• Animal Systematics, Evolution and Diversity
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• v.26 no.2
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• pp.99-104
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• 2010

The subfamily Murinae is a very controversial group concerning their phylogenetic relationship. Previous studies could not resolve phylogeny among four genera Apodemus, Micromys, Mus and Rattus of the Muridae. In the present study, eight rodent species resident in South Korea were collected and phylogenetically analyzed based on sequence data of five mitochondrial and nuclear DNA regions: 12S rRNA, cytochrome b gene (cyt b), cytochrome oxidase II (COII), control region of mitochondrial DNA, and a thyroglobulin (Tg) of nuclear DNA. According to the phylogeny of the concatenated data, M. musculus separated early in Murinae (ML 100%; BA 1.00 pp) and the genus Rattus grouped with the harvest mouse, M. minutes; these were separated from the genus Apodemus with relatively strong support (ML 74%; BA 0.76 pp). The Siberian chipmunk population was also examined using the five genes to obtain better resolution. The phylogeny for Korean rodents determined using the 12S rRNA, cyt b, COII and control regions discriminated the Siberian chipmunk populations from Korea, Russia, and China.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.237-242
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• 2015

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.

• Microbiology and Biotechnology Letters
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• v.11 no.1
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• pp.75-79
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• 1983

To exploit a suitable vector and recipient strain for molecular cloning in Bacillus subtilis, oxytetracycline-resistant plasmic DNA has been prepared from Streptomyces rimosus by phenol-buffer extraction of lysozyme-lysed cells and introduced into B. subtilis KPM 60 [St $r^{R}$-mutant of RM 125 (leu A8, arg 15, hsm $M^{-10}$ , hsr $M^{-10}$ )] by transformation. Oxitetracycline-resistant plasmid was well transferred into B. subtilis KPM 60 with average frequency of 10$^{-4}$ per $\mu\textrm{g}$ of DNA. The highest frequency of plasmid transformation was obtained after 3 hours incubation of recipient cells in the growth medium and 30 to 60 minutes incubation in the competence medium, and then 20 minutes contact of DNA and host cells. Optimum pH for competence was 7.5, and optimum temperature for transformation was 2$0^{\circ}C$.>.

• The Plant Pathology Journal
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• v.31 no.2
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• Journal of Yeungnam Medical Science
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• v.14 no.1
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• pp.155-167
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• 1997

The identification of serum HBV DNA is very important for the assessment of the disease activity in persistent infection, for the evaluation of the infectivity of an individuals blood. The dot blot, however, has limited sensitivity and sometimes inconsistent with other serological markers and clinical settings. Using the most important recent advance in molecular biology, the polymerase chain reaction(PCR), specific DNA sequences can be amplified more than a million-fold in a few hours and with this technique the detection of the extreme low level of DNA is possible. This study was to determine sensitivity of the PCR for the detection of serum HBV DNA in comparison with dot blot analysis and to investigate the serum HBV DNA status and clinical significance of PCR in patients with chronic HBsAg positive liver disease. The subjects of this study were 17 patients with asymptomatic HBsAg carriers(9 HBeAg positive patients, 8 anti-HBe positive patients), 91 chronic hepatitis B(50 HBeAg positive patients, 41 anti-HBe positive patients), 57 liver cirrhosis(21 HBeAg positive patients, 36 anti-HBe positive patients), 27 hepatocellular carcinoma(10 HBeAg positive patients, 17 anti-HBe positive patients). The results were summerized as following; The detection rates of HBV DNA by dot blot, PCR were 58.9%, 72.2% in HBeAg positive patients, 34.3%, 53.9% in anti-HBe positive patients. The detection rates of HBV DNA by PCR in HBeAg negative patients were 25.0% in asymptomatic HBsAg carriers, 61.0% in chronic hepatitis B, 52.8% in liver cirrhosis, 52.9% in hepatocellular carcinoma. The positive rate for HBV DNA is a significant difference between HBeAg positive and negative asymptomatic HBsAg carriers, but not significantly difference in other groups. In conclusions, this study confirmed that the PCR is much more sensitive than the dot blot analysis in detecting the HBV DNA in the sera of patients with chronic liver disease. The presence of HBV DNA in the serum was detected by PCR with higher sensitivity and it suggested that active viral replication is still going on in most patients with chronic HBsAg positive liver disease irrespective of HBeAg/anti-HBe status, and PCR may be used as a prognostic factor in asymptomatic HBsAg carriers.

• Animal Systematics, Evolution and Diversity
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• v.17 no.1
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• pp.49-57
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• 2001

The partial sequences of mtDNA cytochrome b gene in two subspecies of striped field mouse(Apodemus agrarius coreae and A. agrarius manchuricus) from Korea and northeast China were analyzed to determine the degree of genetic diversity in ech subspecies and to confirm their subspecific difference. In 18 specimens of A. agrarius coreae, ten haplotypes were resulted, and in two specimens of A. agrarius manchuricus. one haplotype was revealed. Tamura-Nei nucleotide distance among ten haplotypes in subspecies coreae ranged 0.36 to 1.86%. and nucleotide distance between two subspecies (coreae and manchuricus) was 0.37 to 1.47%: maximum infrasubspecific divergence in coreae was greater than maximum intersubspecific difference between two subspecies. Moreover, no major subgroup was resulted when 11 haplotypes in two subspecies were compared. Our sequence result was not cancordant with the morphological data studied so far, and it is concluded that cytochrome b gene sequence is not a good genetic marker to distinguish two subspecies of A. agrarius. In futurem, mtDNA control region analyses seemded to be necessary to reveal genetic relationship between these two subspecies of A. agrarius.

• Clinical and Experimental Pediatrics
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• v.53 no.3
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• pp.397-407
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• 2010

Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs) and conformation sensitive gel electrophoresis (CSGE) to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA) was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%), whereas for multiplex PCRCSGE screened sequencing, the mutations could be detected in 23 (85.2%). One patient's mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

• Analytical Science and Technology
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• v.31 no.2
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• pp.71-77
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• 2018

Detection of bloodstains is a very important process in scientific investigations, and luminol is often used for the detection of bloodstains that are not visible. Recently, new preparation methods of blood detection reagents based on luminol (BloodFlareA, B) were developed and reported to have higher active persistence and to be more economical than conventional blood detection reagent, BlueStar forensic. In this paper, we tested the specificity and effect of the BloodFlares (A and B) on DNA and compared them with those of BlueStar forensic. False positive results for the BloodFlares were not observed in semen, saliva, vaginal fluids, urine, sweat, and nasal discharge, but were observed in $CuSO_4$, $FeSO_4$, and bleach solutions, and the observed patterns were similar to those of BlueStar forensic. The effect on DNA was determined by analyzing the DNA yield, degradation index, and DNA profiling. Based on these results, we concluded that the BloodFlares based on luminol do not affect DNA stability and are applicable in forensics.

• BMB Reports
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• v.43 no.4
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• pp.250-256
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• 2010

Natural phosphodiester bond CpG-DNA that contains immunomodulatory CpG motifs (PO-DNA) upregulates the expression of proinflammatory cytokines and induces an Ag-driven Th1 response in a CG sequence-dependent manner in mice. In humans, only phosphorothioate backbone-modified CpG-DNA (PS-DNA) and not PO-DNA has immunomodulatory activity. In this study, we found that liposome-encapsulated PO-DNA upregulated the expression of human $\beta$-defensin-2 (hBD-2) and major histocompatibility class II molecules (HLA-DRA) in a CG sequence-dependent and liposome- dependent manner in human B cells. Of the three different liposomes, DOTAP has the unique ability to enhance the immunomodulatory activity of PO-DNA. In contrast, HLA-DRA and hBD-2 promoter activation can be induced by liposome-encapsulated PS-DNA in a CG sequence-independent manner, depending on the CpG-DNA species. Our observations demonstrate that, when encapsulated with a proper liposome in the immune system, natural PO-DNA has the potential to be a useful therapy for the regulation of the innate immune response.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.197-204
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• 2019

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.