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TARGETING OF HUMAN GENES AND ITS APPLICATION TO GENE MAPPING AND CLONING

TARGETING OF HUMAN GENES AND ITS APPLICATION TO GENE MAPPING AND CLONING
人类基因靶向及其在基因图谱和克隆中的应用
批准号:
2574330
负责人:
M KOI
金额:
$0.0万
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:
至

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中文摘要
翻译
我们的工作集中在将新的肿瘤抑制基因(TSG)定位到人类 染色体1,3,11和X。我们还研究了错配的功能 修复基因。定位和克隆有功能的TSG,正常人类染色体 转移到几个肿瘤细胞系中。介绍一种 将1号染色体导入子宫内膜癌细胞系,并将1号染色体转化为 3号染色体导入结直肠癌细胞系诱导生长停滞 端粒酶活性的体外和下调。介绍一种 11号染色体导入膀胱癌细胞系诱导生长停滞 而不下调端粒酶。我们调查了海特的损失- 人卵巢癌X染色体上的杂合性(LOH) Xq25-26.1区域的特定杂合性缺失,提示存在TSG 在这个地区。损失优先发生在非活动的X 染色体。为了便于绘制和克隆这些TSG,我们开发了 利用人染色体在鸡细胞中的人基因打靶系统 同源重组频率高的品系。通过插入一个 将功能端粒序列整合到染色体的特定基因位点 在鸡的细胞中,我们将产生可转移的染色体片段 来自染色体1,3和11。这些片段包含一个定义的区域 一条染色体的一部分将被转移到每个肿瘤细胞系以 绘制遏制增长的活动地图。通过转移人类2号和3号染色体 在错配修复(MMR)缺陷的大肠肿瘤细胞系中,我们 证明了MMR缺陷及其相关表型,包括 微卫星不稳定性和对烷化剂的耐受性是 隐性的。我们还证明了1)MMR可能与G2相连 识别DNA损伤时的细胞周期检查点;2)MMR蛋白 可能参与转录偶联的切除修复途径;3)MMR 系统参与了对顺铂的耐药性。为了证明 MMR基因负责这些表型,我们将产生 缺失mmr基因的染色体(S)并转移到mmr缺陷型 细胞。
英文摘要
Our efforts focused on mapping novel tumor suppressor genes(TSG) to human chromosomes 1,3,11, and X. We also studied the functions of mismatch repair genes. To map and clone functional TSGs, normal human chromosomes were transferred into several tumor cell lines. Introduction of a chromosome 1 into an endometrial carcinoma cell line and that of a chromosome 3 into a colorectal cancer cell line induced growth arrest in vitro and downregulation of telomerase activity. Introduction of a chromosome 11 into a bladder carcinoma cell line induced growth arrest without down-regulation of telomerase. We examined loss of heter- ozygosity(LOH) on the X chromosome of human ovarian carcinoma and found specific LOH at the Xq25-26.1 region, suggesting the presence of a TSG in this region. The loss was preferentially on an inactive X chromosome. To facilitate mapping and cloning of these TSGs, we developed a human gene targeting system using human chromosomes in a chicken cell line with a high frequency of homologous recombination. By inserting a functional telomere sequences into a specific gene loci of chromosomes in the chicken cells, we will generate transferable chromosomal fragments from chromosomes 1,3, and 11. These fragments containing a defined region of a chromosome will be transferred to each of the tumor cell lines to map growth arrest activities. By transferring human chromosomes 2 and 3 into mismatch repair (MMR) deficient colorectal tumor cell lines, we demonstrated that MMR defects and its associated phenotypes, including microsatellite instability and tolerance to alkylating agents are recessive. We also demonstrated that 1) MMR may be connected to the G2 cell cycle check point upon recognition of DNA damage; 2) MMR proteins may be involved in transcription-coupled excision repair pathway; 3) MMR system is involved in resistance to cisplatin. To demonstrate that the MMR genes are responsible for these phenotypes, we will generate chromosome(s) with disrupted MMR genes and transfer them to MMR-deficient cells.
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TARGETING OF HUMAN GENES AND ITS APPLICATION TO GENE MAPPING AND CLONING
TARGETING OF HUMAN GENES AND ITS APPLICATION TO GENE MAPPING AND CLONING
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