• Journal of Animal Science and Technology
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• 제45권5호
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• pp.703-710
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• 2003

본 연구는 재래돼지와 랜드레이스를 기초축으로 이용한 F$_2$ 241두에 대해 Heart Fatty Acid- Binding Protein 유전자와 연관되어 있는 PCR- RFLP를 이용하여 그 다형성을 조사하고 돼지의 성장형질, 도체형질, 육질형질과 그 유전자형간의 연관성을 구명코자 실시하였다. H-FABP PCR-RFLP는 두 쌍의 primer에 의한 850bp와 700bp의 증폭산물을 HaeⅢ와 HinfⅠ제한효소를 사용하여 실시되었다. HaeⅢ을 이용한 PCR-RFLP 유전자형은 DD형/700+150bp, Dd형/700+400+300+ 150bp 그리고 dd형/400+300+150bp의 DNA 단편을 보였으며, Hinf1에 의한 유전자형은 HH형은 350+180+130bp, Hh형은 350+220+180+130bp, hh형/350+220+130bp의 절단된 DNA 단편을 보였다. H-FABP/HaeⅢ 유전자형 중에서 12주령 체중은 DD형에 비해 Dd와 dd형에서 유의적으로 높은 값을 보였으며(p〈0.001), 3주령 체중 (p〈0.01)과 5주령, 30주령 체중 (p〈0.05)에 경우도 Dd와 dd형에서 유의적으로 높게 관찰되었고(Table 3), 특히 ‘d’ 대립유전자가 체중과 연관성이 있음이 관찰되었다. H-FABP/HinfⅠ의 유전자형과 표현형질의 연관성을 보면 12주령, 30주령 체중 및 도체지방 과 등지방 두께에서는 hh형에 비해 HH와 Hh형에서 유의적으로 높게 나타났으며(p〈0.001), 5주령 체중과 근내지방 함량에서도 HH형에서 유의적으로 높게 관찰되었고(p〈0.05), 특히 ‘H’ 대립유전자가 체중과 도체지방, 등지방 두께 및 근내지방 함량과 연관성이 있음이 관찰되었다. 따라서 돼지성장 및 지방축적과 관련한 선발력을 높이기 위해 H- FABP PCR-RFLP(HaeⅢ & HinfⅠ)를 분자생물학적 marker로 사용할 수 있을 것으로 사료된다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.74.1-74
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• 2003

Differential display (DD) of mRNA is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). Using DD-RT-PCR we obtained many genes that expressed differentially in healthy and PVX-infected Nicotiana benthamima, using total RNAs extracted from healthy and PVX-infected N. benthamiana plants. Three hundred and twenty-five DNA fragments isolated from DD-RT-PCR were cloned and sequenced for further characterization. Several host genes including SKPI-like protein, heat shock transcription factor and Avr9/Cf-9 rapidly elicited protein were selected to obtain full-length open reading frame and to characterize their potential involvement in virus disease development and/or host's defense against virus infection employing PVX-based expression vector. Transcrips from wild-type and clones containing each selected gene were inoculated onto N. benthamiana Levels of virus replication were confirmedby RT-PCR and RNA blot analysis, Expression profiles and potential role(s) of selected genes upon PVX infection will be discussed.

• Archives of Pharmacal Research
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• 제28권2호
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• pp.232-237
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• 2005

Mast cells play an important role in allergic inflammation by releasing their bioactive mediators. The function of mast cells is enhanced by stimulation because of the induction of specific genes and their products. While many inducible genes have been elucidated, we speculated that a significant number of genes remain to be identified. Thus, we applied differential display (dd) PCR to establish a profile of the induced genes in bone marrow-derived mast cells (BMMCs) after they were co-cultured with 3T3 fibroblasts. To date, 150 cDNA fragments from the connective-type mast cells (CTMCs) were amplified. Among them, thirty cDNA fragments were reamplified for cloning and sequencing. The ddPCR strategy revealed that serine proteases were the most abundant genes among the sequenced clones induced during the maturation. Additionally, unknown genes from the co-culture of BMMCs with 3T3 fibroblasts were identified. We confirmed their induction in the CTMCs by Northern blot analysis and RT-PCR. Characterization of these induced genes during the maturation processes will provide insight into the functions of mast cells.

• ALGAE
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• 제37권4호
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• pp.281-291
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• 2022

Quantifying the abundance of Chattonella species is necessary to effectively manage the threats from ichthyotoxic raphidophytes, which can cause large-scale mortality of aquacultured fish in temperate waters. The identification and cell counting of Chattonella species have been conducted primarily on living cells without fixation by light microscopy because routine fixatives do not retain their morphological features. Species belonging to the Chattonella marina complex, including C. marina and C. marina var. ovata, had high genetic similarities and the lack of clear morphological delimitations between the species. To estimate the abundance of C. marina complex in marine plankton samples, we developed a protocol based on the droplet digital polymerase chain reaction (ddPCR) assay, with C. marina complex-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA. Cell abundance of the C. marina complex can be determined using the ITS copy number per cell, ranging from 25 ± 1 for C. marina to 112 ± 7 for C. marina var. ovata. There were no significant differences in ITS copies estimated by the ddPCR assay between environmental DNA samples from various localities spiked with the same number of cells of culture strains. This approach can be employed to improve the monitoring efficiency of various marine protists and to support the implementation of management for harmful algal blooms, which are difficult to analyze using microscopy alone.

• Parasites, Hosts and Diseases
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• 제37권4호
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.251-256
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• 2006

The exploration of genes which expressions are changed by exposure to ecotoxicants or pollutants can provide the important information about the reaction mechanisms in the body as well as adaptation to exterior stimulus or environmental changes. Also they can be developed as biomarkers for the detection of environmental pollution. Differential display polymerase chain reaction (DD-PCR) technique has been usefully used to hunt the clones which expressions are up-regulated or down-regulated by exterior changes and this study aimed to search for those clones in diesel oil-exposed rockfish (Sebastes schlegeli) using DD-PCR. The RNA isolated from liver of 20 ppb diesel oil-exposed rockfish was used for screening of the differentially displayed genes and total 44 differentially expressed genes (DEG) are detected then their nucleotide sequences were analyzed. The present data provided the general information about the effect of diesel oil contamination on marine organism and further more the primary step in development of new biomarkers for marine environmental pollution or ecotoxicological stresses.

• The Plant Pathology Journal
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• 제39권1호
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• pp.141-148
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• 2023

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 10³-10⁷ copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.

• BMB Reports
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• 제33권2호
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• pp.184-187
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• 2000

Prostaglandin $E_2$ ($PGE_2$) plays an important role in the regulation of various gastric functions, and the growth-inhibitory activities on tumor cells are studied in vitro and in vivo. Although the mechanisms have attracted many researchers in the past decade, the molecular mechanisms of cell cycle arrest, or induction of apoptosis by $PGE_2$, is unclear. We investigated the effects of $PGE_2$ on the growth of the human gastric carcinoma cell line SNU1 and genes that are regulated by $PGE_2$ and isolated them using differential display RT-PCR (DD RT-PCR). FACS analysis suggested that SNU1 cells were arrested at the G1 phase by $PGE_2$ treatment. This growth inhibitory effect was in a time- and dose-dependent manner. Treatment of SNU1 cells with $10\;{\mu}g/ml$ $PGE_2$, followed by DD RT-PCR analysis, revealed differently expressed bands patterns from the control. Among the differently expressed clones, we found an unidentified cDNA clone (HGP-27) overexpressed in $PGE_2$-treated cells. The full-length cDNA of HGP-27 was isolated using RACE, which consisted of a 30-nt 5'-noncoding region, a 891-nt ORF encoding the 296 amino acid protein, and a 738-nt 3'-noncoding region including a poly(a) signal. This gene was localized on the short arm of chromosome number 11. Using the Motif Finder program, a myb-DNA binding repeat signature was detected on the ORF region. The COOH-terminal half was shown to have similarity with the $NH_3$-terminal domain of thioredoxin (Trx). This relation between HGP-27 and Trx implied a potential role for HGP-27 in modulating the DNA binding function of a transcription factor, myb.

• 자연과학논문집
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• 제11권1호
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• pp.95-98
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• 1999

$CO_2$ gas 처리가 배추 자가불화합성 타파에 미치는 영향을 조사하고자 배추 inbred line의 암술에서 mRNA를 분리하여 DD-PCR을 수행하였다. $CO_2$ gas를 처리한 암술에서 분리한 mRNA와 처리하지 않은 꽃에서 분리한 암술 mRNA 발현과의 특이적인 차이를 DD-PCR로 조사한 결과 세 가지 각기 다른 anchor primer에 대해 모두 18개의 특이적 증폭 DNA fragment를 얻을 수 있었다. 이 결과로 미루어 보아 $CO_2$ gas 처리에 의한 암술 mRNA발현의 변화가 배추 자가불화합성 타파에 영향을 미치는 것으로 생각된다.

• 한국육종학회지
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• 제42권5호
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• pp.565-573
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• 2010

사과의 경우 한 과총에 5개의 꽃이 피는데 그 중 한 가운데의 중심화가 먼저 개화하여 과일로 발달하고 주위의 측과 4개는 스스로 낙과되는 현상을 자가적과성이라고 한다. 적과는 인위적으로 과실의 숫자를 줄여 잎 수와 과실 수의 균형을 맞추는 작업으로 과실의 크기를 증가시키고, 수세, 수형을 유지시켜 안정적인 생산에 도움을 준다. 노동력 절감을 위해 인간에게 유용하게 사용될 수 있는 특성이 자가적과성인데, 자가적과성 품종의 사과에서 측과는 만개 후 30일 이내에 떨어지고 중심과만 남아서 성숙하게 된다. 51개의 사과 품종으로부터 비자가적과성 그룹 20종, 6월 생리적 낙과 그룹 16종, 자가적과성 그룹 15종을 분류하였다. 대표적인 자가적과성 품종인 Aori #9 로부터 중심과와 측과에서 다르게 발현이 되는 유전자들을 DD-PCR 방법으로 확인하였다. 중심과에서 30개의 clones 과 측과에서 24개의 clones을 선발하여 염기서열을 분석하였다. 주로 측과에서 발현되는 유전자들은 pathogenesis, senescence, temperature stress, protein degradation, fruit browning, sorbitol metabolism에 관여하는 유전자들과 높은 상동성을 나타내고, 중심과에서 발현되는 유전자들의 염기서열을 분석해 보면 anthocyanin의 up-regulation이나 flavonol 생합성, ethylene 생합성에 관여하는 유전자들이 분포한다. Cytochrome P450 유전자의 발현양상을 보기 위해 Real time PCR 분석을 한 결과 중심과보다 측과에서 발현량이 높게 나타났다. 중심과와 측과에서 다르게 발현되는 유전자들의 실제 발현양상을 분석하기 위해 Real time PCR을 이용해서 상대정량을 분석할 계획이며 분자수준에서의 자가적과를 조절하는 기작에 대한 앞으로의 연구는 생력 재배가 가능한 품종 육성의 육종 소재 개발에 활용될 수 있을 것이다.