CHROMATIN MAPPING BY FLUORESCENCE IN SITU HYBRIDIZATION

通过荧光原位杂交进行染色质定位

• 批准号: 3333321
• 负责人: BARBARA J. TRASK
• 金额: $ 6.9万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333321
• 关键词: DNA replication origin; cell cycle; chromatin; color blindness; fluorescence; fluorescence microscopy; fragile X syndromes; gene dosage; gene expression; genetic crossing over; genetic mapping; genome; hamsters; human; human genetic material tag; in situ hybridization; method development; nucleic acid sequence; sex chromosomes; sex linked trait; telomere

The long-term goals of this project are 1) to develop fluorescence in situ hybridization (FISH) as a tool for mapping and ordering the genome; 2) to understand the organization of sequences spaced 50 kbp to > 3 Mbp apart in chromatin during the cell cycle; and 3) to construct a long range map of Xq27-Xqter. The five specific aims of the proposed research are as follows: 1) To optimize one- and two-color detection of single copy sequences by FISH in terms of labeling efficiency, signal resolution, and image registration. 2) To compare the proximity of hybridization sites in chromatin of varying compaction. These targets include G1 interphase, prematurely condensed chromosomes, and (pro)metaphase chromosomes. The targets will be treated to extract proteins and decondense the chromatin. Emphasis will be on the selection of target(s) in which the relative spacing and order of sequences 50 kbp to 3 Mb apart can be most efficiently and accurately determined. 3) To describe the relationship between known linear distance and measured interphase and prometaphase distances for sequences separated by 50 kbp to 3 Mbp. This study will demonstrate whether FISH mapping is generally applicable to diverse chromosomal regions (telomeric vs. interstitial; G- dark vs. G-light banded). 4) To determine if the separation between sister chromatids at specific loci as cells progress from G1 to G2 can be used to derive additional map information. Sister chromatid separation will be studied in terms of the relative proximity of sequences to the telomere (human 4p16-pter) and to replication origins (DHFR region, Chinese hamster). 5) To use the FISH mapping techniques developed in aims 1-4 to construct a long range map of human chromosome Xq27-Xqter. FISH will be used a) to rapidly screen for Xq28 region-specific sequences from large-insert libraries; b) to confirm the relative spacing, orientation, and order of previously characterized PFG fragment clusters; c) to produce a fine map over regions of 1-2 Mbp by determining the spacing and order of region- specific sequences using FISH to interphase nuclei; and d) to determine if interphase distance measurements can be used to map disease loci by detecting disease-specific deletions.

该项目的长期目标是：1)发展原位荧光技术 杂交(FISH)作为绘制和排序基因组图的工具；2) 了解相距50 kBP至&gt；3 MBP的序列的组织 细胞周期中的染色质；以及3)构建一个远距离图谱。 Xq27-Xqter。 拟议研究的五个具体目标如下： 1)通过以下方式优化单拷贝序列的单色和双色检测 FISH在标记效率、信号分辨率和图像方面 注册。 2)比较不同核染色质杂交位点的接近程度。 压实。这些靶点包括过早浓缩的G1间期 染色体和(前)中期染色体。这些目标将会被 提取蛋白质并解聚染色质。重点将放在 靶标的选择(S)中的相对间隔和序列顺序 50KBP到3Mb之间的距离可以最有效和准确地确定。 3)描述已知直线距离与被测直线距离之间的关系 相隔50kBP至的序列的相间和前中期距离 3 MBP。这项研究将论证鱼类测绘是否普遍 适用于不同的染色体区域(端粒与间质；G- 深色VS G光带)。 4)确定姐妹染色单体之间的分离是否在特定情况下 细胞从G1向G2发展时的基因座可用于推导额外的图谱 信息。姐妹染色单体分离将从以下方面进行研究 序列与端粒的相对接近(人4p16-PTER)和TO 复制起源(DHFR区，中国仓鼠)。 5)使用AIMS 1-4中开发的鱼类测绘技术来构建 人类染色体Xq27-Xqter的远程图。鱼将被用来a) 从大插入片段中快速筛选Xq28区域特异性序列 库；b)确定相对间距、方向和顺序 先前表征的PFG片段簇；c)产生精细的地图 通过确定区域的间距和顺序，在1-2 MBP的区域上 使用FISH对核进行间期的特定序列；以及d)确定 相间距离测量可用于绘制疾病基因座图 检测特定于疾病的缺失。