• The Plant Pathology Journal
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• v.32 no.3
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• pp.173-181
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• 2016

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

• Molecules and Cells
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• v.26 no.6
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• pp.554-557
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• 2008

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (${\Delta}-908$ to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (${\Delta}-908$ to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

• BMB Reports
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• v.30 no.5
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• pp.320-325
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• 1997

Thioredoxin is a multi-functional protein which is ubiquitous in microorganisms, animals and plants. Previously, expression of the E. coli thioredoxin gene was found to be negatively regulated by cAMP. In the present study, the effect of cAMP on two separate promoters of the E. coli thioredoxin gene was investigated. Cyclic AMP had a repressible effect on P1 and P1P2 promoter activity of the constructs. This effect was also observed in the cya strain. The P2 promoter construct gave very high -galactosidase activity, and its expression was not affected by exogenous cAMP. It was assumed that a cis-acting negative element, probably the cAMP-CRP binding site, might have been deleted in the P1 promoter construct. Repression of the thioredoxin gene expression by cAMP appeared to be independent of ppGpp.

• BMB Reports
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• v.37 no.5
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.138-143
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• 1997

We have designed and constructed a gene encoding novel high essential amino acid encoding protein(HEAAE). The resultant DNA fragment was tested for in vitro and in vivo expression and then cloned into plant expression vector pBI121, under the control of the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens, strain LBA4404, was subsequently transformed with this new construct and Nicotiana tabacum var. Xanthi transgenic plants were obtained. DNA analysis by Southern procedure confirmed the presence of the multi-copy number of genes in the transformed plants. Analysis of RNA and protein synthesized in these transgenic plants demonstrated the stable expression of this gene.

• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.1
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• Journal of Communications and Networks
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• v.7 no.2
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• pp.144-156
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• 2005

Video streaming over wireless networks is challenging due to node mobility and high channel error rate. In this paper, we propose a multi-source video streaming (MUVIS) system to support high quality video streaming service over IEEE 802.1l-based wireless networks. We begin by collocating a streaming proxy with the wireless access point to help leverage both the media server and peers in the WLAN. By tracking the peer mobility patterns and performing content discovery among peers, we construct a multi-source sender group and stream video using a rate-distortion optimized scheme. We formulate such a multi-source streaming scenario as a combinatorial packet scheduling problem and introduce the concept of asynchronous clocks to decouple the problem into three steps. First, we decide the membership of the multisource sender group based on the mobility pattern tracking, available video content in each peer and the bandwidth each peer allocates to the multi-source streaming service. Then, we select one sender from the sender group in each optimization instance using asynchronous clocks. Finally, we apply the point-to-point rate-distortion optimization framework between the selected sender-receiver pair. In addition, we implement two different caching strategies, simple caching simple fetching (SCSF) and distortion minimized smart caching (DMSC), in the proxy to investigate the effect of caching on the streaming performance. To design more realistic simulation models, we use the empirical results from corporate wireless networks to generate node mobility. Simulation results show that our proposed multi-source streaming scheme has better performance than the traditional server-only streaming scheme and that proxy-based caching can potentially improve video streaming performance.

• Mycobiology
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• v.50 no.5
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• pp.302-316
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• 2022

Many Apiospora species have been isolated from bamboo plants - to date, 34 bambusicolous Apiospora species have been recorded. They are known as saprophytes, endophytes, and plant pathogens. In this study, 242 bambusicolous Apiospora were isolated from various bamboo materials (branches, culms, leaves, roots, and shoots) and examined using DNA sequence similarity based on the internal transcribed spacer, 28S large subunit ribosomal RNA gene, translation elongation factor 1-alpha, and beta-tubulin regions. Nine Apiospora species (Ap. arundinis, Ap. camelliae-sinensis, Ap. hysterina, Ap. lageniformis sp. nov., Ap. paraphaeosperma, Ap. pseudohyphopodii sp. nov., Ap. rasikravindrae, Ap. saccharicola, and Ap. sargassi) were identified via molecular analysis. Moreover, the highest diversity of Apiospora was found in culms, and the most abundant species was Ap. arundinis. Among the nine Apiospora species, two (Ap. hysterina and Ap. paraphaeosperma) were unrecorded in Korea, and the other two species (Ap. lageniformis sp. nov. and Ap. pseudohyphopodii sp. nov.) were potentially novel species. Here, we describe the diversity of bambusicolous Apiospora species in bamboo organs, construct a multi-locus phylogenetic tree, and delineate morphological features of new bambusicolous Apiospora in Korea.

• BMB Reports
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• v.55 no.9
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• pp.417-428
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• 2022

Herbal medicine, a multi-component treatment, has been extensively practiced for treating various symptoms and diseases. However, its molecular mechanism of action on the human body is unknown, which impedes the development and application of herbal medicine. To address this, recent studies are increasingly adopting systems pharmacology, which interprets pharmacological effects of drugs from consequences of the interaction networks that drugs might have. Most conventional network-based approaches collect associations of herb-compound, compound-target, and target-disease from individual databases, respectively, and construct an integrated network of herb-compound-target-disease to study the complex mechanisms underlying herbal treatment. More recently, rapid advances in high-throughput omics technology have led numerous studies to exploring gene expression profiles induced by herbal treatments to elicit information on direct associations between herbs and genes at the genome-wide scale. In this review, we summarize key databases and computational methods utilized in systems pharmacology for studying herbal medicine. We also highlight recent studies that identify modes of action or novel indications of herbal medicine by harnessing drug-induced transcriptome data.

• BMB Reports
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• v.39 no.3
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• pp.329-334
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• 2006

RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides-based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the onestep ligation of short oligonucleotides, it is simple, less error-prone, and economical.