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

1. 各种已建立的高分辨率染色体带的方法被重新评估和部分改进，以应用于不同的细胞培养系统。(1)溴化乙锭（EB）法通过提高EB和Colcemid的剂量和治疗时间，适用于培养的生长良好的肿瘤细胞。(2)用甲氨蝶呤（MTX）细胞同步和随后的BrdU处理方法应用于犬（Canis familiaris）淋巴细胞培养，获得了足够的高分辨率复制r带结果。(3)建立了一种获得淋巴母细胞样细胞系（LCL）高分辨率r带染色体的可靠方法，即EB添加（1-1.5小时）和过量胸腺嘧啶诱导的细胞同步，然后BrdU处理（6.5-7小时）。通过将EB方法应用于外周血淋巴细胞培养，精确定义了各种结构染色体异常的断点，从而对以下疾病位点进行了区域定位：唐氏综合征相关区域21q22.2->qter)， HMC综合征（1q31.2或7p15.1-p15.3）， 2型神经纤维瘤病（22q12.2）。利用重复序列（端粒DNA、rDNA α卫星DNA和含有Alu重复序列的DNA片段）的DNA探针，采用荧光原位杂交技术对部分结构染色体异常（环状、假双心性等）进行分子细胞遗传学鉴定。此外，通过FISH分析和高分辨率r带分析，对来自人类21号染色体的20个DNA片段进行了精确的区域定位。其中包括13个NotI连接克隆和7个sfi连接克隆。

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.