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비특이적 DNA 결합력을 보이는 람다파지 Xis 단백질 돌연변이체에 대한 연구
조은희,남찬은,유정아 한국유전학회 1999 Genes & Genomics Vol.21 No.4
The bacteriophage λ exicisionase (Xis) is an architectural protein which is required for excisive recombination of λ. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites, X1 and X2, that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host encoded factor for inversion stimulation (FIS) at the X1 and F sites, respectively. Since Xis protein is very unstable and is difficult to be purified, we constructed His-tagged Xis and confirmed that the His-tagged version carried the same activity as the wild-type Xis protein. We then analyzed the effect of a glutamic acid substitution to an alanine or a lysine at codon 40 in Xis functions. In gel binding assays, XisE40K and XisE40A did not discriminate specific Xis binding sequences and that they only showed non-specific DNA binding activity. In spite of non-specific DNA binding phenotype of these variants, they were still able to assist excisive recombination in vivo if FIS protein is present. This suggests that XisE40K and XisE40A still interact with FIS protein and these two mutant proteins can be loaded onto the Xis binding sites possibly through direct protein-protein interactions with FIS. Taken these together, we concluded that substitution of glutamic acid 40 results in a local structural change in sequence-specific DNA binding domain of Xis.
악성 혈액질환에서 성공적인 동종골수이식 후 숙주 기질 미세환경의 구축
조상희,이제중,남찬은,최경상,정익주,이일권,김진희,박종태,김형준 대한조혈모세포이식학회 2002 대한조혈모세포이식학회지 Vol.7 No.1
인체의 골수는 간엽모세포를 함유하고 있으며 이들은 골수미세환경의 주된 세포들로 분화가 가능하여 조혈기능을 지지한다. 본 연구에서는 성별이 다른 동종조혈모세포이식 환경에서 조혈모세포의 완전 생착을 보이고, 이식 후 1년에서 8년이 지난 11예의 재생불량성 빈혈 및 백혈병 환자들을 대상으로 하여 골수에서 MSC를 분리하고 체외 확장을 통해 배양된 MSC에서 X 염색체 탐식자를 이용한 FISH 및 microsatellite polymorphism PCR 기법으로 그 기원을 확인하였다. 그 결과 조혈모세포는 완전히 공여자 기원으로 대치되었음에도 불구하고 MSC는 모두 수여자 기원임을 알 수 있어, 동종조혈모세포이식에서 미세환경의 구축은 수여자의 자가 생산에 의한 골수 간질세포에 의한 것으로 생각된다. Background: Human bone marrow (BM) contains mesenchymal stem cells (MSC) that can differentiate into various cells of mesenchymal origin. It remains a matter of controversy whether donor-derived stromal cells are capable of engraftment following hematopoietic stem cell transplantation (HSCT) or not. Methods: To determine if donor-derived stromal cells are transferred to the recipients of allogeneic HSCT, we investigated the characterization of MSC in 11 patients 1 to 8 years after sex mis-matched allogeneic HSCT in severe aplastic anemia and leukemia. Results: All patients had complete engraftment with donor- derived stem cells as shown by detection of donor type DNA in peripheral blood mononuclear cells. Following culture, MSC showed the expression of SH2 and SH4, but none of the hematopoietic markers of CD14, CD34, or CD45. MSC which can be differentiated to osteogenic lineage showed the genotype of recipient completely using FISH or PCR analysis. Conclusion: This study confirmed that MSC isolated from recipients of allogeneic HSCT in severe aplastic anemia and leukemia are not of donor genotype despite of full hematopoietic engraftment with donor type. Donor cells did not contribute to reconstitute the marrow microenvironment.