• 제목/요약/키워드: Display cloning

검색결과 34건 처리시간 0.026초

Identification and Characterization of a Novel Angiostatin-binding Protein by the Display Cloning Method

  • Kang, Ha-Tan;Bang, Won-Ki;Yu, Yeon-Gyu
    • BMB Reports
    • /
    • 제37권2호
    • /
    • pp.159-166
    • /
    • 2004
  • Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of $3.4{\times}10^{-7}\;M$. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.

역전사 연쇄중합반응에 의한 착상전 생쥐난자에서의 상이한 mRNA의 발현조사에 의한 새로운 유전자의 크로닝법 (Differential Display of mRNA in the Preimplantation Mouse Embryos by Reverse Transcriptase Polymerase Chain Reaction)

  • 김진회;박흠대;이훈택;정길생
    • 한국가축번식학회지
    • /
    • 제18권3호
    • /
    • pp.199-206
    • /
    • 1994
  • 본 연구는 생쥐 배 발생과정의 상이한 발현을 RT-PCR법에 의해 무작위 증폭함으로서 새로운 유전자를 손쉽게 크로닝하기 위해 수행되었다. mRNA의 상이한 display법은 Ling 과 Pardee (Science 257, 1992)에 의해 개발되었으며, 최근 Zimermann과 Schultz (PNAS USA 91, 1994)에 의해 재증명되었다. 이 방법은 특정 유전자의 일시적 발현의 변화가 maternal 제어로부터 접합체 제어로의 이행에 따른 발현전이, 다정자 침입과 단일 정자 침입에 의한 배발생의 기능적 차이, 성공적으로 부화한 배반포기 배와 부화에 실패한 배반포기 배에서의 발현의 차이는 물론 세포주기에 따른 유전자 발현 양식의 변화에 따른 새로운 유전자의 크로닝을 가능케 한다. 이 방법에 의해, 2세포기 특이 발현 유전자를 크로닝 하였으며, 이 유전자는 EcoRI제한 효소 처리후 Southern blot을 행한 결과 약 15kb genomic size를 가진 것으로 나타났다. 이 새로운 유전자는 간장 특이적 발현을 나타내었다. 또한, 적어도 2개의 mRNA가 존재하였으며, 이는 RNA splicing에 의한 것으로 추정되었다. (PCR, RT-PCR, cloning, preimplantation, mouse)

  • PDF

cDNA Cloning of Farnesoic Acid-Induced Genes in Candida albicans by Differential Display Analysis

  • CHUNG SOON-CHUN;LEE JI-YOON;OH KI-BONG
    • Journal of Microbiology and Biotechnology
    • /
    • 제15권5호
    • /
    • pp.1146-1151
    • /
    • 2005
  • The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Previously, we reported that the yeast-to-hypha transition in this organism is suppressed by farnesoic acid, a morphogenic autoregulatory substance that accumulates in the medium as the cells proliferate. In this study, using a differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique, we have identified several genes induced in C. albicans by farnesoic acid treatment. These observations indicate that farnesoic acid can alter the expressivity of multiple genes, including the DNA replication machinery and cell-cycle-control proteins.

Approach for Cloning and Characterization of Blue/White Flower Color Specific cDNA Clones from Two Commelina Species

  • Lee Gunho;Yeon Mooshik;Hur Yoonkang
    • Journal of Plant Biotechnology
    • /
    • 제7권1호
    • /
    • pp.45-50
    • /
    • 2005
  • To clone blue and white flower color specific genes, mRNA differential display was carried out with two different Commelina species, C. communis Linne for blue color and C. coreana Leveille for. leucantha Nakai for white color. Fifty two and 100 cDNA clones specific for blue or white flower color, respectively, were ranging from 200 to 700 bp in size. From the reverse northern blot analysis, 12 and 7 positive clones were selected for blue and white flower, respectively. These clones appear to be novel cDNAs for these Commelina plants, but not color specific. This finding was supported by the northern blot analysis. However, two clones, B18 and B19, derived from blue flowered Commelina were highly expressed than in the white Commelina species, implying that further study will be valuable. The results indicated that both mRNA display experiment and dot blot analysis may not sensitive enough to clone color-determining gene from the plant, leading to explore more advanced method, like high-density colony array study (HDCA).