CAFFEINE-ENHANCED ANALYSIS OF CHROMOSOME BAND 11Q23

该项目有两个长期目标：1)开发一个 改进的简单高分辨率条带(HRB)技术 对人类染色体进行详细的细胞遗传学分析；以及2) 精确确定11q23染色体区域的断裂点 在广泛可用的人类细胞系的库中供其他人使用 调查人员。一种简单有效的人力资源平衡技术将是 遗传性骨质疏松症的临床诊断 染色体疾病和自发性肿瘤，以及在医学上 人类基因图谱和染色体突变的研究。这个 11q23区域在医学上具有重要意义，因为它在一些 髓系白血病与Ets-1原癌基因和 这是一个脆弱的位置，也是其他几个基因的可能位置。 具有精确定位的11q23断裂点的细胞系文库 将有助于研究人员快速绘制基因图谱和 对这一重要的染色体区域进行分子分析。 这项赠款提案提出了一项分两个阶段的试验计划，以 实现这些目标。第一阶段将检验这一假设 咖啡因提高了高分辨率条带的效率 利用放线菌素-D、吖啶橙、5‘- 胰酶G显带中的溴脱氧尿苷或溴化乙锭 四种人成纤维细胞和淋巴样细胞系的分析。如果 咖啡因被证明可以增加有丝分裂指数和比例。 具有高分辨率带状染色体的细胞，然后简单地 咖啡因增强方案将用于培养的 人类细胞。这些咖啡因效应的可逆性 环己亚胺也将接受测试。第二阶段将适用于 这种改进的HRB技术对11q23进行了详细的分析 NIGMS人类中已有9个人类细胞系发生易位 遗传突变细胞库。高分辨率显带分析 将能够识别每个组件中涉及的特定子带 LINE的11q23易位，以及每个染色体的详细核型 细胞系。

This project has two long-term objectives: 1) to develop an improved simple high resolution banding (HRB) technique for the detailed cytogenetic analysis of human chromosomes; and 2) to precisely define breakage points in the 11q23 chromosomal region in a library of widely-available human cell lines for use by other investigators. A simple and efficient HRB technique would be medically valuable both in clinical diagnosis of inherited chromosomal disorders and spontaneous tumors, and in medical research in human gene mapping and chromosomal mutation. The 11q23 region is medically significant for its breakage in some myeloid leukemias, and the locus of the ets-1 proto-oncogene and a fragile site, and as the probable locus for several other genes. A library of cell lines with precisely-localized 11q23 breakpoints would be useful to investigators for rapid gene mapping and molecular analysis of this important chromosomal region. This grant proposal presents a two-phase experimental plan to achieve these objectives. The first phase will test the hypothesis that caffeine increases the efficiency of high resolution banding techniques utilizing actinomycin-D, acridine orange, 5'- bromodeoxyuridine or ethidium bromide, in the trypsin G-banding analysis of four human fibroblast and lymphoid cell lines. If caffeine is shown to increase the mitotic index and the proportion of cells with high resolution banded chromosomes, then a simple caffeine-enhanced protocol will be designed for use on cultured human cells. The reversibility of these caffeine effects by cycloheximide will also be tested. The second phase will apply this improved HRB technique to the detailed analysis of 11q23 translocations already in 9 human cell lines in the NIGMS Human Genetic Mutant Cell Repository. High resolution banding analysis will enable identification of the specific sub-band involved in each line's 11q23 translocation, as well as detailed karyotyping of each cell line.