• 미생물학회지
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• 제35권3호
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• pp.205-210
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• 1999

Vibrio cholerae 는 사람에게 설사를 일으키는 병원성 세규닝며 본 연구에 이용된 V.cholerae KNIH002 는 국내의 설사질환 환자로부터 분리하였다. 콜레라 독소 검출용 프라이머를 이용하여 PCR 로 증폭한 산물을 탐침자로 이용하여 Southern hybridization을 실시한 결과 PstI 및 BglII로 이중절단된 4.5-kb 절편내에서 ctx 유전자가 존재함을 확인하였다. 따라서 염색체 DNA를 PstI 및 BglII로 절단 후 V. cholerae KNIH002 의 유전자 mini-libraries를 제조하였다. 그리고 동일 탐침자를 이용하여 colony hybridization을 실시한 결과 제조된 유전자 mini-libraries 로부터 신호를 나타내는 한 개의 클론을 선발하였다. 선발된 클로닝 지니는 플라스미드를 pCTX75 라 명명하였으며, 이 클론은 CHO 세포에 대한 세포 독력이 나타남을 확인하였다. 염기서열을 결정한 결과 클로닝된 플라스미드에는 ace 와 zot 유전자들은 각각 ATG 개시코돈과 TGA 종결코돈을 포함하여 291 bp와 1,200 bp 로 구성되어져 있었다. ace 유전자의 염기서열은 V.cholerae E7946 EI Tor Ogawa strain 이 것과 100% 일치하였다. 그러나 zot 유전자의 염기서열 및 아미노산 서열은 V. cholerae 395 Classical Ogawa strain 의 것과 각각 99% 및 98.8% 의 상동성을 보였다. 특히, V.cholerae 395 Classicale Ogawa strain 의 Zot 폴리펩타이드에서 100번, 272번, 281번째 alanine 은 V.cholerae KNIH002에서 모두 valine 으로 치환되어져 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제38권3호
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• pp.126-134
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• 2011

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence $in-situ$ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

• Asian-Australasian Journal of Animal Sciences
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• 제16권12호
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• pp.1837-1841
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• 2003

In an attempt to isolate novel molecules that may play a regulatory role in adipocyte differentiation, we devised an experimental strategy to identify adipose tissue-specific genes by modifying cDNA microarray technique. We used genefilter membranes containing approximately 15,000 rat non-redundant EST clones of which 4,000 EST were representative clones of known genes and 11,000 ESTs were uncharacterized clones. A series of hybridization of genefilter membranes with cDNA probes prepared from various rat tissues and nucleic acids sequence analysis allowed us to identify two adipose-tissue specific genes, adipocyte-specific secretory factor (ADSF) and H-rev107. Verification of tissue-specific expression patterns of these two genes by Northern blot analysis showed that ADSF mRNA is exclusive expressed in adipose tissue and the H-rev107 mRNA is predominantly expressed in adipose tissue. Further analysis of gene expression of ADSF and H-rev107 during 3T3-L1 adipocyte differentiation revealed that the ADSF and H-rev107 gene expression patterns are closely associated with the adipocyte differentiation program, indicating their possible role in the regulation of adipose tissue development. Overall, we demonstrated an application of modified cDNA microarray technique in molecular cloning, resulting in identification of two novel adipose tissue-specific genes. This technique will also be used as a useful tool in identifying novel genes expressed in a tissue-specific manner.

• Genomics & Informatics
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• 제14권1호
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• pp.34-40
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• 2016

Due to advances in omics technologies, numerous genome-wide studies on human samples have been published, and most of the omics data with the associated clinical information are available in public repositories, such as Gene Expression Omnibus and ArrayExpress. While analyzing several public datasets, we observed that errors in gender information occur quite often in public datasets. When we analyzed the gender description and the methylation patterns of gender-specific probes (glucose-6-phosphate dehydrogenase [G6PD], ephrin-B1 [EFNB1], and testis specific protein, Y-linked 2 [TSPY2]) in 5,611 samples produced using Infinium 450K HumanMethylation arrays, we found that 19 samples from 7 datasets were erroneously described. We also analyzed 1,819 samples produced using the Affymetrix U133Plus2 array using several gender-specific genes (X (inactive)-specific transcript [XIST], eukaryotic translation initiation factor 1A, Y-linked [EIF1AY], and DEAD [Asp-Glu-Ala-Asp] box polypeptide 3, Y-linked [DDDX3Y]) and found that 40 samples from 3 datasets were erroneously described. We suggest that the users of public datasets should not expect that the data are error-free and, whenever possible, that they should check the consistency of the data.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.194-199
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• 1995

A portion (7.4 kb) of ribosomal DNA tandem repeat unit from a genome of the mushroom T. matsutake has been cloned. A 1.75 kb EcoRI fragment was cloned first using S. cerevisiae 255 rRNA gene as a probe, and this was then used for further cloning. A chromosomal walking experiment was carried out and the upstream region of the 1.75 kb fragment was cloned using SmaI/BamHI enzyme, the size was estimated to be 5.2 kb in length. Part of the downstream region of the 1.75 kb fragment was also cloned using XbaI/BamHI enzymes. Restriction enzyme maps of three cloned DNA fragments were constructed. Northern hybridization, using total RNA of T. matsutake, and the restriction fragments of three cloned DNAs as probes, revealed that all four ribosomal RNA genes (large subunit[LSU], small subunit [SSU], 5.85 and 5S rRNA genes) are present in the cloned region. The gene organization of the rDNA are regarded as an intergenic spacer [IGS]2 (partial) - SSU rRNA - internal transcribed spacer [ITS]1 - 5.8S rRNA - ITS2 - LSU rRNA - IGS1 -5S rRNA - IG52 (partial).

• Food Science and Biotechnology
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• 제18권5호
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Journal of Microbiology
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• 제35권1호
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• pp.40-46
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• 1997

The pWHM220 cosmid with a 24-kb insert cloned from Streptomyces albus ATCC 21838 induces the biosynthesis of a polysther antibiotic similar to salinomycin in Streptomyces invidans. We have analyzed this region by DNA sequencing as well as Southern blot hybridization with type I and type II polyketide synthase (PKS) probes. Surprisingly, we found another set of type II SKS genes only 10-kb from the original PKS genes, salABCDE. The DNA sequence revealed two complete open reading frames (ORFs) named salB2 and salC2, and one partial ORF that does not resemble any known DNA or deduced protein sequence. The salC2 should code for chain length determining factor while the deduced amino acid sequence encoded by salB2 exhibits high similarity to .betha.-ketoacyl synthase from different PKS gene clusters. The highest identity was found for .betha.-keetoacyl synthases from S. argillaceus (MtmP. 59.1% identity), the mithramycin producer and from S. venezuelae ISP5230 (JadA, 52.3% identity), the jadomycin producer. The SalC2 protein clearly resembles its counterparts in order aromatic PKS gene clusters that are believed to influence the length of the polyketide chain. The highest identities observed were to that of S. argillaceus (MtmK, 62.3%) and S. venezuelae ISP 5230 (JadB, 55.1%) proteins, Moreover, the deduced amino acid sequences of the salB2 and salC2 products were 29.0% identical.

• Tuberculosis and Respiratory Diseases
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• 제58권2호
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• pp.129-134
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• 2005

배 경 : 에탐부톨의 내성여부는 결핵 환자 처방 결정에 있어서 중요 변수의 하나가 된다. 에탐부톨 내성의 상당부분이 embB 유전자의 돌연변이와 관계가 있으므로 역교잡반응법으로 이 유전자의 돌연변이를 신속하게 검출하고자 하였다. 방 법 : 에탐부톨 내성에 관련된 embB 유전자의 306번, 406번, 497번 아미노산의 정상적인 염기서열과 돌연변이 염기서열에 대한 probe를 합성하였고, 약제감수성검사에서 에탐부톨 내성균으로 나타난 149균과 전약제 감수성으로 나타난 50개균을 대상으로 조사하였다. 결 과 : 149개의 에탐부톨 내성균 중에서 embB 유전자 전체에서 돌연변이가 나타난 균은 100균주(67.1%)였으며, 그 중 embB 유전자 중 306번 돌연변이를 가진 균주가 75주(50.3%), 406번 돌연변이를 가진 균주가 16주(10.7%), 497번 돌연변이가 있는 균주가 13주(8.7%)였다. 이 중 4균주는 306번과 406번 돌연변이를 동시에 가지고 있었다. 406번 돌연변이 하나만 가지고 있는 균은 12균주(8.1%)로 이는 다른 조사에서 볼 수 없었던 비교적 높은 수치이었다. 한편 에탐부톨 및 11가지 항결핵약제에서 감수성인 50개균에서는 embB 유전자의 돌연변이를 발견하지 못하였다. 결 론 : 역교잡반응법으로 에탐부톨 내성에 관련되어 있는 것으로 알려진 embB 유전자 돌연변이를 찾는 것이 가능하였으며, 에탐부톨 내성기전 및 관련 유전자가 더 발견되어 민감도가 향상된다면 신속한 에탐부톨내성균 검출이 가능할 것으로 본다.

• 환경생물
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• 제20권3호
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• pp.189-196
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• 2002

The direct detection of intestinal pathogens and viruses often requires costly, tedious, and time-consuming procedures. These requirements developed a test to show that the water was contaminated with sewage-borne pathogens by assessing the hygienic quality of water based on indicator microorganisms whose presence indicates that pathogenic microorganisms may also be present. Various groups of microorganisms have been suggested and used as indicator microorganisms. Proposed and commonly used microbial indicators are total coliforms, fecal coliforms, fecal streptococci, Clostridium perfringens, heterotrophic plate count, bacteriophage, and so on. Unfortunately, most, if not all, of these indicators are not ideal because of the sensitivity and resistance to environment stresses and disinfection. However, the development of gene probes and PCR technology may give hope for the discovery of rapid and simple methods toy detecting small number of fecal pathogens in various environments.

• 대한수의학회지
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• 제32권3호
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• pp.389-398
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• 1992

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus(BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone(No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3'-end. $^{32}P$-labeled DNA probes of 300~1,800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EcoR I, Sst I, Hin d III and Pst I restriction enzymes in the DNA fragment.