PROVIDE SEQUENCE ANALYSIS OF TK AND HPRT GENE MUTATIONS

我们建议使用新的分子技术来调查光谱的 在小鼠L5178 Y细胞中在tk-1时发生的诱变事件。 我们将比较 这一光谱与人类癌症中发生的异常范围， 验证小鼠淋巴瘤assy（MLA）作为事件的准确反映 人类的意义。 具体来说，大量的突变体将被 通过Southern印迹筛选tk-1等位基因缺失。 小病灶将 通过手动和/或自动测序进行分析， 单链构象多态性（SSCP）分析定位 突变并简化测序工作。 SSCP的可行性 筛选突变体的分析在I期得到证实。二期将 提供了这种技术的全面使用，以简化突变分析。 我们还建议开发一种小鼠染色体II绘画探针，使用 我们实验室开发的独特技术， 杂交方法 这样的探针将允许简化的染色体分析。 TK突变体中的事件。 SSCP和测序中使用的引物和方案 分析可以作为试剂盒或服务商购。 绘画 探针将在TK突变体的分析中以及在染色体中具有实用性。 鉴定，并最终形成新的遗传毒理学的基础 染色体断裂素测定。

We propose using new molecular techniques to survey the spectrum of mutagenic events occurring at tk-l in mouse L5178Y cells. We will compare this spectrum with the range of abnormalities occurring in human cancer, to validate the mouse lymphoma assy (MLA) as an accurate reflection of events of human significance. Specifically, a large collection of mutants will be screened for tk-l allele loss by Southern blotting. Small lesions will be analyzed by manual and/or automated sequencing, following prescreening by single strand conformational polymorphism (SSCP) analysis to localize mutations and streamline the sequencing effort. The feasibility of SSCP analysis for screening mutants was demonstrated in Phase I. Phase II will provide full-scale use of this technique to streamline mutation analysis. We also propose developing a mouse chromosome Il painting probe, using a unique technology developed in our laboratory, preparative in situ hybridization. Such a probe will allow simplified analyses of chromosomal events in tk mutants. Primers and protocols employed in SSCP and sequence analysis may be made commercially available as kits or services. Painting probes will have utility in analysis of tk mutants, and in chromosome identification, and eventually form the basis for a new genetic toxicology assay for clastogens.