• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.197-206
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• 2016

Listeria monocytogenes is a foodborne pathogen of considerable genetic diversity with varying pathogenicity. Initially, we found that the strain M7 was far less pathogenic than the strain Lm850658 though both are serovar 4a strains belonging to the lineage III. Comparative genomic approaches were then attempted to decipher the genetic basis that might govern the strain-dependent pathotypes. There are 2,761 coding sequences of 100% nucleotide identity between the two strains, accounting for 95.7% of the total genes in Lm850658 and 92.7% in M7. Lm850658 contains 33 specific genes, including a novel 20K prophage whereas strain M7 has 130 specific genes, including two large prophages (38K and 44K). To examine the roles of these specific prophages in pathogenicity, the 20K and 38K prophages were deleted from their respective strains. There were virtually no differences of pathogenicity between the deletion mutants and their parent strains, although some putative virulent factors like VirB4 are present in the 20K region or holin-lysin in the 38K region. In silico PCR analysis of 29 listeria genomes show that only strain SLCC2540 has the same 18 bp integration hotspot as Lm850658, whereas the sequence identity of their 20K prophages is very low (21.3%). The 38K and 44K prophages are located in two other different hotspots and are conserved in low virulent strains M7, HCC23, and L99. In conclusion, the 20K and 38K prophages of L. monocytogenes serovar 4a strains Lm850658 and M7 are not related to virulence but contribute to genetic diversity.

• 원예과학기술지
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• 제33권2호
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• pp.242-250
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• 2015

기능 유전체 연구에서 이동유전자 또는 T-DNA 도입을 이용한 삽입돌연변이체 분석은 미지의 유전자 기능을 분석할 수 있게 한다. 이에 따라 본 연구에서는 배추(Brassica rapa ssp. pekinensis)와 같은 고등 식물에서 genomic DNA조각을 분리할 수 있는 효과적인 PCR 방법을 개발하였다. 본 연구에서 개발한 variable argument thermal asymmetric interlaced PCR(VA-TAIL PCR)은 single-step annealin-gextension PCR 방법과 길게 구성된 유전자 특이적 primer 및 touch-up PCR 방법이 적용되었으며, 증폭 효율을 증가시키기 위하여 autosegment extension 증폭 방법이 적용되었다. 또한 개발된 VA-TAIL PCR 방법은 배추에 특화된 변성 primer를 Integr8 단백질체 데이터베이스를 분석하여 작성하였으며 대량의 배추 T-DNA 삽입 돌연변이 집단에서 각각의 T-DNA 인접 염기 서열을 분석하는 데 매우 정확하고 효과적인 것으로 분석되었다. 따라서 본 연구에서 개발된 VA-TAIL PCR 방법은 기존에 확인된 염기 서열을 이용하여 양쪽 방향을 모두 분석할 수 있기 때문에, 유전체 연구에서 T-DNA 또는 기 밝혀진 염기 서열에 인접한 미지의 염기서열을 확인하는데 매우 효과적일 것으로 기대된다.

• 한국전자통신학회논문지
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• 제8권12호
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• pp.1875-1882
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• 2013

마이크로 어레이 기술은 유전자 네트워크의 순서 와 게놈의 통합과 같은 많은 업적을 기여하고 있으며, 이러한 기술은 유전자 발현의 패턴을 조사하기 위한 수단 등으로 잘 확립 되어있다. DNA 마이크로배열은 Affymetric 칩을 이용하여 대량의 DNA 서열을 합성 할 수 있는데 기존의 DNA 어레이 스포팅에는 일반적으로 접촉방식과 압전전자 방법등 두가지 유형이 있다. 접촉방법은 유리 슬라이드 표면과 접촉하도록 스포팅핀을 사용하는데 이 방법은 표면 매트릭스의 손상이나 상처가 발생할 수 있어 단백질이 오염 되거나 특정 결합을 방해할 위험이 있다. 반면에 압전전자 방법은 대량 생산이 가능함에도 불구하고 결과를 인쇄할 분석기가 필요하므로 현재 실험실 내에서만 수행 가능한 실정이다. 본 논문에서 유리 슬라이드 표면에 닿지 않고 지속적으로 일관성 있게 스포팅이 가능하도록 하는 진보된 방법을 제시한다.

• Molecular & Cellular Toxicology
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• 제4권3호
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• pp.260-266
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• 2008

Recently, there has been scientific discussion on the utility of -omics techniques such as genomics, proteomics, and metabolomics within toxicological research and mechanism-based risk assessment. Toxicogenomics is a novel approach integrating the expression analysis of genes (genomic) or proteins (proteomic) with traditional toxicological methods. Since 1999, the toxicogenomic approach has been extensively applied for regulatory purposes in order to understand the potential toxic mechanisms that result from chemical compound exposures. Therefore, this article's purpose was to consider the utility of toxicogenomic profiles for improved risk assessment, explore the current limitations in applying toxicogenomics to regulation, and finally, to rationalize possible avenues to resolve some of the major challenges. Based on many recent works, the significant impact toxicogenomic techniques would have on human health risk assessment is better identification of toxicity pathways or mode-of-actions (MOAs). In addition, the application of toxicogenomics in risk assessment and regulation has proven to be cost effective in terms of screening unknown toxicants prior to more extensive and costly experimental evaluation. However, to maximize the utility of these techniques in regulation, researchers and regulators must resolve many parallel challenges with regard to data collection, integration, and interpretation. Furthermore, standard guidance has to be prepared for researchers and assessors on the scientifically appropriate use of toxicogenomic profiles in risk assessment. The National Institute of Toxicological Research (NITR) looks forward to an ongoing role as leader in addressing the challenges associated with the scientifically sound use of toxicogenomics data in risk assessment.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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• pp.19-26
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• 2001

Transformed Spodoptera frugiperda (Sf9) cells expressing baculovirus very late factor (VLF-1) were constructed by using Autograha nuclear polyhedrosis virus (AcNPV) immediate earthy gene (ie1). Neomycin-resistance gene as a selectable marker was introduced under the control of AcNPV ie1 promoter, and Bombyx mori nuclear polyhedrosis (BmNPV-K1) vlf-1 gene was introduced under the control of the Drosophila heat shock protein gene (hspr70) promoter to yield dual expression plasmid with two independent transcription units. It was transfected into Sf9 cells and cell clones expressing vlf-1 were selected by G4l8 treatment. Genomic DNA from transformed cells was isolated and integration of AcNPV iel harboring vlf-1 was confirmed by PCR using AcNPV iel-specific primers and Southern blot analysis. The transformed cells expressing VLF-1 in an infection-independent manner expressed foreign gene product of recombinant baculovirus in the earlier stage of infection compared with control Sf9 cells. These results suggest the possible to develop highly efficient transformed insect cells for baculovirus expression vector system.

• Journal of Plant Biotechnology
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• 제7권2호
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• pp.97-103
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• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2489-2493
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• 2015

Background: Loss of heterozygosity (LOH) on chromosomal regions is crucial in tumor progression and this study aimed to identify genome-wide LOH in pancreatic cancer. Materials and Methods: Single-nucleotide polymorphism (SNP) profiling data GSE32682 of human pancreatic samples snap-frozen during surgery were downloaded from Gene Expression Omnibus database. Genotype console software was used to perform data processing. Candidate genes with LOH were screened based on the genotype calls, SNP loci of LOH and dbSNP database. Gene annotation was performed to identify the functions of candidate genes using NCBI (the National Center for Biotechnology Information) database, followed by Gene Ontology, INTERPRO, PFAM and SMART annotation and UCSC Genome Browser track to the unannotated genes using DAVID (the Database for Annotation, Visualization and Integration Discovery). Results: The candidate genes with LOH identified in this study were MCU, MICU1 and OIT3 on chromosome 10. MCU was found to encode a calcium transporter and MICU1 could encode an essential regulator of mitochondrial $Ca^{2+}$ uptake. OIT3 possibly correlated with calcium binding revealed by the annotation analyses and was regulated by a large number of transcription factors including STAT, SOX9, CREB, NF-kB, PPARG and p53. Conclusions: Global genomic analysis of SNPs identified MICU1, MCU and OIT3 with LOH on chromosome 10, implying involvement of these genes in progression of pancreatic cancer.

• 정보처리학회논문지D
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• 제18D권2호
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• pp.89-100
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• 2011

인간 유전체에 존재하는 유전적 구조 변이(genetic structural variation) 중 하나인 유전체 단위반복변이(Copy Number Variation, CNV)은 유전자의 기능 발현과 밀접한 관련이 있다. 특히 특정 유전 질병이 있는 사람들을 대상으로 CNV와 유전자발현의 관계를 밝히는 연구가 계속 진행되고 있지만, 정상인 유전체에 대한 CNV의 기능적 분석은 아직 활발히 이루어지고 있지 않다. 본 논문에서는 다수의 정상인 샘플에서 찾아낸 공통된 CNV에 대하여 유전자들과의 기능적 관계를 유전자의 분자적 위치와 상관없이 밝힐 수 있는 분석 방법을 제시한다. 이를 위해 서로 다른 이질적인 생물학데이터를 통합하는 방법을 제시하고 공통된 CNV와 유전자와의 연관성을 분자적 위치와 상관없이 계산할 수 있는 새로운 방법을 제시한다. 제안된 방법의 유의성을 보이기 위해서 유전자 온톨로지 (Gene Ontology) 데이터베이스를 이용한 다양한 검증 실험들을 수행하였다. 실험결과 새롭게 제안된 연관성 측정방법은 유의성이 있으며 공통된 CNV와 강한 연관성을 갖는 유전적 기능의 후보들을 시스템적으로 제시할 수 있는 것으로 나타났다.

• Fisheries and Aquatic Sciences
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• 제18권1호
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• 한국작물학회지
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• 제49권1호
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• pp.61-68
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• 2004

A reproducible transformation system via optimized regeneration media for Korean rice cultivars was established using Agrobacterium tumefeciens LBA4404 (pSBM-PPGN; gusA and bar). Although japonica rice genotypes were easier to produce transgenic plants compared to Tongil type cultivars, transformation efficiencies were not always correlated with regeneration efficiencies of non-transgenic callus on the control medium. Regeneration efficiencies of Donganbyeo, Ilmibyeo, and Manchubyeo were over 50% in non-transgenic control, however, transformation efficiencies were significantly low when only sucrose was added to the media as a carbon source. However, the medium, MSRK5SS-Pr (or MSRK5SM-Pr), that contains $5\textrm{mgL}^{-1}$ kinetin, $0.5\textrm{mgL}^{-1}$ NAA, 2 % sucrose (or maltose), 3% sorbitol, and $500\textrm{mgL}^{-1}$ proline, was the most efficient not only for regeneration of non-transgenic callus but also for regeneration of transgenic callus in the presence of L-phosphinotricin (PPT). Average transformation efficiencies of 16 Korean rice cultivars were significantly enhanced by using the optimized medium from 1.5% to 5.8% in independent callus lines and from 2.9% to 19.4% in tromsgenic plants obained. Approximately 98.9% (876 out of 885) transgenic plants obtained on optimized media showed basta resistance. Stable integration, inheritance and expression of gusA and bar genes were continued by GUS assay and PCR and Southern analysis of the bar gene. With Pst1 digestion of genomic DNA of transgenic plants, one to five copies of T-DNA segment were observed; however, 76% (19 out of 25 transgenic plants) has low copy number of T-DNA. The transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.