• Title/Summary/Keyword: DNA-DNA hybridization

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Cytogenetic Characteristics of Chinese Hamster Ovarian Cell CHO-K1

  • Sohn, Sea-Hwan;Cho, Eun-Jung;Jang, In-Surk
    • Reproductive and Developmental Biology
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    • v.30 no.4
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    • pp.263-270
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    • 2006
  • The Chinese Hamster Ovarian cells CHO-K1 are one of the most extensively used cells for the evaluation of gene expression and toxicology. However, these cells are frequently used for biomedical research without consideration of their cytogenetic characteristics. Therefore, we carried out to investigate the karyologic profiles, the frequency and type of chromosome aberration, and the distribution of telomeric DNA on chromosomes of the CHO-K1 cells. The GTG banding and fluorescence in situ hybridization on CHO-K1 cells were performed to characterize the karyotype and the distribution of telomeric DNA The present study revealed that the chromosome modal number of CHO-K1 cells was 2n=20; eight chromosomes appeared to be identical with those of the normal Chinese hamster, whereas the remaining 12 chromosomes were shown to be translocated, deleted, inversed, or rearranged from Chinese hamster chromosomes. The telomeric DNA on CHO-K1 chromosomes was intensively distributed at the centromeres rather than the ends of chromosomes. In addition, three chromosomes had interstitial telomeres and one marker chromosome entirely consisted of telomeric DNAs. The frequency and type of chromosome aberrations in CHO-K1 cells were examined. Of the 822 metaphase spreads, 68 (8.3%) cells resulted in chromosome aberrations of which the chromosome breakage was the most frequently occurred.

Molecular and Cellular Studies of Seed Storage Proteins from Rice and Wheat

  • Kim, Woo-Taek
    • Applied Biological Chemistry
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    • v.32 no.1
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    • pp.64-72
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    • 1989
  • Near full length cDNA clones encoding the rice seed storage protein, prolamine, were isolated and divided into two homology classes based on cross-hybridization and DNA sequencing analysis. These cDNA clones contain a single open reading frame encoding a putative rice prolamine precursor(M.W.=17,200) possessing atypical 14 amino acid signal peptide. Clones of these two homology classes diverge mainly by insertions/deletions of short nucleotide stretches and point mutations. The deduced primary structures of both types of prolamine polypeptides are devoid of any major tandem repetitive sequences, a feature prevalent in other cereal prolamines. No significant homology teas detected between the rice prolamine and other cereal prolamines, indicating that the rice gene evolved from a different ancestor that gave rise to other cereal prolamine genes. Developing wheat and rice endosperms were examined using ultrathin sections prepared from tissues harvested at various days after flowering. By immunocytochemical localization techniques, wheat prolamines are localized within vesicles from Golgi apparatus and in homogeneous regions of protein bodies. The involvement of the goli apparatus in the packaging of wheat prolamines into protein bodies indicates a pathway which differs from the mode of other cereal prolamines and resembles the mechanism employed for the storage of rice glutelin and legume globulins.

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Characterization of a Low Molecular Weight Heat-Shock Protein cDNA Clone from Nicotiana tabacum

  • Park, Soo-Min;Joe, Myung-Kuk;Hong, Choo-Bong
    • Proceedings of the Botanical Society of Korea Conference
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    • 1999.04a
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    • pp.18-18
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    • 1999
  • We characterized a cDNA clone for a low molecular weight heat-shock protein (LMW HSP) from tobacco named TLHS-l. Nucleotide sequence determination of TLHS-1 identified an open reading frame for 159 amino acids. To the upstream of the open reading frame, a sequence of 124 nucleotides was determined. To the 3' downstream of the open reading frame, 212 nucleotides were identified which carried poly(A)-tail. Comparison of the open reading frame and hydropathy plot of TLHS-1 with the previously reported class I LMW HSPs showed high identity which classified TLHS-1 as a class I LMW HSP cDNA clone. We proposed that there are six consensus regions in class I LMW HSPs. RNA blot hybridization for TLHS-1 showed a typical expression pattern of heat-shock-inducible gene from three common tobacco cultivars. The open reading frame of TLHS-1 was overexpressed in Escherichia coli. TLHS-1 protein confers thermal protection of other proteins in vitro and in vivo. Thermal induced aggregation of citrate synthase was reduced by purified TLHS-1 protein, and thermal death rate at $50^{\circ}C$ was reduced in E. coli expressing TLHS-l. From these data, we can expect that TLHS-1 acts as a molecular chaperone.perone.

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Cloning and Characterization of Highly Repetitive Sequences in the Genome of Allium sativum L. (마늘(Allium sativum L.) 게놈의 고반복서열의 분이와 특성 조사)

  • 이동희
    • Journal of Plant Biology
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    • v.39 no.1
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    • pp.49-55
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    • 1996
  • We have studied the DNA of Allium sativum L. with respect to highly repetitive sequences. Fast reassociated DNA fragments expected to be highly repetitive sequences based on $C_{o}t$ curve were isolated and characterized. Their copy numbers were approximately $10^{5}~10^{7}$ per haploid genome. Nucleotide sequences analysis of six candidates reveals that their G/C content were low, 25-40% and typical patterns of repeating sequences exist. Repeat sequences were used as probes to access restriction fragment length polymorphism (RFLP) of genomic DNAs of four local clones, Tanyang, Mungyong, So san, and Uisong. The hybridization pattern were very similar among these four local clones.clones.

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Nucleotide sequence analysis of a second set of the polyketide synthase .betha.-ketoacyl synthase and chain length factor genes from the salinomycin-producing streptomyces albus

  • Hyun, Chang-Gu;Park, Kwan-Hyung;C.Richard Hutchinson;Suh, Joo-Won
    • Journal of Microbiology
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    • v.35 no.1
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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.

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Genetic testing in clinical pediatric practice

  • Yoo, Han Wook
    • Clinical and Experimental Pediatrics
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    • v.53 no.3
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    • pp.273-285
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    • 2010
  • Completion of the human genome project has allowed a deeper understanding of molecular pathophysiology and has provided invaluable genomic information for the diagnosis of genetic disorders. Advent of new technologies has lead to an explosion in genetic testing. However, this overwhelming stream of genetic information often misleads physicians and patients into a misguided faith in the power of genetic testing. Moreover, genetic testing raises a number of ethical, legal, and social issues. Diagnostic genetic tests can be divided into three primary but overlapping categories: cytogenetic studies (including routine karyotyping, high-resolution karyotyping, and fluorescent in situ hybridization studies), biochemical tests, and DNA-based diagnostic tests. DNA-based testing has grown rapidly over the past decade and includes preandpostnatal testing for the diagnosis of genetic diseases, testing for carriers of genetic diseases, genetic testing for susceptibility to common non-genetic diseases, and screening for common genetic diseases in a particular population. Theoretically, once a gene's structure, function, and association with a disease are well established, the clinical application of genetic testing should be feasible. However, for routine applications in a clinical setting, such tests must satisfy a number of criteria. These criteria include an acceptable degree of clinical and analytical validity, support of a quality assurance program, possibility of modifying the course of the diagnosed disease with treatment, inclusion of pre-and postnatal genetic counseling, and determination of whether the proposed test satisfies cost-benefit criteria and should replace or complement traditional tests. In the near future, the application of genetic testing to common diseases is expected to expand and will likely be extended to include individual pharmacogenetic assessments.

Molecular Characterization of an Apple cDNA Encoding Cinnamyl Alcohol Dehydrogenase

  • Kim, Sung-Hyun;Lee, Jae-Rin;Shin, Yong-Uk;An, Gyn-Heung;Kim, Seong-Ryong
    • Journal of Microbiology and Biotechnology
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    • v.9 no.4
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    • pp.475-481
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    • 1999
  • The study of lignin, a major component of secondary cell wall, has been partly focused on its removal from the woody part in the kraft pulping industry. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.l95) catalyzes the synthesis of cinnamyl alcohols from corresponding cinnamaldehydes. A cDNA clone, MdCADl, encoding putative CAD from apples (Malus domestica Borkh. cv Fuji) was characterized in this study. The clone contains an open reading frame of 325 amino acid residues, which shows a greater than 80% identity with Eucalyptus CADl. MdCADl mRNA was detectable in vegetative tissues and was strongly expressed in the fruit. The expression pattern of MdCADl mRNA in the fruit peel after light exposure was also examined. The mRNA was rapidly increased until 1 day after light exposure and remained stable thereafter, suggesting that MdCADl is light inducible. The inducibility of the MdCADl gene was examined using several environmental stresses. Mechanical wounding of leaves increased the MdCADl mRNA level and the induction was further increased by salicylic acid. Southern blot hybridization showed that there is either one or a few copies of CAD genes in apples. To our knowledge, it is believed that MdCADl is the first CAD clone expressed predominantly in fruit.

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Quantitative analysis using decreasing amounts of genomic DNA to assess the performance of the oligo CGH microarray

  • Song Sunny;Lazar Vladimir;Witte Anniek De;Ilsley Diane
    • Proceedings of the Korean Society for Bioinformatics Conference
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    • 2006.02a
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    • pp.71-76
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    • 2006
  • Comparative genomic hybridization (CGH) is a technique for studying chromosomal changes in cancer. As cancerous cells multiply, they can undergo dramatic chromosomal changes, including chromosome loss, duplication, and the translocation of DNA from one chromosome to another. Chromosome aberrations have previously been detected using optical imaging of whole chromosomes, a technique with limited sensitivity, resolution, quantification, and throughput. Efforts in recent years to use microarrays to overcome these limitations have been hampered by inadequate sensitivity, specificity and flexibility of the microarray systems. The oligonucleotide CGH microarray system overcomes several scientific hurdles that have impeded comparative genomic studies of cancer. This new system can reliably detect single copy deletions in chromosomes. The system includes a whole human genome microarray, reagents for sample preparation, an optimized microarray processing protocol, and software for data analysis and visualization. In this study, we determined the sensitivity, accuracy and reproducibility of the new system. Using this assay, we find that the performance of the complete system was maintained over a range of input genomic DNA from 5 ug down to 0.15 ug.

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Genotyping of Brucella abortus isolated from cattle in Gyeongbuk province by MLVA (MLVA를 이용한 경북지역 소에서 분리된 Brucella abortus의 유전형별)

  • Kim, Seong-Guk;Kim, Young-Hoan;Lee, Hong-Young;Choi, Jeong-Hye;Choi, Seong-Kyoon
    • Korean Journal of Veterinary Service
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    • v.34 no.3
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    • pp.227-234
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    • 2011
  • Brucella (B.) abortus is a facultative intracellular pathogen that infects a wide variety of animal species and human. Brucellosis is the zoonosis and an extremely important disease around the world. Although the eight species can be differentiated by conventional phenotypic tests, these species display a high degree of DNA homology in DNA-DNA hybridization assay (>90%). Various methods have been established for genotyping of Brucella species, but most of analytical methods are lack reproducibility and limited capability to differentiate them. The attempt of this study was to evaluate multiple-locus VNTR analysis (MLVA) for use of epidemiological trace-back analysis in bovine brucellosis. Ninety-four B. abortus isolates from Gyeongbuk province during 2006~2010 were analyzed using 16 VNTR locus. High resolution automatic capillary electrophoresis system was used for more throughput, simpleer, faster, and better discriminable than conventional gel electrophoresis. As a result, 13 different genotypes were identified from 94 B. abortus isolates. MLVA could contribute to epidemiological trace-back analysis of bovine brucellosis.

Distribution of Electrochemically Active Bacteria in Activated Sludge Characteristics (활성슬러지내의 전기화학적활성 박테리아 분포 특성)

  • Son, Hyeng-Sik;Son, Hee-Jong;Kim, Mi-A;Lee, Sang-Joon
    • KSBB Journal
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    • v.26 no.5
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    • pp.407-411
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    • 2011
  • Microbial fuel cell (MFC) wes enriched using sludge in wastewater treatment. The microbial community of activated sludge and enriched MFC were analyzed by FISH (fluorescent in situ hybridization) and 16S rDNA sequencing. Bacteroidetes group were pre-dominant in activated sludge by FISH. ${\alpha}$ group, ${\gamma}$ group and Acintobacter group were dominant and they were similar to distribution. The average value of 10 peak of MFC is 0.44C. When MFC wase enriched by sludge, ${\gamma}$-Proteobacteria, Plantomycetes group increased 70% and 60%, respectively. In results of 16S rDNA sequencing, Sphiringomonas sp. was comprised in ${\alpha}$ proteobacteria and Enterobacter sp., Klebsiella sp., Acinetobacter sp., Bacillus sp. were comprised in ${\gamma}$ proteobacteria and Chryseobacterium sp. was comprised in Flavobacteria were isolated from sludge.