• Molecules and Cells
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• 제42권2호
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• pp.123-134
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• 2019

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as $LPA_{1-6}$. For one of its receptors, $LPA_1$ (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

• BMB Reports
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• 제56권3호
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• pp.153-159
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• 2023

Neuronal differentiation is highly coordinated through a cascade of gene expression, mediated via interactions between trans-acting transcription factors and cis-regulatory elements of their target genes. However, the mechanisms of transcriptional regulation that determine neuronal cell-fate are not fully understood. Here, we show that the nuclear transcription factor Y (NF-Y) subunit, NFYA-1, is necessary and sufficient to express the flp-3 neuropeptide gene in the IL1 neurons of C. elegans. flp-3 expression is decreased in dorsal and lateral, but not ventral IL1s of nfya-1 mutants. The expression of another terminally differentiated gene, eat-4 vesicular glutamate transporter, is abolished, whereas the unc-8 DEG/ENaC gene and pan-neuronal genes are expressed normally in IL1s of nfya-1 mutants. nfya-1 is expressed in and acts in IL1s to regulate flp-3 and eat-4 expression. Ectopic expression of NFYA-1 drives the expression of flp-3 gene in other cell-types. Promoter analysis of IL1-expressed genes results in the identification of several cis-regulatory motifs which are necessary for IL1 expression, including a putative CCAAT-box located in the flp-3 promoter that NFYA-1 directly interacts with. NFYA-1 and NFYA-2, together with NFYB-1 and NFYC-1, exhibit partly or fully redundant roles in the regulation of flp-3 or unc-8 expression, respectively. Taken together, our data indicate that the NF-Y complex regulates neuronal subtype-specification via regulating a set of terminal-differentiation genes.

• 한국수정란이식학회지
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• 제31권4호
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• pp.335-347
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• 2016

Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), a new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome and to examine expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. These transcrips could be regulated through caudal-related homeobox-2 (CDX2) and ETS2 and/or AP1 (JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on day 17, 20 and 22 bovine conceptuses. The expression level of bIFNT1 was higher on day 17 transcripts were gradually weakly detectable on day 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on day 20 and transcripts were weakly detectable on day 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had very effect on activity by alone ETS2, and AP1 (JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not identified. We demonstrated that the activities of bIFN genes are regulated by differential, tissue-specific and developmental competence during pregnancy.

• Journal of Animal Science and Technology
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• 제45권2호
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• pp.169-182
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• 2003

ADRP 유전자가 24개월령 한우 등심조직에서 발현량이 급격히 증가하여 30개월령 등심조직에서는 발현량이 다소 감소하는 발현양상 분석결과로부터 이전 연구에서는 ADRP 유전자를 한우 성장단계 특이발현 유전자로 선정하였다. 본 연구에서는 ADRP 유전자의 발현조절 기작을 분석하기 위하여 promoter 영역을 포함하는 ADRP 유전자 전체영역을 cloning하였으며, 구조를 분석하고 promoter의 특성을 조사하였다. 한우 ADRP cDNA 단편을 probe로 합성하여 Southern blot 분석을 실시한 결과로부터 ADRP 유전자가 한우 genome 상에서 single copy로 존재하고 크기는 대략 12 kb에 해당하는 것을 확인하였다. Genomic DNA library screening을 실시하여 promoter 영역을 포함하는 ADRP 전체 유전자에 해당하는 clone을 확보하고 HwADRPg-1으로 명명한 후, 염기서열을 결정하고 분석하였다. 한우 ADRP 유전자, HwADRPg-1은 8개의 exon과 7개의 intron으로 구성되어 있으며 모든 exon-intron 경계는 GT/AG 원칙을 따르고 있었고, coding 영역은 7,633 bp로서 6개의 intron에 의해 7개의 exon으로 나누어져 있었다. HwADRPg-1의 promoter 영역에서는 TATAA box는 발견되지 않았으며, -70 위치에 근육 특이적 transcription activator인 Myo G 서열이 존재하였고, -629 위치에는 지방세포의 분화를 유도하는 것으로 알려진 C/EBP (CCAAT/enhancer binding protein) 서열이 존재하였다. HwADRPg-1의 조절영역에 있는 Myo G factor가 근육조직에서 ADRP 유전자가 발현될 수 있도록 하며, 근육의 발달정도를 신호로써 감지하여 근육조직에서 성장단계에 따른 ADRP 유전자의 발현량을 조절할 것으로 추정되고, 다른 종류의 지방세포 특이적인 전사인자 및 지방세포의 분화정도를 신호로 인식하는 전사단계 조절인자를 조사하기 위하여 promoter 영역의 추가분석이 이루어져야 할 것으로 사료된다.

• Journal of Microbiology
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• 제45권2호
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• pp.139-145
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• 2007

The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

• 한국미생물·생명공학회지
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• 제25권6호
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• pp.571-579
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• 1997

For the overproduction of pectate lyase (PL), the recombinant plasmid pl2BS fl which has strong promoter from alkali-tolerent Bacillus sp. YA-14 was used. In order to overexpress the pectate lyase by the action of overlapping strong promoter in pl2BS$\Delta$fl, 1.6 kb of PL gene was inserted into pl2BS$\Delta$fl to form pl2BS$\delta$f1-PL and the enzyme was expressed. But decreased expression efficiency of the PL gene was observed and it was due to the presence of the transcription terminator region on the upstream of the PL gene. The transcription terminator of the PL gene in pl2BS$\delta$f1-PL was removed and the resulting plasmid p12BS$\Delta$fl$\Delta$PL was formed. Bacillus subtilis 207-25 harboring the recombinant plasmid, p12BS$\Delta$fl$\Delta$PL, revealed increased expression efficiency with chloramphenicol induction when cat-86 was used as a reporter gene.

• Fisheries and Aquatic Sciences
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• 제14권4호
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• pp.303-310
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• 2011

We characterized a cytoplasmic actin gene locus in greenling Hexagrammos otakii (Scorpaeniformes). Genomic clones isolated from the greenling DNA library contained two homologous cytoplasmic actin gene copies (HObact2.1 and HObact2.2) in a tail-to-head orientation. Their gene structure is characterized by six translated exons and one non-translated exon. Exon-intron organization and the nucleotide sequences of the two actin gene isoforms are very similar. However, only the HObact2.1 isoform contains microsatellite-like, dinucleotide repeats in the 5'-flanking region (named HOms2002) and intron 1 following the non-translated exon 1 (named HOms769). One microsatellite locus (HOms769) was highly polymorphic while the other (HOms2002) was not. Based on bioinformatic analysis, different transcription factor binding motifs are related to stress and immune responses in the two actin isoforms. Semiquantitative and real-time reverse transcription-PCR assays showed that both isoform transcripts were detectable ubiquitously in all the tissues examined. However, the basal expression levels of each isoform varied across tissues. Overall, the two isoforms showed a similar, but not identical, expression pattern. Our data suggest that the cytoplasmic actin genes may be the result of a recent duplication event in the greenling genome, which has not experienced significant subfunctionalization in their housekeeping roles.

• 한국동물학회지
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• 제38권2호
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• pp.196-203
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• 1995

The c-myc proto-oncogene, one of the immediately earlY genes, is expressed in various mammalian cell types and heavily involved in the regulation of cell proliferation and differentiation. To determine endogeneous expression pattern of c-myc gene in preimpBantation mouse embwos, we employed a reverse transcription coupled to polvrnerase chain reaction (RT-PCR). Transcript of c-myc was detected at fertilized embryos as a maternal transcript. At the early two-cell stave, transcript of c-myc gene was hardly detected, bu, appeared at late two-cell embryos as a zygotic transcript. The level of c-myc expresion was increased at later stases and peaked at blastocvst stage. To examine the functional role of promoter region for c-myc gene transcription, we fused the 5'upstream region (1.8 kb) including econ 1 of c-myc genomic DNA with E. coli lacE gene fnamed as pcMYC-laczl. pcMYC-lacZ was microiniected into the pronscleus of mouse one-cell embryovs, and p·salactosidase activity was determined tv histochemical staining with X-gal at different stases. f-galactosidase activity was detected only at blastocyst, but not at the earlier stage embryos. This result indicates that c-myc gene is transcriptionallv active during mouse preimplantation development.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.18-24
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• 2001

누에 핵다각체병 바이러스의 gp64 프로모터를 이용한 transient 발현 벡터를 제작하기 위해서 gp64 유전자의 구조를 분석하였다. Southern blotting 분석을 통해 genome DNA에서 gp64 유전자를 탐색하기 gp64 구조유전자를 포함하는 2,277 nucleotide의 염기를 분석하였으며 gp64의 early, late 프로모터 발현을 조절하는 인자들을 확인하였다. gp64프로모터를 이용한 transient 발현 벡터를 제작하고 외래유전자로써 lacZ 유전자를 Bm5 세포주에서 transient 발현시켰다. 세포주 내에 도입된 플라스미드 DNA의 안정성을 확인하였으며, gp64 프로모터의 외래유전자 발현성 여부를 조사하기 위하여 gp64 프로모터 하에 laxZ 유전자를 가지는 재조합 바이러스를 제작하고 $\beta$-galactosidase in 냐셔 staining을 수행한 결과 전체적인 발현량은 매우 약한 것으로 판단되었다. BmNPV-K1의 gp64 프로모터를 이용한 벡터는 더욱 민감한 표지 유전자를 발현시켜 재조합 바이러스의 분리에 이용하거나 숙주세포에 독성을 보이는 유전자 산물의 소량 발현에 더욱 유용할 것으로 판단된다.

• Molecular & Cellular Toxicology
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• 제1권4호
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• pp.248-255
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• 2005

To understand the response of cancer cells to anticancer drugs at the gene expression level, we examined the gene expression changes in response to five anticancer drugs, 5-fluorouracil, cytarabine, cisplatin, paclitaxel, and cytochalasin D in NCI-H460 human lung cancer cells. Of the five drugs, 5-fluorouracil had the most distinctive gene expression signature. By clustering genes whose expression changed significantly, we identified three clusters with unique gene expression patterns. The first cluster reflected the up-regulation of gene expression by cisplatin, and included genes involved in cell death and DNA repair. The second cluster pointed to a general reduction of gene expression by most of the anticancer drugs tested. A number of genes in this cluster are involved in signal transduction that is important for communication between cells and reception of extracellular signals. The last cluster represented reduced gene expression in response to 5-fluorouracil, the genes involved being implicated in DNA metabolism, the cell cycle, and RNA processing. Since the gene expression signature of 5-fluorouracil was unique, we investigated it in more detail. Significance analysis of microarray data (SAM) identified 808 genes whose expression was significantly altered by 5-fluorouracil. Among the up-regulated genes, those affecting apoptosis were the most noteworthy. The down-regulated genes were mainly associated with transcription-and translation-related processes which are known targets of 5-fluorouracil. These results suggest that the gene expression signature of an anticancer drug is closely related to its physiological action and the response of caner cells.