• 제목/요약/키워드: Genomic Cloning

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Reprogramming of Cloned Embryos During Early Embryogenesis (초기 발생에 있어서 복제수정란의 리프로그래밍)

  • Han, Yong-Mahn;Kang, Yong-Kook;Koo, Deog-Bon;Lee, Kyung-Kwang
    • 대한생식의학회:학술대회논문집
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    • 2002.05a
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    • pp.11-17
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    • 2002
  • Animal clones derived from somatic cells have been successfully produced in a variety of mammalian species such as sheep, cattle, mice, goats, pigs, cat and rabbits. However, there are still many unsolved problems in the present cloning technology. Somatic cell nuclear transfer has shown several developmental aberrancies including high rate of abortion in early gestation and increased perinatal death. These developmental failures of cloned embryos may arise from abnormal reprogramming of donor genome and/or incomplete cloning procedure. We have found that overall genomic methylation status of cloned bovine embryos is quite different from that of normal embryos in various genomic regions, suggesting that the developmental failures of cloned embryos may be due to incomplete reprogramming of donor genomic DNA. Many of the advances in understanding the molecular events for reprogramming of donor genome will more clarify the developmental defects of cloned embryos.

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Cloning and Heterologous Expression of Acetyl Xylan Esterase from Aspergillus ficuum

  • Jeong, Hye-Jong;Park, Seung-Mun;Yang, Mun-Sik;Kim, Dae-Hyeok
    • 한국생물공학회:학술대회논문집
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    • 2000.04a
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    • pp.153-156
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    • 2000
  • Xylan, the major hemicellulose component of many plants, occurs naturally in a partially acetylated form and lignin, the most resistant component in plant cell wall degradation, is also attached to ${\beta}-1,4-linked-D-xylose$ backbone through the ester linkage. Esterases are required to release the esterified substituent and acetyl esterases are important in the complete degradation of acetylated polysaccharides, like pectins and xylans. The gene(Axe) encoding acetyl xylan estarase(AXE) was isolated from genomic ${\lambda}$ library from Aspergillus ficuum. Nucleotide sequencing of the Axe gene indicated that the gene was separated with two intervening sequences and the amino acid sequence comparison revealed that it was closely related to that from A. awamori with the 92 % indentity. Heterologous expression of AXE was conducted by using YEp352 and Saccharomyces cerevisae 2805 as a vector and host expression system, respectively. The Axe gene was placed between GAL1 promoter and GAL7 terminator and then this recombinant vector was used to transform S. cerevisiae 2805 strain. Culture filtrate of the transformed yeast was assayed for the presence of AXE activity by spectrophotometry and, comparing with the host strain, four to five times of enzyme activity was detected in culture filtrate of transformed yeast.

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Molecular Cloning of Chitinase Genes Family from Serratia marcescens

  • Song, Young-Hwan;Kweon, Oh-Gun
    • Journal of fish pathology
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    • v.6 no.2
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    • pp.103-110
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    • 1993
  • Total genomic DNA library of Serratia marcescens was prepared by inserting Sau3AI partial digesting fragments(above 5 kb) into the dephosphorylated BamHl site of pUC19. In primary screening, two colonies were selected by observing the halo around E. coli transformants grown on the swollen colloidal chitin media. Secondary screening was performed by soaking two colonies with a few drops of 4-methylumbelleliferryl N-acetyl-$\beta$-D-glucocosaminide(4-MuNGlcNAc). As 4-MuNGlcNAc is a specific, fluorogenic substrate for chitinase, the positive clones produce light fluorescence by the exposure under the long wave U.V. light(360 nm). From genomic DNA library derived from pUC19, we have isolated two different chitinase clones, pCH1(11.0Kb) and pCH2(7.5Kb), which show completely different restriction map to each other. The cross-hybridization of pCH1EA and pCH2 have not revealed any hybridization signals to each other.

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Molecular Cloning and Characterization of a Gene for Cyclodextrin Glycosyltransferase from Bacillus sp. E1 (Bacillus sp. E1 의 cyclodextrin 생산효소 유전자 분리 및 구명)

  • Yong, Jeong-Sik;Choi, Jin-Nam;Park, Sung-Soon;Park, Cheon-Seok;Park, Kwan-Hwa;Choi, Yang-Do
    • Applied Biological Chemistry
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    • v.40 no.6
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    • pp.495-500
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    • 1997
  • To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

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Molecular Cloning of Antagonistic Genes in Pseudomonas maItophiliQ B-14 (토양병해 길항성 Pseudomonas maltophilia B-14의 길항유전자탐색)

  • 구본성;서영우;윤상홍;박경수;은무영;김용환;오상우;류진창;은무영
    • Microbiology and Biotechnology Letters
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    • v.20 no.6
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    • pp.619-624
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    • 1992
  • Tn5 lac 삽입으로 채소입고병원균에 길항력이 약화된 T-67 및 고추역병균과 참깨역병균에 길항력이 약화된 T-81의 Tn5 lac 유전자 일부와 오른쪽 말단에 있는 길항관련 유전자의 flanking sequence가 cloning된 pAG67 및 pAG81 clone을 선발하였고, pAG67 및 pAG81 clone된 길항관련 유전자의 flanking sequence를 야생 길항균 Pseudomonas maltophilia B-14의 DNA를 probe로 사용하여 Southern hybridization으로 확인하였으며, 제한효소 지도를 작성하여 8Kb 및 4Kb 크기의 flanking sequence가 cloning되었음을 확인하였다. pAG6 및 pAG81의 flanking sequence를 EcoRi-BglII와 EcoRI-MpaI으로 분리하여 유전자 은행으로부터 길항관련 유전자가 cloning된 cosmid clone 7개주를 선발하였다.

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