• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.130-135
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• 2004

The marine dinoflagellate genus Alexandrium, which produces PSP toxins, has a global distribution. As human-assisted dispersal of the species has been suggested, it is important to develop molecular tools to trace the dispersal pathway. To screen population-specific DNA sequences that differentiate Korean and Japanese A. catenella, we targeted the area downstream of the chloroplast psbA gene using PCR with population-specific DNA primers followed by RFLP (restriction fragment length polymorphism) analysis and sequencing. The RFLP patterns of the PCR products divided Korean and Japanese A. catenella regional isolates into three types: Korean, Japanese, and type CMC3, isolated from Korea. We sequenced the PCR products, but found no similar gene in a homology search. The molecular phylogeny inferred from the sequences separated the Korean and Japanese A. catenella strains, as did the RFLP patterns. However, the Japanese isolates included two slightly different sequences (types J and K), while the Korean sequence was the same as the Japanese K type. In addition, a unique sequence was found in the Korean strains CMC2 and CMC3. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers designed from the type J sequence yielded PCR products for Japanese strains only, showing that the unknown gene can be used for a population analysis of Korean and Japanese A. catenella.

• 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.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.357-363
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• 2013

The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.

• Restorative Dentistry and Endodontics
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• v.20 no.2
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• pp.577-609
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• 1995

Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.

• Journal of fish pathology
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• v.23 no.3
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• pp.313-322
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• 2010

Edible tunicate Halocynthia roretzi, one of the most commercially important aquatic organisms in Korea, has been killed by tunic softness syndrome since last decade. The intracellular protistan parasite observed by the transmission electron microscope in hemocytes of the tunicate was considered to be the causative agent of the mass mortality. The goal of the present work is to examine the characteristic features of the parasite by identifying the 18S rDNA sequences of the parasite. The experiments conducted include amplification of presumptive 18S rDNA from diseased tunicate tissues with UNonMet-PCR and sequencing the product. A preliminary phylogenetic analysis was performed on the presumptive parasite rDNA. A digoxigenin labeled DNA probe was designed on the basis of the sequences of rDNA. Dig-ISH assay was conducted to diagnose the protistan parasite. A PCR using UNonMet-PCR primer generated 595 bp SSU rDNA fragment. Subsequently, PCRs with primer pair expended this sequence to 1542 bp. This is the first partial sequences of SSU rDNA gene to be published on the protistan parasite that has presumed causing the mass mortality of tunicate. Since the Dig-ISH technique demonstrated the presence of infection in hemocytes on the all host tissues, the fragment was confirmed to be the intracellular protistan parasite SSU rDNA. A phylogenetic analysis suggested that the protistan parasite may be a unique eukaryote that is closely related to Apicomplexa.

• The Korean Journal of Zoology
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• v.35 no.2
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• pp.219-225
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• 1992

The genetic variation in mitochondrial DNA (mtDNA) was analysed within and between two species of tree frogs. Hyla japonica and H. suweonensis from South Korea. Purified mtDNAs were digested with each of 11 restriction enLvmes which cleave at six base recognition sequences. The genome size of H. iaponica revealed ho types (20.0 $\pm$ 0.3 and 19.6 $\pm$ 0.3 kb) and this difference is explained by either addition or deletion of about 0.4 kb fragment. On the other hand, the genome sire of H. suueonensis was about 19.0 $\pm$ 0.4 kb only. For the analysis, level of fragment homology (F) and nucleotide sequence divergence (p) were estimated from comparisons of digestion profiles. Among four populations of H. iaponica, substantial mean sequence divergence was 0.017 (range 0.001-0.026); between identical types, 0.001 IslilaRl type) and 0.004 (Large type) respectively; between different ones, 0.024 (range 0.023-0.026). The level of sequence divergence between he species was 0.142 (range 0.131-0.146). This result suggested that he species ㅂwere distinctly differentiated species. The divergence time between ko species was estimated 7.1 million years.

• Journal of Plant Biology
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• v.39 no.4
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• pp.279-286
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• 1996

To know the changes of rbcL mRNA level by illumination, Northern hybridization analysis was performed with maize (Zea mays L.cv. Golden X Bantam). The average level of rbcL. mRNA in the light-grown shoots was 3.1 times higher than that of the dark-grown shoots after 6 to 10 growth days. The maximum difference of rbcL mRNA level between the dark-grown and the light-grown shoots was 5.1 folds. These results indicate that accumulation of rbcL mRNAin maize shoots is induced by light. Since the transcriptional DNA binding proteins and their cognate promoter elements, we carried out gel-retardation assays to elucidate the specific binding proteins on the rbcL promoter. It was found that plastid proteins of light-grown shoots bound to the R2 DNA fragment (-33 to -229) and R3 DNA fragment (-230 to -418 from ATG) of the rbcL promoter. From the results of competitive binding assays and heat or protease treatments, it was demonstrated that the bindings were sequence-specific DNA-protein interactions. Therefore, it could be concluded that the rbcL promoter region has at least two specific recognition sites for plastid proteins.

• Fisheries and Aquatic Sciences
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• v.9 no.3
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• pp.101-106
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• 2006

The cytochrome c oxidase subunit I (COI) gene region of mitochondrial DNA (mtDNA) was examined in four abalone species to estimate its utility as a genetic marker using restriction fragment length polymorphism (RFLP) analysis. The utility was evaluated in terms of genetic divergence and relationships among Haliotis discus hannai, H. rufescens, H. rubra, and H. midae in both hemispheres of the world. There was clear genetic divergence in the mtDNA COI region between all pairs of the four species. Moreover, relationships among the abalone species were reflected in their geographical distributions and morphological characteristics. Therefore, RFLP analysis of the mtDNA COI region is a suitable genetic marker for the estimation of genetic divergence and relationships among abalone species. However, it is not effective for the evaluation of genetic differences within abalone species.

• Journal of Genetic Medicine
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• v.2 no.1
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• pp.23-26
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• 1998

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which has been clinically shown to cause progressive weakness and result in atrophy of the facial muscles, shoulder girdle and upper arm muscles. The responsible gene for the FSHD has been located on chromosome 4q35-qter. The probes p13E-11 and pFR-1 detect DNA rearrangements associated with FSHD as under 28 kb DNA fragment in genomic southern analysis digested with EcoRI and the fragment contains 3.3 kb Kpn I tandem repeats. In this study, 4 fetuses with a family history of FSHD were analysed by genomic southern hybridization analysis with probes to determine whether they carried the deleted region. Of the 4 fetuses, three of them had mothers who were FSHD patients and the other one had a father affected with FSHD. After 10-11 weeks of gestation, we performed chorionic villi sampling and extracted DNA from uncultured and cultured tissue cells for the direct DNA analysis. The result of the southern analysis showed two fetuses having received about 15-18 kb of deleted genes from the father and the mother respectively, and found to be FSHD patients. The other two fetuses were shown to have two normal alleles from the parents and found to be normal. Two pregnancies which were determined to be normal were carried to term delivering two healthy babies.

• Environmental Analysis Health and Toxicology
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• v.29
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• pp.7.1-7.8
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• 2014

Objectives The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. Methods A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.