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

我们的工作重点是定位新的肿瘤抑制基因（TSG）到人类

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.