• Journal of Life Science
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• v.22 no.3
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• pp.293-297
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• 2012

The movement of vesicles from the neuronal cell body to specific destinations requires molecular motors. The squid giant axon represents a powerful model for studies of the axonal transport mechanism because the axoplasm can readily be separated from the sheath by simple extrusion. In a previous study, vesicular movements in the axoplasm of the squid giant axon were inhibited by the kinesin antibody. In the present study, we cloned and sequenced the cDNAs for squid brain KIFs. Amplification of the conserved nucleotide sequences of the motor domain by polymerase chain reaction (PCR) using first-strand cDNAs of the squid optic lobe identified six new KIF proteins. Motif analysis of the motor domains revealed that the squid KIFs are homologous to the consensus sequences of the mouse KIFs. The phylogenetic tree generated by using the maximum parsimony (MP) method, the neighbor-joining (NJ) method, the minimum evolution (ME) method, and the maximum likelihood (ML) method showed that squid KIFs are closest to mouse KIFs. These data prove the phylogenetic relationships between squid KIFs and mouse ones.

• Mycobiology
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• v.49 no.5
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• pp.461-468
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• 2021

The genus Laccaria (Hydnangiaceae, Agaricales) plays an important role in forest ecosystems as an ectomycorrhizal fungus, contributing to nutrient cycles through symbiosis with many types of trees. Though understanding Laccaria diversity and distribution patterns, as well as its association with host plants, is fundamental to constructing a balanced plant diversity and conducting effective forest management, previous studies have not been effective in accurately investigating, as they relied heavily on specimen collection alone. To investigate the true diversity and distribution pattern of Laccaria species and determine their host types, we used four different approaches: specimen-based analysis, open database search (ODS), NGS analysis, and species-specific PCR (SSP). As a result, 14 Laccaria species have been confirmed in Korea. Results regarding the species distribution pattern were different between specimen-based analysis and SSP. However, when both were integrated, the exact distribution pattern of each Laccaria species was determined. In addition, the SSP revealed that many Laccaria species have a wide range of host types. This study shows that using these four different approaches is useful in determining the diversity, distribution, and host of ECM fungi. Furthermore, results obtained for Laccaria will serve as a baseline to help understand the role of ECM fungi in forest management in response to climate change.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.514-531
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• 2018

Tomato spotted wilt virus (TSWV; Genus Orthotospovirus: Family Tospoviridae) is one of the most destructive viruses affecting a wide range of horticultural crops on a worldwide basis. In 2015 and 2016, 171 leaf and fruit samples from tomato (Solanum lycopersicum) plants with viral symptoms were collected from the fields in various regions of Iran. ELISA test revealed that the samples were infected by TSWV. The results of RT-PCR showed that the expected DNA fragments of about 819 bp in length were amplified using a pair of universal primer corresponding to the RNA polymerase gene and DNA fragments of ca 777 bp and 724 bp in length were amplified using specific primers that have been designed based on the nucleocapsid (N) and non-structural (NSs) genes, respectively. The amplified fragments were cloned into pTG19-T and sequenced. Sequence comparisons with those available in the GenBank showed that the sequences belong to TSWV. The high nucleotide identity and similarities of new sequences based on the L, N, and NSs genes showed that minor evolutionary differences exist amongst the isolates. The phylogenetic tree grouped all isolates six clades based on N and NSs genes. Phylogenetic analysis showed that the Iranian isolates were composed a new distinct clade based on a part of polymerase, N and NSs genes. To our knowledge, this is the first detailed study on molecular characterization and genetic diversity of TSWV isolates from tomato in Iran that could be known as new clade of TSWV isolates.

• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.309-313
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• 2006

Discovery of single nucleotide polymorphisms (SNPs), including small insertions and deletions, is one of the hot topics in genetic research. The most common type of sequence variant consists of single base differences or small insertions and deletions at specific nucleotide positions. Significance of SNPs in rice is increasing for genetic research, positional cloning and molecular breeding. $F_2$ 170 lines and $F_3$ 194 lines derived from Sangjuchalbyeo/HR13721-53-3-1-3-3-2-2 Were used for Searching SNP markers related to bacterial blight resistance. Sangjuchalbyeo is susceptible to bacterial blight, but HR13721-53-3-1-3-3-2-2 has Xa1 gene resistant to bacterial blight. Individual lines were inoculated with $K_1$ race of bacterial blight and resistant or susceptible was evaluated after 3 weeks from inoculation. The genotypes of population were analysed by PCR-RFLP for SNP marker developing. The segregation of $F_2\;and\;F_3$ population showed almost 3:1, 1:1 ratio, respectively. Analysis of genotype using SNP marker is capable of confirming resistance for $K_1$ race and genotype through amplifying the gene using 16PFXal primer and digested the PCR product with Eco RV. There were close relation between resistance test for $K_1$ race and SNP marker genotype. Especially, DNA analysis using SNP marker is capable of judging homozygote/heterozygote in $F_2$ population compared with resistant test for Kl race. So, it seems to improve the selection efficiency in disease resistant breeding.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.

• Journal of agriculture & life science
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• v.44 no.6
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• pp.91-99
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• 2010

We used a multiplex PCR primer set composed of 11 microsatellite (MS) markers and two sexing markers for gender detection. Genomic DNA extracted from hair roots of 3,510 Hanwoo were genotyped. Based on the 11MS markers, no animals had identical genotypes(TGLA227, BM2113, TGLA53, ETF10, SPS115, TGLA122, ETH3, ETH225, BM1824 and INRA23). The expected probability of identity among genotypes of random individuals (PI), the probability of identity among genotypes from random half-sibs ($PI_{half-sibs}$) and among genotypes of random individuals, and the probability of identity among genotypes from random sibs ($PI_{sibs}$) were estimated as $1.31{\times}10^{-23}$, $2.52{\times}10^{-16}$and $1.09{\times}10^{-6}$, respectively using the API-CALC program, version 1.0. We successfully completed the genotype analysis of 3,510 Hanwoo with a 3.93% genotyping failure rate. It was revealed that extracting DNA from the hair root was a time-efficient and cost-effective method to collect specimens for DNA isolation from live animals. This method also minimized stress for the animals during specimen collection. Among the hair roots from the back, belly, upper tail and lower tail, 5~13 hair roots of the lower tail led to the best genotype analysis results. Finally, we established a 96-well-format method of DNA preparation applicable for high- throughput genotype analysis.

• Clinical and Experimental Pediatrics
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• v.50 no.7
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• pp.686-693
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• 2007

Purpose : Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), is a potent inhibitor of inosine-monophosphate dehydrogenase (IMPDH), a new immunosuppressive drug used. It was reported that MPA protected neurons after excitotoxic injury, induced apoptosis in microglial cells. However, the effects of MPA on hypoxic-ischemic (HI) brain injury has not been yet evaluated. Therefore, we examined whether MPA could be neuroprotective in perinatal HI brain injury using Rice-Vannucci model (in vivo) and in rat brain cortical cell culture induced by hypoxia (in vitro). Methods : Cortical cells were cultured using a 18-day-pregnant Sprague-Dawley (SD) rats and incubated in 1% $O_2$ incubator for hypoxia. MPA ($10{\mu}g/mL$) before or after a HI insult was treated. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 hours of hypoxic exposure (8% $O_2$). MPA (10 mg/kg) before or after a HI insult were administrated intraperitoneally. Apoptosis was measured using western blot and real-time PCR for Bcl-2, Bax, caspase-3. Results : H&E stain revealed increased brain volume in the MPA-treated group in vivo animal model of neonatal HI brain injury. Western blot and real-time PCR showed the expression of caspase-3 and Bax/Bcl-2 were decreased in the MPA-treated group In in vitro and in vivo model of perinatal HI brain injury, Conclusion : These results may suggest that the administration of MPA before HI insult could significantly protect against perinatal HI brain injury via anti-apoptotic mechanisms, which offers the possibility of MPA application for the treatment of neonatal HI encephalopathy.

• Restorative Dentistry and Endodontics
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• v.32 no.2
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• pp.102-110
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• 2007

The purpose of this study was to obtain the basic information for the improvement of dental environment by investigating the presence of methicillin- or vancomycin-resistant Staphylococcus aureus (MRSA or VRSA) isolated from dental health care workers (DHCWs) and environment of the Chosun University Dental Hospital (CUDH) and a private dental clinic (control group). Staphylococcus aureus (S. aureus) was isolated from anterior nares of 42 DHCWS and 38 sites, unit chairss, x-ray devices, computers, etc., at 10 departments of the CUDH and 20 DHCWs and 11 sites at the private dental clinic. S. aureus was isolated on mannitol salt agar plate and confirmed by PCR with S. aureus species-specific primer. Antimicrobial susceptibility test of clinical isolates of S. aureus against several antibiotics including methicillin (oxacillin) was performed by investigating minimum inhibitory concentration (MIC) using broth microdilution assay. In addition, PCR was performed to detect the methicillin- or vancomycin-resistant gene. The data showed that one strain of S. aureus was isolated from DHCWs of the CUDH and three strains of S. aureus was isolated from 3 samples of the private dental clinic, respectively. All of the isolates from the CUDH and the private dental clinic had resistance to penicillin G, amoxicillin and vancomycin and susceptibility to oxacillin and ciprofloxacin. The S. aureus strains were already obtained the resistance to penicillin G and amoxicillin. These results suggest that two dental clinics were under relatively safe environment.

• Korean journal of applied entomology
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• v.57 no.4
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• pp.339-345
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• 2018

The sugar beet cyst nematode (SBCN), Heterodera schachtii first detected in Taebaek, Gangwon-do in 2011, is one of the major plant parasitic nematodes that cause economic damage to the Chinese cabbage in highland regions. In addition, the distribution of clover cyst nematode (CCN), H. trifolii was confirmed in the highland Chinese cabbage cultivated regions in 2017. In order to investigate the spread of cyst nematodes, this study has been conducted since 2013 in the highland Chinese cabbage cultivation area. In addition, in 2017, the Real-Time PCR technique with the species-specific primer was used to investigate those two cyst nematodes and the soybean cyst nematode (SCN), H. glycines which is known for its distribution in Korea, focusing on the main production regions of highland Chinese cabbage cultivation. The number of infected fields in the Chinese cabbage plantation in highland increased every year to confirm distribution in Taebaek, Samcheok, Jeongseon and Gangneung in 2017, and the cumulative number of infection fields reached 245 by 2017. Of the 41 possible cyst nematode samples for PCR analysis, 61% were CCN, only 9.8% of the SBCN and 29.3% of the SCN were identified. Therefore, some of the previously known SBCN or CCN discoveries are likely to have been infected with SCN. It is believed that the CCN needs to be controlled in the future as CCN have been found to be dominant species in the highland Chinese cabbage plantation regions.

• Journal of Life Science
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• v.18 no.8
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• pp.1023-1030
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• 2008

NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.