• 생명과학회지
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• 제22권11호
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• pp.1470-1476
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• 2012

본 연구는 유럽에서 식용버섯으로 선호도가 높은 Lepista nuda (민자주방망이버섯)에 대하여 random amplified polymorphic DNA (RAPD)와 internal transcribed spacer (ITS) 염기서열을 이용하여 종내 및 종간의 유전적 변이를 분석하였다. RAPD 분석 결과 40개의 random primer 중 다형성을 나타내는 primer는 22개 였으며, 증폭된 밴드는 355개, DNA 단편의 크기는 200~4,000 bp의 사이에 위치하였다. RAPD band들을 marker로 하여 Nei-Li's의 방법을 이용한 비유사도 지수행렬을 조사한 결과 L. nuda 종내 유전적 변이는 0~21.60%로 나타났으며, L. nuda와 L. sordida의 종간에는 16.93~24.82%, L. irina와는 20.62~25.54%로 나타났으며, L. sordida와 L. irina와의 종간 변이는 23.49%로 나타났다. ITS I 과 II 영역의 673 bp의 염기서열을 분석하여 비유사도 지수행렬을 조사한 결과, L. nuda의 종내 변이는 1.58~11.47%였으며, L. nuda와 L. sordida와는 3.83~12.88%로 나타났다. 그리고 L. nuda와 L. irina는 7.11~15.61%였으며, L. sordida와 L. irina와의 종간 변이는 4.79%로 나타났다. 본 실험결과 RAPD와 ITS실험을 통해 확인된 primer와 연기서열은 Lepista속의 종을 검색 및 분류 시 유전적 표지 marker로서 이용 할 수 있을 것으로 생각된다.

• 한국축산식품학회지
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• 제31권5호
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• pp.658-662
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• 2011

In this study, 543 samples of press hams, sausages, processed ground meat and processed cheese acquired from retail markets in Seoul and Gyeonggi province in Korea from 2005 to 2010 were monitored using a one-step multiplex polymerase chain reaction (PCR) method that involves the amplification of specific soya or maize endogenous genes and the amplification of 35S promoter (p35S) and nopaline synthase terminator (tNOS) for GMO detection. Among the 543 samples, 477 samples were amplified for maize and/or soybean endogenous genes. Although one sausage sample collected in 2008 showed amplification of tNOS, the result was assumed to be false positive based on the results from further tests of other sausage samples of the same brand. Our results demonstrate the absence of GM soya and/or maze of livestock products in the Korean market during 2005-2010. In addition, the one-step multiplex PCR using previously constructed primer sets appears to be useful as a screening method for the detection of GMOs in processed livestock products. However, more specific methods should be established and employed to detect the event-specific GM gene for positive reaction samples by screening tests in processed livestock products.

• 미생물학회지
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• 제38권4호
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• pp.230-230
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• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1586-1592
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• 2017

Some promoters were isolated and characterized from the genome of Leuconostoc mesenteroides SY2, an isolate from kimchi, a Korean traditional fermented vegetable. Chromosomal DNA of L. mesenteroides SY2 was digested with Sau3AI and ligated with BamHI-cut pBV5030, a promoter screening vector containing a promoterless cat-86. Among E. coli transformants (TFs) resistant against Cm (chloramphenicol), 17 were able to grow in the presence of $1,000{\mu}g/ml$ Cm and their inserts were sequenced. Transcription start sites were examined for three putative promoters (P04C, P25C, and P33C) by primer extension. Four putative promoters were inserted upstream of a promoterless ${\alpha}$-amylase reporter gene in $pJY15{\alpha}$. ${\alpha}$-Amylase activities of E. coli TFs containing $pJY15{\alpha}$ (control, no promoter), $pJY03{\alpha}$ ($pJY15{\alpha}$ with P03C), $pJY04{\alpha}$ (with P04C), $pJY25{\alpha}$ (with P25C), and $pJY33{\alpha}$ (with P33C) were 66.9, 78.7, 122.1, 70.8, and 99.3 U, respectively. Cells harboring $pJY04{\alpha}$ showed 1.8 times higher activity than the control. Some promoters characterized in this study might be useful for construction of food-grade expression vectors for Leuconostoc sp. and related lactic acid bacteria.

• Journal of Veterinary Science
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• 제23권1호
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• pp.14.1-14.11
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• 2022

Background: Carnivore protoparvovirus 1, also known as canine parvovirus type 2 (CPV-2), is the main pathogen in hemorrhagic gastroenteritis in dogs, with a high mortality rate. Three subtypes (a, b, c) have been described based on VP2 residue 426, where 2a, 2b, and 2c have asparagine, aspartic acid, and glutamic acid, respectively. Objectives: This study examined the presence of CPV-2 variants in the fecal samples of dogs diagnosed with canine parvovirus in Bogotá. Methods: Fecal samples were collected from 54 puppies and young dogs (< 1 year) that tested positive for the CPV through rapid antigen test detection between 2014-2018. Molecular screening was developed for VP1 because primers 555 for VP2 do not amplify, it was necessary to design a primer set for VP2 amplification of 982 nt. All samples that were amplified were sequenced by Sanger. Phylogenetics and structural analysis was carried out, focusing on residue 426. Results: As a result 47 out of 54 samples tested positive for VP1 screening, and 34/47 samples tested positive for VP2 980 primers as subtype 2a (n = 30) or 2b (n = 4); subtype 2c was not detected. All VP2 sequences had the amino acid, T, at 440, and most Colombian sequences showed an S514A substitution, which in the structural modeling is located in an antigenic region, together with the 426 residue. Conclusions: The 2c variant was not detected, and these findings suggest that Colombian strains of CPV-2 might be under an antigenic drift.

• Journal of Plant Biotechnology
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• 제29권3호
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• pp.151-159
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• 2002

작물의 신품종 육성 과정에 있어서 선발은 육종의 성패를 좌우하는 중요한 과정이다. 하지만 개체가 나타내는 표현형은 유전적 요인과 환경적 요인이 동시에 작용하여 나타나기 때문에 유전적인 효과만 구분하여 선발하는 것은 매우 어렵다. 최근에 분자생물학 분야의 연구가 급속도로 발전함에 따라 분자 수준의 표지 인자를 유용 유전자의 간접선발 지표로 활용하여 선발 효율을 높일 수 있게 되었다. 작물의 유전자 지도 및 분자 표지인자는 작물 육종에 매우 유용하게 활용될 수 있다. 따라서 본 연구에서는 무에서 양친인 '835'와 'B$_2$'에서 유래한 여교잡 집단 82개체를 이용하여 RAPD 유전자군 지도를 작성하고 관련 마커를 탐색하여 육종에 이용하고자 연구를 수행하였다. Primer 375종류를 이용하여 양친, 835와 B$_2$ 사이의 다형화 밴드 128개를 찾았다. BC$_1$F$_1$집단 조사를 통해 MAPMAK ER/EXP를 이용하여 연관군 지도를 작성하였다. 분리 분석된 RAPD 표지인자 128개 중 126개는 멘델의 이론 분리비 1:1에 적합하였으며. 2개는 동형접합체 (모본형) 쪽으로 편중되어 분리되었다. LOD 3.0 수준에서 128개의 표지인자가 9개의 연관군으로 나뉘어졌고 전체거리는 1,688.3 cM이었으며, 표지인자 간 평균거리는 13.8 cM으로 Lefebvre 등 (1996)이 발표한 자료를 참고하여 무 genome 전체의 유전적 거리를 계산한 결과 무의 유전자지도는 무 genome전체의 68.7%~80.1%를 포함하는 것으로 추정할 수 있었다. RAPD 마커에서 문제시되는 재현성 문제를 검정하기 위해 이를 STS 마커로 변환하고자 OPE10 primer에 의해 증폭된 특정 밴드를 클로닝하고 염기서열을 분석한 다음 얻어진 염기서열을 기본으로 하여 primer를 제작하여 PCR 하였다. 그 결과 10mer인 OPE10을 이용하여 분석했을 때와 동일하였으며 목적 밴드 외 다른 밴드는 생성되지 않아 앞으로 분자 마커로서 충분히 이용될 수 있음을 보여주었다.

• Molecules and Cells
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• 제24권1호
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• pp.60-68
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• 2007

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The $(AG)_n$ motif was most common (23.1%), followed by the $(AAC)_n$ motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.

• 대한의생명과학회지
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• 제13권4호
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• pp.319-324
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• 2007

One-tube nested polymerase chain reaction (PCR) was evaluated for its efficacy in detecting Mycobacterium leprae in biopsy samples from leprosy patients. Primers were derived from the M leprae-specific element (RLEP) sequences which yield a 230 bp fragment. The specificity and the sensitivity of the one-tube nested PCR were compared with those of single PCR for detecting M leprae. The results showed that the one-tube nested PCR was about 100 times more sensitive than that of the single indicating the one-tube nested primer sets developed in this study can be an effective screening tool for the detection of M leprae in clinical diagnostic laboratories.

• Asian-Australasian Journal of Animal Sciences
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• 제17권3호
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• pp.423-427
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• 2004

Seventy-six animals including cattle, sheep, horses, 6 species of zoo animals were employed for collection of fresh feces in order to detect verotoxigenic Esherichia coli (VTEC) by safe, quick and sensitive PCR-based molecular methods. Bacterial cell disruption with bead-beating followed by bacterial DNA purification with hydroxyapatide chromatography and gel filtration allowed DNA preparation from animal feces with high recovery and purity. A mountain goat was firstly shown by PCR and sequencing to shed verotoxin 2 gene (vt2) that was used to generate vt2 probe and second primer set for nested PCR to attempt more sensitive detection. Most sensitive nested PCR revealed that 45% of tested cattle and 47% of tested zoo animals were VTEC-positive, while least sensitive normal PCR detected VTEC from none of these animals except a mountain goat. Moderately sensitive detection by PCR in combination with hybridization suggested that the VTEC density varied between the VTEC-positive cattle.

• 대한바이러스학회지
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• 제27권1호
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.