• 농업과학연구
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• 제46권3호
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• pp.471-488
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• 2019

Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.256-260
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• 1991

In B. subtilis, $\alpha$-amylase synthesis is regulated by amyR located directly on the upstream of amyE. Three different amyR alleles have been reported, amyR1, amyR2 and amyR3. Strains bearing the gra-10 mutation which confers derepression for catabolite repression has GlongrightarrowA transition mutation at ＋5 of amyR1. S1 nuclease mapping demonstrated that transcription initiated at 8 bases downstream from the -10 region of putative E$\sigma^{A}$ promoter P1 in amyR1 and gra-10. In amyR2, the major transcription initiatd at the same place and the minor, 10 bases downstream from -10 of P2. The transcript from P2 contributed approximately 15-20% of total amyE mRNA. S1 nuclease protection experiment indicated that amyE mRNA levels corresponded to the rate of synthesis assumed by specific activities of $\alpha$-amylase in culture supernatants, suggesting that $\alpha$-amylase synthesis is regulated at the level of transcription.n.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.237-244
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• 1994

The vector systems for the expression and secretion of human lipocortin-l (LC1) from Saccharomyces cerevisiae were constructed with GAL10 promoter and the prepro leader sequence of mating factor-$\alpha$1. They were further constructed to contain three different transcription terminators; GAL7 terminator, LCl terminator and a fused form of these two terminators. The expression and secretion levels of LCl were compared to investigate the effect of transcription terminators on the LCl gene expression. For the expression cassettes employing the GAL7 terminator or the terminator of fused form, the expression levels of LCl were measured by scanning the immunoreactive LCl protein bands, and were found to be 0.27 g/l and 0.32 g/l, respectively. The highest expression level of 0.54 g/l was obtained with the expression vector containing the LCl transcription terminator. In all expression cassettes, the majority of LCl proteins expressed were retained intracellularly, indicating a low secretion efficiency of about 5%. The high expression level of LCl was explained by the great content and stability of LCl mRNA transcribed from the LCl terminator-employing vector. The results of this study demonstrate that the LCl transcription terminator functions for the expression of LCl in S. cerevisiae better than the GAL7 terminator.

• Genomics & Informatics
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• 제5권3호
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• pp.107-112
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• 2007

MicroRNAs (miRNAs) are known to negatively control protein-coding genes by binding to messenger RNA (mRNA) in the cytoplasm. In innate immunity, the role of miRNA gene silencing is largely unknown. In this study, we performed microarray-based experiments using lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type, MyD88 knockout (KO), TRIF KO, and MyD88/TRIF double KO mice. We employed a statistical approach to determine the importance of the commonality and specificity of miRNA binding sites among groups of temporally co-regulated genes. We demonstrate that both commonality and specificity are irrelevant to define a priori groups of co-down regulated genes. In addition, analyzing the various experimental conditions, we suggest that miRNA regulation may not only be a late-phase process (after transcription) but can also occur even early (1h) after stimulation in knockout conditions. This further indicates the existence of dynamic interactions between miRNA and signaling molecules/transcription factor regulation; this is another proof for the need of shifting from a 'hard-wired' paradigm of gene regulation to a dynamical one in which the gene co-regulation is established on a case-by-case basis.

• Genomics & Informatics
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• 제20권3호
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• pp.26.1-26.19
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• 2022

Diabetes and its related complications are associated with long term damage and failure of various organ systems. The microvascular complications of diabetes considered in this study are diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. The aim is to identify the weighted co-expressed and differentially expressed genes (DEGs), major pathways, and their miRNA, transcription factors (TFs) and drugs interacting in all the three conditions. The primary goal is to identify vital DEGs in all the three conditions. The overlapped five genes (AKT1, NFKB1, MAPK3, PDPK1, and TNF) from the DEGs and the co-expressed genes were defined as key genes, which differentially expressed in all the three cases. Then the protein-protein interaction network and gene set linkage analysis (GSLA) of key genes was performed. GSLA, gene ontology, and pathway enrichment analysis of the key genes elucidates nine major pathways in diabetes. Subsequently, we constructed the miRNA-gene and transcription factor-gene regulatory network of the five gene of interest in the nine major pathways were studied. hsa-mir-34a-5p, a major miRNA that interacted with all the five genes. RELA, FOXO3, PDX1, and SREBF1 were the TFs interacting with the major five gene of interest. Finally, drug-gene interaction network elucidates five potential drugs to treat the genes of interest. This research reveals biomarker genes, miRNA, TFs, and therapeutic drugs in the key signaling pathways, which may help us, understand the processes of all three secondary microvascular problems and aid in disease detection and management.

• Molecules and Cells
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• 제26권2호
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• pp.212-215
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• 2008

The GC-rich discriminator sequence between the -10 region and the transcription start site of the rnpB promoter is responsible for stringent control of M1 RNA synthesis. The rnpB promoter also contains a G nucleotide at the previously identified transcription start site. In this study, we examined by mutagenesis of G to A whether this +1G nucleotide is involved in the stringent response. We found that the change did not alter the stringent response. Since the +1 mutation might alter transcription initiation, we compared the transcription start sites of the wt and mutant promoters by primer extension analysis. Surprisingly, we found that wild type rnpB transcription starts at both the +1G position (70%) and the -1C position (30%), and that the +1A mutation led to transcription initiation exclusively at the -1C position. We also generated two transversion mutations at the -1 position, both of which led to transcription starting exclusively at that position. The -1G mutant promoter gave a stringent signal similar to the wild-type, whereas the -1A mutant generated a significantly less stringent signal. Base on these results, we propose that a short sequence, up to 7 bp downstream of the -10 region, is involved in the stringent response of the rnpB promoter.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권1호
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• pp.23-34
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• 2004

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2'(3')-O-( N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6${\times}$10$\^$6/ M$\^$-1/ sec$\^$-1/ whereas the second nucleotide binds at a slower rate of 4.7${\times}$10$\^$5/ M$\^$-1/ sec$\^$-1/ at 18$^{\circ}C$, RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.

• BMB Reports
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• 제43권2호
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• pp.110-114
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• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.

• 한국균학회지
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• 제49권3호
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• pp.285-294
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• 2021

In general, mycoviruses remain latent and rarely cause visible symptoms in fungal hosts; however, some viral infections have demonstrated abnormal mycelial growth and fruiting body development in commercial macrofungi, including Lentinula edodes. Compared to other cultivated mushrooms, L. edodes is more vulnerable to viral infections as it is still widely cultivated under near-natural conditions. In this study, we investigated whether Korean wild strains of L. edodes were infected by RNA mycoviruses that have previously been reported in other parts of the world (LeSV, LePV1, LeV-HKB, LeNSRV1, and LeNSRV2). Using specific primer sets that target the RNA-dependent RNA polymerase genes of each of the RNA mycovirus, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral infection. Viral infection was detected in about 90% of the 112 wild strains that were collected in Korea between 1983 and 2020. Moreover, multiple infections with RNA mycoviruses were detected in strains that had normal fruiting bodies. This work contributes to our understanding of the distribution of RNA mycoviruses in Korea and the impact of multiple viral infections in a single strain of L. edodes.

• 자연과학논문집
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• 제14권1호
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• pp.25-37
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• 2004

T7 RNA 중합효소는 두 종류의 인자 독립형 전사 종결신호를 인지하여 전사연장을 종결하게 되는데 이 중 머리핀 구조가 만들어지지 않는 PHT와 CJ 전사종결 부위는 ‘ATCTGTT' non-template 염기서열 다음에 T염기가 많은 부분이 존재하는 형태로 이루어져 잇다. 이와 같은 전사종결을 민감하게 인지하는 돌연변이 T7 RNA 중합효소인 X4, X19, BG8과 이와 반대로 둔감하게 인지하는 R173C를 제조하여 전사활성도를 측정하였다. 이결과 야생형 T7 RNA 중합효소에 비해 X4는 8%, X19는 33%, BG8은 34%의 전사활성도를 보여 전사활성도가 상당히 감소하는 결과를 보였다. 반면에 R173C는 야생형에 비해 112%의 전사활성도를 보여 야생형과 거의 유사한 전사활성도를 보였다. 또한 PTH 및 CJ 전사종결 부위를 전사주형으로 이용하여 전사하였을 때 X4, X19, BG8은 야생형 RNA중합효소에 비해 종결 부위에서의 종결이 증가하였으나 R173C는 거의 PTH 및 CJ 전사종결부위에서 전사종결이 일어나지 않고 전사가 진행되는 것을 확인하였다.