Hak Yong Kim

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation.

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.

Mining Frequent Contiguous Sequence Patterns in Biological Sequences

Biological sequences such as DNA and amino acid sequences typically contain a large number of items. They have contiguous sequences that ordinarily consist of more than hundreds of frequent items. In biological sequences analysis (BSA), a frequent contiguous sequence search is one of the most important operations. Many studies have been done for mining sequential patterns efficiently. In recent years, the MacosVSpan algorithm was proposed based on the idea of the prefixSpan algorithm to significantly reduce its recursive process. However, the algorithm is inefficient for mining frequent contiguous sequences from long biological data sequences. In this paper, we propose an efficient method to mine maximal frequent contiguous sequences in large biological data sequences by constructing the spanning tree with a fixed length. To verify the superiority of the proposed method, we perform experiments in various environments. The experiments show that the proposed method is much more efficient than MacosVSpan in terms of retrieval performance.

Characterization of salicylic acid-induced genes in Chinese cabbage

Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.

Development and Evaluation of a Rapid Diagnostic Test for Plasmodium falciparum , P. vivax , and Mixed-Species Malaria Antigens

Plasmodium falciparum and P. vivax malaria are endemic to many parts of the world and humans can be co-infected with both species. Because each Plasmodium species has different biological and clinical characteristics, accurate differentiation of the infecting species is essential for effective treatment. Therefore, we produced three monoclonal antibodies that recognize the lactate dehydrogenase of P. falciparum, P. vivax, or both to develop the first P. falciparum, P. vivax, and mixed-species infections malaria antigen detection kit. The detection limits of this kit were 150 and 250 parasites/μL for P. falciparum and P. vivax, respectively, and the kit was able to detect mixed-species infections. The sensitivity and specificity of this kit was assessed with 722 clinical specimens. Our results showed that its sensitivities for P. falciparum, P. vivax, and mixed-species infection were 96.5%, 95.3%, and 85.7%, respectively. In addition, its specificity was high (99.4%).

A droplet-based microfluidic immunosensor for high efficiency melamine analysis

We report a droplet-based microfluidic immunosensor for the rapid and accurate detection of melamine, an organic base that has been implicated in widescale adulteration of food products such as milk. Our melamine assay is based on the competitive reaction between native melamine and a melamine-fluorescein isothiocyanate (FITC) conjugate against an anti-hapten antibody. The adoption of fluorescence polarization, allows the quantification of melamine in a more direct and rapid manner than established heterogeneous methods based on liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). The detection protocol provides a limit of detection of 300 ppb, which is below the maximum allowable melamine levels (2.5 ppm) defined by the U.S. Food and Drug Administration and the European Commission to a significant extent.

The Pro/Hel region is indispensable for packaging non-replicating turnip yellow mosaic virus RNA, but not replicating viral RNA

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA. The genomic RNA has a tRNA-like structure (TLS) at the 3′-end. The 3′-TLS and hairpins in the 5′-untranslated region supposedly serve as packaging signals; however, recent studies have shown that they do not play a role in TYMV RNA packaging. In this study, we focused on packaging signals by examining a series of deletion mutants of TYMV. Analysis of encapsidated viral RNA after agroinfiltration of the deletion constructs into Nicotiana benthamiana showed that the mutant RNA lacking the protease (Pro)/helicase (Hel) region was not encapsidated by the coat proteins provided in trans, implicating that a packaging signal lies in the Pro/Hel region. Examination of two Pro−Hel− mutants showed that protein activity from the Pro/Hel domains was dispensable for the packaging of the non-replicating TYMV RNA. In contrast, the mutant TYMV RNA lacking the Pro/Hel region was efficiently encapsidated when the mutant TYMV was co-introduced with a wild-type TYMV, suggesting that packaging mechanisms might differ depending on whether the virus is replicating or not.

The effect of phytochrome action on the activity of cytosolic cholinesterase in oat cells.

Cholinesterases in the oat cell were found to be distributed in the cell wall (50%) and cytoplasm (42%). Activity of the cytosolic enzyme was inhibited about 80% by 1 mM Ca2+. The enzyme activity was also inhibited by Mn2+, but no inhibition by Mg2+ was observed. Effects of red light and calcium ion on the enzyme activity were investigated in vivo to confirm the involvement of phytochrome action in the regulation process of this enzyme via Ca2+. It was observed that inhibition by red light only occurs when external Ca2+ existed in the cell medium. Based on a previous report (8) that red light stimulates the influx of Ca2+ into the cytosol of oat cell, inhibition of the enzyme activity by irradiation of red light can be suggested to occur via the influx of Ca2+.

Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA–protein interactions

Fluorescence polarization (FP) is a sensitive, robust, and homogeneous assay format, able to probe a diversity of biological molecules and their interactions. Herein, we describe a new FP strategy based on the use of streptavidin as a signal amplifier. Such signal amplified fluorescence polarization (SAFP) was used to monitor the binding affinity of human angiogenin and a single-stranded DNA aptamer. Streptavidin was bound to a biotinylated single-stranded DNA aptamer and the interaction between this complex and Alexa Fluor 488 labelled human angiogenin was measured. A dissociation constant of 135.3 ± 32.9 nM and a limit of detection of 6.3 nM were successfully extracted only when the FP signal was increased (without binding hindrance) via streptavidin. Moreover, the demonstrated approach was specific to target molecules without any non-specific binding. The streptavidin-triggered SAFP method unlike amplification strategies that utilize nanomaterials (such as graphene oxides, carbon nanotubes, and metal nanoparticles) is not compromised by fluorescence quenching, and it is able to operate within nanomolar concentration regimes. Furthermore, unlike the other FP signal amplification strategies that use dual binding DNA probes, the presented method is simple to implement with signal amplification only requiring the binding of streptavidin with biotinylated DNA. This method could be expanded to analyze molecular interactions and it may be a useful tool for FP measurement by reducing the concentration of rare and expensive protein samples.

Discovery of cancer-related new drug target proteins from re-constructed human disease network based on protein-protein interaction netowk

Experimental methods such as high-throughput screening have widely used for discovery of the new drug targets. We employed an idea that disease-related proteins tend to be work as an important factor for architecture of the disease network in this study. To determine new drug target proteins, we proposed a new methodology that analyzes the human disease network by using informational technology. We re-constructed cancer-related human disease network based on the protein-protein interaction network. We derived 105 cancer related new drug-target proteins from the disease network. To evaluate our method, first of all, we extracted the cancer-related target genes that independently obtained from the human gene network introduced by Barabasi et al [1]. The cancer-related target genes and proteins compared with cancer-related target gene dataset obtained from DrugBank. Our results from disease-related network have 81% identity with the cancer-related target genes (164) from the DrugBank. In contrast, the target genes from the human gene network have only 71% identity. These results suggest that our new methodology will contribute for discovering drug target proteins.

Epigenetic Characterization of Aging Related Genes

Gene expression is regulated by a wide range of mechanisms at the DNA sequence level. In addition, gene expression is also regulated by epigenetic mechanisms through DNA methylation, histone modification, and ncRNA. To understand the regulation of gene expression at the epigenetic level, we constructed aging related gene database and analyzed epigenetic properties that are focused on DNA methylation. The DNA methylation of promoter or upstream region of the genes induces to repress the gene expression. We compared and analyzed distribution between whole human genes and aging related genes in the epigenetic properties such as CGI distribution, methylation motif pattern, and TFBS (transcription factor binding site) distribution. In contrast to methylation motif pattern, CGI and TFBS distributions are positively correlated with epigenetic regulation of aging related gene expression. In this study, the epigenetic data about DNA methylation of the aging genes will provide us to understand phenomena of the aging and epigenetic mechanism for regulation of aging related genes.