Improvement of high-resolution chromosome banding methods, and its application to human gene mapping.

高分辨率染色体显带方法的改进及其在人类基因图谱中的应用。

基本信息

批准号：
02454492
负责人：
IKEUCHI Tatsuro
金额：
$ 2.88万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

1. Various established methods for high-resolution chromosome banding were re-evaluated and in part improved for their application to different cell culture systems.(1) The ethidium bromide (EB) method was found to be applicable, by improving the dose and duration of treatment with EB and Colcemid, to the well-grown tumor cells in culture.(2) The method of cell synchronization with methotrexate (MTX) and subsequent BrdU treatment which was applied to lymphocyte cultures from the dog (Canis familiaris) yielded sufficient results for high-resolution replication R-banding.(3) A reliable method to obtain high-resolution R-banded chromosomes from lymphoblastoid cell lines (LCL) was established by combination of EB addition (1-1.5 hr) and excess thymidine-induced cell synchronization followed by the BrdU treatment (6.5-7 hrs).2. By applying the EB method to peripheral lymphocyte cultures, the break points of various structural chromosome abnormalities were precisely defined, leading to the regional mapping of the following disease loci : Down syndrome-related region 21q22.2->qter), HMC syndrome (1q31.2 or 7p15.1-p15.3), neurofibromatosis type 2 (22q12.2).3. Fluorescent in situ hybridization was performed for molecular cytogenetic characterization of some structural chromosome abnormalities (rings, pseudodicentrics, etc.) by using DNA probes of repeated sequences (telomere'DNA, rDNA alpha satellite DNA and DNA segments containing Alu repeats). Furthermore, precise regional mapping of a total of 20 DNA segments derived from human No.21 chromosomes were performed by FISH analysis and high-resolution R-banding. They include 13 NotI- and 7 SfiI-linking clones.

1。对高分辨率染色体带的各种已建立的方法进行了重新评估，并部分改善了它们在不同的细胞培养系统中的应用。（1）发现，通过改善EB和Colcemid治疗的剂量和持续时间，发现了溴化乙锭（EB）方法适用于培养物中的EB和COLCEMID的剂量和持续时间，并在培养物中进行了良好的BRENTRIANE（2）（2）（2）（2）（2）（2）（2）（Mort）。狗（Canis familis）适用于淋巴细胞培养的治疗方法可为高分辨率复制R带r带来。（3）通过通过EB添加（1-1.5 HR）的组合确定了一种可靠的方法，从淋巴细胞样细胞系（LCL）中建立了一种可靠的方法来获得高分辨率的R带染色体（LCL）。（6.5-7小时）.2。通过将EB方法应用于外围淋巴细胞培养物，精确定义了各种结构性染色体异常的断裂点，从而导致以下疾病基因座的区域映射：唐氏综合症相关的区域21q22.2-> QTER），HMC综合征（1Q31.2或7p15.1-P15.1-P15.3），NEUREFORTONT HMC综合征（22q12.2）.3。通过使用重复序列的DNA探针（telomere'tna，rDNA alpha satellite DNA和DNA段包含ALU重复序列）），对某些结构染色体异常（环，假性介质等）进行了荧光原位杂交进行分子细胞遗传学表征。此外，通过鱼类分析和高分辨率的R伴侣进行了总共20个源自人类染色体的DNA段的精确区域映射。它们包括13个Noti-和7个SFII链接克隆。