• 식물조직배양학회지
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• 제28권5호
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• pp.255-261
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• 2001

VVTL1은 포도 과육에서 특이적으로 다량 발현되는, PR5 계열의 thaumatin과 높은 상동성을 나타내는 단백질로서, 품종에 따라 염기서열의 차이를 나타낸다고 알려져 있다. 현재까지 포도의 VVTL1에 대해서 몇몇 연구가 진행되었지만, 우리나라에서 가장 많이 재배되는 품종인 캠벨에서는 전혀 이루어지지 않았다. 본 연구에서는 캠벨 품종으로부터 VVTL1-homolog 유전자의 게놈 DNA를 분리하여, 염기서열을 분석하였다. VVTL1-homolog 유전자는 일반적으로 PR5 유전자의 구조와 동일한 구조인, intron이 없는 하나의 exon으로만 구성되어 있었다. 염기서열로부터 추론된 VVTL1-homolog 단백질의 아미노산 서열은 VVTL1을 비롯한 다른 품종의 포도에서 분리된 TLP와는 달리 염기성의 등전점을 가지고 있었다. Primer extension 분석을 통해 전사개시 부위를 결정하였고, promoter영역을 포함하는 5'upstream 지역에 전사에 중요한 TATA box (4개)와 CAAT box (1개)가 존재하였으나, 이들의 위치와 수는 다른 PR5 유전자의 promoter와는 다랐다. 이러한 연구결과는 VVTL1-homolog 유전자의 발현이 과육 성숙과정동안 abscisic acid와 스트레스 또는 자극에 의해 발현이 유도되고 있음을 제시해준다. 포도 과육특이발현 promoter인 VVTL1-homolog 유전자의 promoter 분리는, 유전자의 도입에 의해 유용형질을 과육에 나타내는 포도품종을 개발하고자 할 때 효율적으로 사용될 수 있을 것으로 사료된다.

• Animal cells and systems
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• 제7권1호
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• pp.63-68
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• 2003

Until recently, interest in human complement receptor type I (CR1) has focused on immune complex processing, which contributed to our understanding of regulatory mechanism of complement activation. However, the promoter structure and transcriptional regulation of human CR1 gene has not been clear. To study the unique regulation of human CR1 gene expression, we assessed promoter activity of the $5^1$-flanking region of human CR1 gene using transient transfection and gel mobility shift assays. In this study we demonstrated that NF-Y binds to the inverted CCAAT element and that the functional interaction with protein(s) which bind to the GC-rich motif may be necessary for optimal transcription of human CR1 gene. We also show that sequence elements which located at-95/58 and +45/+50 are important for optimal transcription of CR1 gene.

• BMB Reports
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• 제30권1호
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• pp.41-45
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• 1997

The present study was performed to analyze the molecular mechanism which dictates the transcription regulation of the $O^6$-methylguanine-DNA methyltransferase (MGMT) gene in Saccharomyces cerevisiae. Previously we identified one possible upstream repressing sequence (URS) in MGMT promoter by promoter deletion and competition analysis. In this paper we report another regulatory element (UAS: upstream activating sequence. -213 to -136) which affects the transcription activity of MGMT promoter. Gel mobility shift assay and Southwestern blot analysis using UAS probe showed several specific proteins which were able to bind to this sequence.

• 한국미생물·생명공학회지
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• 제29권2호
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• pp.72-77
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• 2001

Strong promoter로 밝혀진 alginate lyase 유전자의 promoter 부위에 대한 특성을 검토하기 위해 alginate lyase 유전자의 46개 N-terminal amino acid가 포함된 promoter 부분과, 같은 균으로부터 분리한 $\beta$-agarase의 유전자를 연결시켜 agarase의 activity를 평판배지상에서 보다 쉽게 확인하는 방법으로 promoter의 활성을 측정한 결과 alginate lyase 유전자 promoter에 의해서 $\beta$-agarase 유전자의 대량발현이 유도되고 있었으며 glucose의 존재하에서 $\beta$-agarase 유전자 발현이 일어나지 않는 catabolite repression 양상을 나타내고 있다. PCR로써 alginate lyase의 46개 N-terminal amino acid 부분이 순차적으로 제거된 plasmid를 제조하여 대량발현을 조사한 결과 46개의 아미노산이 제거된 후에도 $\beta$-agarase의 활성에는 변화가 없어 46개의 N-말단이 정상적인 상태에서 발현에는 영향을 미치고 있지 않음을 확인할 수 있었다. 또한 alginate lyase 유전자의 promoter region에 존재하는 가능한 2개의 promoter consensus sequence PI, PII를 subcloning한 결과 promoter PII만이 존재할 때도 대량발현이 유도되고 있음을 확인할 수 있었으며 동시에 glucose가 존재할 때 catabolite repression이 역시 나타나고 있어 이 부분이 발현 및 glucose에 의한 regulation에 매우 중요하게 작용하는 부분이라는 것을 확인할 수 있었다.

• BMB Reports
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• 제32권4호
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• pp.345-349
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• 1999

We fused the promoter region of an H3.2 chicken histone gene, whose expression is dependent on the cell cycle, to the 5' coding region of an H3.3 chicken histone gene, which is expressed constitutively at a low level throughout the cell cycle. This fusion gene showed a cell cycle-regulated pattern of expression, but in a different manner. The mRNA level of the fusion gene increase during the S phase of the cell cycle by about 3.7-fold at 6 h and 2.7-fold at 12 h after the serum stimulation. The mRNA level of the intact H3.2 gene, however, increased by an average of 3.6-fold at 6 h and 8.7-fold at 12 h. This different expression pattern might be due to the differences in their 3' end region that is responsible for mRNA stability. The 3' end of the H3.2 mRNA contains a stem-loop structure, instead of a poly(A) tail present in the H3.3 mRNA. We also constructed a similar fusion gene using a H3.3 histone gene whose introns had been eliminated to rule out the possibility of involvement of the introns in cell cycle-regulated expression. The expression of this fusion gene was almost identical to the fusion gene made previously. These results indicate that the promoter region of the H3.2 gene is only partially responsible for its expression during the S phase of the cell cycle.

• 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.

• 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.

• Fisheries and Aquatic Sciences
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• 제15권4호
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• pp.325-335
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• 2012

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

• Journal of Plant Biology
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• 제38권3호
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• pp.259-266
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• 1995

To investigate how the dyad symmetry region (DSR) and the distance between vir box and -35 sequence of the virE promoter plays a role in virE gene expression, two mutants were constructed by base substitution and insertional mutagenesis. The base substitutional mutation, a AAlongrightarrowCG substitution at positions -39 and -40 on the DSR, showed the level of $\beta$-galactosidase activity approximately 91% of the wild type virE promoter activity. Therefore, the native structure of the DSR seems to be not essential for virE expression. The insertional mutation, constructed by inserting 8 bp ClaI linker between -49 and -50, displayed the $\beta$-galactosidase activity at 12% of the native virE promoter activity. However, this striking reduction appears to be not caused by destruction of the native DSR structure, but by shifting the vir box far from putative -35 sequence.

• Journal of Microbiology
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• 제34권1호
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• pp.40-48
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• 1996

We have examined the chromatin structure of the HMRE/HSP82 and HMRa/HSP82 allels using three complementary approaches : DNase I chromating footprinting, micrococcal nuclease (MNase) nucleosome-protected ladder assay, and an in vivo E. coli dam methylase accessibility assay. The footprinting results indicate that the promoter and silencer sequences are assembled into nucleoprotein complexes which exhibit no detectable change in structure, despite a 70-fold range in expression levels. In addition, the promoter region of the HMRa/HSP82 allele is cleaved randomly by MNase in all cases, indicating the absence of anonical nucleosomes over this region irrespective of SIR4 or heat-shock. Finally, no discernible difference in the accessibility of the HMRE/HSP82 locus to dam methylase in SIR4 vs. sir4 cells was seenm which again suggests that the chromatin structure of HMRE/HSP82 allele is identical regardless of SIR4. Altogether, our results indicate that in contrast to other observations of the silent mating-type loci, no discernible structural alteration is detected at either HMR/HSP82 allele regardless of SIR genetic background or transcriptional state of the gene.