인한 결과, 유도 효율이 2∼6배로 나타났으며 WPRE 서열이 rtTA 유전자의 downstream에 위치한 조건에서 가장 높은 유도 효율을 나타내었다. 이러한 PGH 유전자의 유도적인 발현의 조절은 고급육 생산의 형질전환 돼지 연구에 있어서 가장 큰 문제점이 되는 PGH 유전자의 과다한 발현에 의한 생리적인 부작용을 최소화할 수 있는 해결 방안으로 제시될 수 있을 것이다.

• BMB Reports
• /
• 제33권5호
• /
• pp.396-401
• /
• 2000

A differential display method is used to identify novel genes whose expression is affected by treatment with ferric ammonium citrate (FAC) or desferrioxamine (DFO), an iron chelating agent in the human hepatoblastoma cell line (HepG2). These chemicals are known to deplete or increase the intracellular concentration of iron, respectively. Initially, we isolated seventeen genes whose expressions are down- or up regulated by the treatment of the chemicals, as well as their four differentially expressed genes that are designated as clone-1, -2, -3, and -4. These are further characterized by cDNA sequencing and Northern blot analysis. Through the cDNA sequencing, as well as comparing them to genes published using the NCBI BLAST program, we identified the sequence of the clone-1 that is up-regulated by the treatment of DFO. It is identical to the human insulin-like growth factor binding protein-1 (IGFBP-1). This suggests that the IGFBP-1 gene in the HepG2 cell is up-regulated by an iron depletion condition. Also, the expression of the clone-3 and -4 is up-regulated by FAC treatment and their eDNA sequences are identical to the human ferritin-fight chain and human NADH-dehydrogenase, respectively. However, the sequence of the clone-2 has no significant homology to any other known gene. Therefore, we suggest that changes of the cellular iron level in the HepG2 cell affects the transcription of cellular genes. This includes human IGFBP-1, ferritin-fight chain, and NADH-dehydrogenase. Regulation of these gene expressions may have an important role in cellular functions that are related to cellular iron metabolism.