• Title/Summary/Keyword: DNA isolation

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Isolation and Characterization of New Family Genes of DNA Damage in Fission Yeast

  • Choi, In-Soon
    • Environmental Mutagens and Carcinogens
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    • v.19 no.1
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    • pp.28-33
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    • 1999
  • The SNF2 family includes proteins from a variety of species with roles I cellular processes such as transcriptional regulation, recombination and various types of DNA repair. Several proteins with unknown function are also included in this family. Here, we report the cloning and characterization of hrp 2+ gene (helicase related gene from S. pombe) which was isolated by PCR amplication using the conserved domain of SNF2 motifs within the ERCC6 gene which encodes a protein involved in DNA excision repair. The hrp2+ gene was isolated by screening with yeast S. pombe genomic library. The isolated cloned contained 6.5 kb insert DNA. Southern blot analysis confirmed that S. pombe chromosome contains the same DNA as hrp2+ gene and this gene exists as a single copy in S. pombe genome. The 4.7 kb transcript of mRNA was identified by Northern blot. To examined the transcriptional regulation of hrp2+ gene, DNA damaging agents were treated. These results indicated that the hrp2+ gene may not be directly involved in DNA replication, but may be involved in damage response pathway.

Cloning of Bovine Macrophage Colony-stimulating Factor

  • Kim, Tae-Yung;Kim, Cheol-Ho;Lee, Sang-Gil;Kang, Chung-Boo
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.6
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    • pp.892-897
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    • 2005
  • Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

Isolation of a cDNA Encoding a Chloroplast Triosephosphate Isomerase from Strawberry

  • Kim, In-Jung;Lee, Byung-Hyun;Jinki Jo;Chung, Won-Il
    • Journal of Plant Biotechnology
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    • v.2 no.3
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    • pp.115-121
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    • 2000
  • A cDNA clone encoding chloroplast triosephosphate isomerase (TPI-cp) was isolated from strawberry fruit cDNA library. Sequence analyses indicated that the cDNA contains an open reading frame of 314 amino acids (33.5 kDa) composed of a transit peptide (59 amino acids) in amino terminal region and mature protein (255 amino acids). The existence of transit peptide in the deduced amino acid sequence implies that it encodes a chloroplast isoform. The protein sequence is more similar to other plant chloroplast isoforms than cytosolic isoforms. RNA blot analysis indicated that its expression is ubiquitous in examined five tissues, flowers, leaves, petioles, roots and fruits, and shows differential pattern according to fruit ripening. Genomic DNA blot analysis showed that TPI-cp is encoded by multiple genes in strawberry. Through sequence comparison and phylogenetic tree construction, TPI-cp is distinctively grouped into dicot and chloroplast isoforms.

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Mitochondrial DNA polymorphism in Fourteen Geographical Strains of Drosophila melanogoater (세계 14지역 계통에 대한 초파리 미토콘드리아 DNA의 다형현상)

  • 김봉기
    • The Korean Journal of Zoology
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    • v.31 no.3
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    • pp.218-224
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    • 1988
  • Restriction endonucleases were used to search for intraspecific variation at 32 cleavage sites in mitochondrial DNA(mtDNA) purified from fourteen strains of Drosophila melanogaster helonging to different localities of the world. mtDNA of D. melanogaster was displayed site variation(Hpall, Haelll and Seal endonucleases) and length variation(maxirnum 550bp). Six genotypes, Ml, M2, M3, M4, M6 and M7, could be distinguished based on ihe site types witti a low average of intraspecific substitution rate (1.88%),but M5 type of Ogasawara strain in Japan was not detected in this study. A possible explanation for the low divergence was that mtDNA variation of fourteen strains in D. melanogaster could not he accumulated sufficiently owing to recent divergence of few individuals, and that sequence divergence was prevented by frequent migration in spite of the geographical isolation.

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Isolation, Identification and Characterization of a Antidementia Acetylcholinesterase Inhibitor-Producing $Yarrowia$ $lipolytica$ S-3

  • Kang, Min-Gu;Yoon, Min-Ho;Choi, Young-Jun;Lee, Jong-Soo
    • Mycobiology
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    • v.40 no.1
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    • pp.42-46
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    • 2012
  • This report describes the isolation and identification of a potent acetylcholinesterase (AChE) inhibitor-producing yeasts. Of 731 species of yeast strain, the S-3 strain was selected as a potent producer of AChE inhibitor. The selected S-3 strain was investigated for its microbiological characteristics. The S-3 strain was found to be short-oval yeast that did not form an ascospore. The strain formed a pseudomycelium and grew in yeast malt medium containing 50% glucose and 10% ethanol. Finally, the S-3 strain was identified by its physiological characteristics and 26S ribosomal DNA sequences as $Yarrowia$ $lipolytica$ S-3.

Isolation of Proteinase Inhibitor II Genes from Potato (감자로부터 단백질분해효소 억제제 II 유전자의 분리)

  • 이종섭
    • Journal of Plant Biology
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    • v.32 no.2
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    • pp.79-87
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    • 1989
  • Southern hybridization of genomic DNAs with radioactively labeled cDNA of tomato proteinase inhibitor II revealed that proteinase inhibitor II proteins in potato plants are encoded by a family of about 10 related sequences. Screening of potato EcoRI genomic library with the cDNA resulted in isolation of 13 recombinant phage clones which carry 3 different genomic regions. Of these clones, clones 8, 18, and 39 were subjected to restriction mapping and subcloning. Further characterization of the subclones of clones 8, 18 and 39 indicated that two inhibitor II genes are present on a 8.0 kb EcoRI fragment of clone 8, one on 3.3 and 0.8 kb EcoRI fragments of clone 18 and two genes on a 13.5 kb EcoRI fragment of clone 39.

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Comparison of Two PCR Assays for Trichomonas vaginalis

  • Noh, Chang-Suk;Kim, Sang-Su;Park, Sung-Yul;Moon, Hong-Sang;Hong, Yeonchul;Ryu, Jae-Sook
    • Parasites, Hosts and Diseases
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    • v.57 no.1
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    • pp.27-31
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    • 2019
  • PCR is known to be the most sensitive method for diagnosing Trichomonas vaginalis infections. This study aimed to compare the sensitivity of a PCR assay for trichomoniasis (HY-PCR) developed in Hanyang University with the use of a Seeplex Ace Detection $Kit^{(R)}$, using urine collected from four Korean men with prostatic disease. Overall, HY-PCR was more sensitive than the Seeplex Kit. The use of Chelex 100 is recommended for DNA isolation in order to increase the sensitivity of the PCR test.

Development of a Highly Efficient Isolation Protocol for Mitochondrial DNA and RNA Using Small Scale Plant Tissues (식물의 초경량 조직을 이용한 미토콘드리아의 DNA와 RNA 정제)

  • Kim Kyung-Min;Lim Yong-Suk;Shin Dong-Ill;Sul Ill-Whan
    • Journal of Life Science
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    • v.16 no.2 s.75
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    • pp.240-244
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    • 2006
  • We present a fast and simple protocol for purification of mitochondria, mitochondrial DNA, and RNA from small amounts of tomato leaves. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. Mitochondria was confirmed by MitoTracker. The mitochondrial DNA was not contaminated by plastid DNA, was successfully used for PCR. Similarly, the isolated mitochondrial RNA was not contaminated only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for RT-PCR analysis

Study on isolation of Prevotella nigrescens 9336- specific DNA probes using random cloning method (무작위 클로닝법을 이용한 Prevotella nigrescens 9336 특이 DNA 프로브의 개발에 관한 연구)

  • Gang, Soon-Won;Kim, Se-Hoon;kim, Dong- Ki;Seong, Jin-Hyo;Kim, Byung-Ock;Kim, Jung- Ki
    • Journal of Periodontal and Implant Science
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    • v.32 no.2
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    • pp.269-280
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    • 2002
  • The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.