The establishment of chromosome engineering for the gene cloning

确定一个候选抑制基因在chr上的位点。1是本研究建立基因克隆染色体工程的典范案例。一种带有间质q臂缺失（del-1q）或源自chr的亚染色体可转移片段（30A3 STF）的新标记的人类1号染色体。通过微细胞融合将1q转移到人子宫内膜癌细胞HHUA中。del-1q和30A3 STF具有诱导细胞生长停滞和类似衰老细胞的形态变化的潜力。这些结果表明，对HHUA细胞具有诱导衰老活性的靶基因定位于chr。1q区域由D1S510-D1S237或d1s103 - d15s547定义。为了进一步确定候选位点，还研究了利用端粒靶向载体或染色体粉碎法制备更精细的染色体片段。这看起来很有用，但实际使用还需要更多的改进。另外，我们筛选了61个手术切除的子宫内膜癌样本，以寻找重排或缺失。最后，我们发现由STS D1S225和D1S459定义的1q41-42区域LOH发生率较高。在确定了一个与该区域相关的YAC （Research Genetics的748H11）后，我们将其转入HHUA。YAC克隆也能诱导细胞衰老。利用该YAC DNA，从正常人子宫内膜cDNA文库中分离出多个cDNA克隆。其中，我们发现了一个在许多子宫内膜癌中原发性序列改变的候选基因。

To identify the locus of a candidate suppressor gene(s) on chr. 1 was a model case for the establishment of chromosome engineering for the gene cloning in this study. A neo-tagged human chromosome 1 that carried an interstitial q arm deletion (del-1q) or a subchromosomal transferable fragment (30A3 STF) derived from a chr. 1q was transferred into human endometrial cancer cell line, HHUA via microcell fusion. The del-1q and the 30A3 STF had the potential to induce the growth arrest and the morphological changes analogous to senescent cell. These results indicate that the target gene that has senescence-inducing activity for HHUA cells is localized to the chr. 1q region defined by D1S510-D1S237 or D1S103-D1S547. To further define the candidate locus, the utilization of telomere targeting vector or chromosome pulverization to make finer chromosome fragment was also studied. It seemed useful but more improvements is necessary for the actual usages. Alternatively, we screened 61 surgically removed endometrial cancer samples for rearrangements or deletions. Finally, we found a high incidence of LOH in the 1q41-42 region defined by the STS D1S225 and D1S459. After identifying a YAC (748H11 by Research Genetics) involved in this region, we transferred it into HHUA. The YAC clone also induce the cellular senescence. By using this YAC DNA, several cDNA clones were isolated from the normal human endometrial cDNA library. Among them, we found one candidate which primary sequence was altered in many endrometrial cancers.