• The Plant Pathology Journal
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• v.26 no.4
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• pp.313-320
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• 2010

Genetic instability of the rice blast fungus Magnaporthe oryzae has been suggested as a major factor underlying the rapid breakdown of host resistance in the field. However, little information is available on the mechanism of genetic instability. In this study, we assessed the stability of repetitive DNA elements and several key phenotypic traits important for pathogenesis after serially transferring two isolates though rice plants and an artificial medium. Using isolate 70-15, we obtained a total of 176 single-spore isolates from 10 successive rounds of culturing on artificial medium. Another 20 isolates were obtained from germ tubes formed at the basal and apical cells of 10 three-celled conidia. Additionally, 60 isolates were obtained from isolate KJ201 after serial transfers through rice plants and an artificial medium. No apparent differences in phenotypes, including mycelial growth, conidial morphologies, conidiation, conidial germination, appressorium formation, and virulence, or in DNA fingerprints using MGR586, MAGGY, Pot2, LINE, MG-SINE and PWL2 as probes were observed among isolates from the same parent isolate. Southern hybridization and sequence analysis of two avirulence genes, AVR-Pita1 and AVR-Pikm, showed that both genes were also maintained stably during 10 successive generations on medium and plants. However, one reversible loss of restriction fragments was found in the telomere-linked helicase gene (TLH1) family, suggesting some telomere regions may be more unstable than the rest of the genome. Taken together, our results suggest that phenotype and genotype of M. oryzae isolates do not noticeably change, at least up to 10 successive generations on a cultural medium and in host plants.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• The Plant Pathology Journal
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• v.33 no.5
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• pp.508-513
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• 2017

The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semiquantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08-0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection.

• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.409-413
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• 2015

To enhance phytoremediation, which removes heavy metal from soil, transgenic plants were applied to contaminated soil. We constructed a transformation vector expressing both $TgMTP_1$ (T. goesingense metal tolerance protein):HA and TgMTP:GFP genes. Transgenic plants were generated using an Agrobacterium-mediated transformation system that expressed the two vectors. Screening and analysis confirmed the incorporation of foreign genes into the Arabidopsis thaliana genome. Callus was induced in the 116 T3 line. These transgenic plants and calli were used for further analyses on the accumulation of Ni. The 116 T3-line plants and calli from selected lines were resistant to heavy metals and accumulated Ni in their leaves. The expression level of TgMTP RNA was equal in all leaves, but protein stability increased in the leaves with Ni treatment. According to these results, we suggest that $TgMTP_1$-overexpressing plants may be useful for phytoremediation of soil.

• ALGAE
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• v.26 no.1
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• pp.21-32
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• 2011

Studies of the red algal genus Bostrychia over the last 15 years have made it a model system for many evolutionary processes within red algal species. The combination of newly developed, or first employed methods, in red algal species studies has made Bostrychia a pioneer genus in intraspecific studies. Bostrychia was the first genus in which a mitochondrial marker was used for intraspecific red algal phylogeny, and the first for which a 3-genome phylogeny was undertaken. The genus was the first red alga used to genetically show maternal plastid and mitochondria inheritance, and also to show correlation between cryptic species (genetically divergent intraspecific lineages) and reproductive incompatibility. The chemotaxonomic use, and physiological function of osmolytes, has also been extensively studied in Bostrychia. Our continuous studies of Bostrychia also highlight important aspects in algal species studies. Our worldwide sampling, and resampling in certain areas, show that intensive sampling is needed to accurately assess the genetic diversity and therefore phylogeographic history of algal species, with increased sampling altering evolutionary hypotheses. Our studies have also shown that long-term morphological character stability (stasis) and character convergence can only be correctly assessed with wide geographic sampling of morphological species. While reproductive incompatibility of divergent lineages supports the biological species nature of these lineages, reproductive incompatibility is also seen between isolates with little genetic divergence. It seems that reproductive incompatibility may evolve quickly in red algae and the unique early stages of fertilization (e.g., gametes covered by walls, active movement of spermatium nuclei to the distant egg nucleus), also well investigated in Bostrychia,. may be key to our understanding of this process.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• Journal of Microbiology and Biotechnology
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• v.19 no.2
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• pp.128-135
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• 2009

Acinetohacter baumannii BD5 was isolated from waters of Baek-du mountain, and the lipase gene was cloned using a PCR technique. The deduced amino acid sequence of the lipase and lipase chaperone were found to encode proteins of 325 aa and 344 aa with a molecular mass of 35 kDa and 37 kDa, respectively. The lipase gene was cloned and expressed in Escherichia coli BL21(trxB) as an inclusion body, which was subsequently solubilized by urea, and then purified using Ni-affinity chromatography. After being purified, the lipase was refolded by incubation at $4^{\circ}C$ in the presence of a 1:10 molar ratio of lipase:chaperone. The maximal activity of the refolded lipase was observed at a temperature of $35^{\circ}C$ and pH 8.3 when p-NP caprate(C10) was used as a substrate; however, 28% of the activity observed at $35^{\circ}C$ was still remaining at $0^{\circ}C$. The stability of the purified enzyme at low temperatures indicates that it is a cold-adapted enzyme. The refolded lipase was activated by $Ca^{2+},\;Mg^{2+},\;and\;Mn^{2+}$, whereas $Zn^{2+}\;and\;Cu^{2+}$ inhibited it. Additionally, 0.1% Tween 20 increased the lipase activity by 33%, but SDS and Triton X-100 inhibited the lipase activity by 40% and 70%, respectively.

• Korean Journal of Microbiology
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• v.51 no.4
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• pp.435-439
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• 2015

THO/TREX complex plays an important role in transcriptional elongation, mRNA processing, nuclear RNA export, and genome stability. A fission yeast, Schizosaccharomyces pombe, SPBC577.04 gene encoding the ortholog of THOC5, a component of THO/TREX complex, was identified and characterized. The S. pombe thoc5 (spthoc5) is not essential for both growth and mRNA export, but deletion of the spthoc5 gene caused growth defect and slight accumulation of $poly(A)^+$ RNA in the nucleus. And the functional spThoc5-GFP protein is localized mainly in the nucleus. Co-immunoprecipitation analysis showed that the Hpr1(THOC1) protein, an evolutionally well-conserved component of THO/TREX complex, interacted with spThoc5 as well as Tho2(THOC2), another subunit of THO complex. These results suggest that S. pombe Thoc5 as a component of THO/TREX complex is also involved in mRNA export from the nucleus.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9241-9247
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• 2014

Background: Rs31489 in the cleft lip and palate transmembrane1-like gene (CLPTM1L) has been identified to be associated with lung cancer through genome-wide association studies (GWAS). However, some recent replication studies yielded inconclusive results. Thus, we undertook this study to investigate the precise effect of rs31489 on lung cancer susceptibility. Materials and Methods: A hospital-based case-control study in 1,673 Chinese subjects (611 individuals with lung cancer and 1,062 controls) and a meta-analysis among 32,199 subjects (16,364 cases and 15,835 controls) were performed in this study. Results: In our case-control study, rs31489 was inversely associated with lung cancer (AC versus CC: OR=0.68, 95%CI=0.52-0.88; additive model: OR=0.68, 95%CI=0.54-0.85; dominant model: OR=0.65, 95%CI =0.51-0.84). Stratification analysis by smoking status showed a significant association and strong genetic effect in non-smokers but not in smokers. Our meta-analysis further confirmed the association, although with significant heterogeneity contributed by study design and source of controls, as shown by stratified analysis. Sensitive and cumulative analyses both indicated robust stability of our results. In addition, there was no observable publication bias in our meta-analysis. Conclusions: Overall, the findings from our replication study and meta-analysis demonstrated that CLPTM1L gene rs31489 is significantly associated with lung cancer.

• Journal of Plant Biotechnology
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• v.36 no.2
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• pp.115-123
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• 2009

In recent years, several genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnological companies. Commercialization of GM crops will be required the assesment of risks associated with the release of GM crops. In advance of the commercial release of GM crops, developer should submit the several information on GM crops for approval. In this study, we carried out to provide the molecular data for the risk assessment of GM rice containing insect-resistant gene, modified Cry1Ac (CryIAc1). Through the molecular analysis with CryIAc1 induced GM rice, we confirmed the steady integration and expression of transgene, the transgene copy number, the adjacent region sequences of inserted gene into rice genome, and the transgene stability in progenies. For the qualitative PCR detection methods, specific primer pairs were designed on the basis of integration sequences, and construct- and event-specific detection markers were developed for leaf folder-resistant rice, Cr7-1 line. From these results, we demonstrated that the molecular data and the PCR detection methods of leaf folderresistant GM rice could be acceptable to conduct the biosafety and environment risk assessment.