• Bulletin of the Korean Chemical Society
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• 제29권10호
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• pp.2026-2028
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• 2008

• BMB Reports
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• 제41권11호
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• pp.778-783
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• 2008

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in its 5' untranslated region (5'-UTR). To investigate the role of the hairpins in replication of TYMV, mutants lacking one or both of the two hairpins were constructed. The TYMV constructs were introduced into Chinese cabbage by an Agrobacterium-mediated T-DNA transfer method, called agroinfiltration. Analysis of total RNA from agroinfiltrated leaves showed that replication of the mutant TYMV RNA lacking both hairpins was about 1/100 of wild type. This mutant was also impaired in systemic spread. Deletion analysis of each hairpin revealed that both hairpins were needed for maximal replication. The deletion analysis along with sequence modification of the hairpin structure indicates that the second hairpin plays a role in efficient long-distance systemic movement of TYMV.

• Molecules and Cells
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• 제26권1호
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• pp.26-33
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• 2008

The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5'-TTTTTGTNNNNTAANNNNNNNNNATG-3', and the ctRNA is complementary only to the 5' conserved sequence 5'-TTTTTGT-3'. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.

• 원예과학기술지
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• 제35권3호
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.271-276
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• 2009

Functional gene studies in the cultivated white button mushroom Agaricus bisporus have been constrained by the absence of effective gene-silencing tools. Using two endogenous genes from A. bisporus, we have tested the utility of dsRNA hairpin constructs to mediate downregulation of specific genes. Hairpin constructs for genes encoding orotidine 5'-monophosphate decarboxylase (URA3) and carboxin resistance (CBX) were introduced into A. bisporus using Agrobacteriummediated transfection. Although predicted changes in phenotype were not observed in vitro, quantitative-PCR analyses indicated unambiguously that transcripts in several transformants were substantially reduced compared with the non-transformed controls. Interestingly, some hairpin transformants exhibited increased transcription of target genes. Our observations show that hairpin transgenic sequences can mediate downregulation of A. bisporus endogenous genes and that the technology has the potential to expedite functional genomics of the mushroom.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Bioinformatics and Biosystems
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• 제2권1호
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• pp.17-23
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• 2007

Short interfering RNA(siRNA)는 특별한 gene의 발현을 막는데 사용될 수 있고 그 gene의 기능과 치료의 적용에 많은 가능성을 가지고 있지만, 효과적인 siRNA를 디자인하는 방법은 아직까지 명확하지 않다. 효과적인 siRNA는 서열적인 경향을 가지고 있는데 낮은 G/C content, Sense strand의 3' 끝에 적은 안정성과 1번 위치에는 G/C, 19번 위치에는 A/U의 존재 여부를 들 수 있다. 이러한 특성 말고도 최근에는 mRNA의 2차구조가 RNAi 작용에 중요한 역할을 하게 되는데 복잡한 구조(hairpin, multi loop)를 가지고 수소결합을 많이 하여 안정한 상태에 있는 부분은 siRNA의 기능을 크게 줄어들게 한다. 또한, siRNA가 특정한 mRNA에 작동하도록 BLAST 검색을 하여 부작용의 가능성을 배제한다.

• Molecules and Cells
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• 제27권6호
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• pp.657-665
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• 2009

The ovomucoid pre-mRNA has been folded into mini-hairpins adaptable for the RNA recognition motif (RRM) protein binding. The number of mini-hairpins were 372 for pre-mRNA and 83-86 for mature mRNA. The spatial arrangements are, in average, 16 nucleotides per mini-hairpin which includes 7 nt in the stem, 5.6 nt in the loop and 3.7 nt in the inter-hairpin spacer. The constitutive splicing system of ovomucoid-pre-mRNA is characterized by preferred order of intron removal of 5/6 > 7/4 > 2/1 > 3. The 5' splice sites (5'SS), branch point sequences (BPS) and 3' splice sites (3'SS) were identified and free energies involved have been estimated in 7 splice sites. Thermodynamic barriers for splice sites from the least (|lowest| -Kcal) were 5, 4, 7, 6, 2, 1, and 3; i.e., -18.7 Kcal, -20.2 Kcal, -21.0 Kcal, -24.0 Kcal, - 25.4 Kcal, -26.4 Kcal and -28.2 Kcal respectively. These are parallel to the kinetic data of splicing order reported in the literature. As a result, the preferred order of intron removals can be described by a consideration of free energy changes involved in the spliceosomal assembly pathway. This finding is consistent with the validity of hnRNP formation mechanisms in previous reports.

• Molecules and Cells
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• 제38권11호
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• pp.959-965
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• 2015

Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

• BMB Reports
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• 제41권4호
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• pp.294-299
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• 2008

Elevated expression of SMYD3 is a frequent genetic abnormality in several malignancies. Few studies knocking down SMYD3 expression in cervical carcinoma cells have been performed to date. In this paper, we established an inducible short hairpin RNA expression system to examine its role in maintaining the malignant phenotype of HeLa cells. After being induced by doxycycline, SMYD3 mRNA and protein expression were both reduced, and significant reductions in cell proliferation, colony formation and migration/invasion activity were observed in the SMYD3-silenced HeLa cells. The percentage of cells in sub-G1 was elevated and DNA ladder formation could be detected, indicating potent induction of apoptosis by SMYD3 knockdown. These findings imply that SMYD3 plays crucial roles in HeLa cell proliferation and migration/invasion, and that it may be a useful therapeutic target in human cervical carcinomas.