DISTAL XQ DNA--ANALYSIS WITH YACS

长期目标是制造一种酵母人工染色体 基于(YAC)的Xq24-q28人类DNA图谱(50Mb；约占基因组的1.5%)。 已经开始使用重叠的YAC/Probe地图，几乎所有 区域，现在将继续绘制更精细的结构图 和功能层面。 结构分析将重点放在放置在 染色体，包括Xq28端粒、脆性X和普通 Xq27.2-27.3中的脆性部位，将对其进行详细分析。在……里面 此外，确定定义的序列元素Win的分布 在YAC和YAC跨细胞遗传学带的重叠群中。这些实验是 旨在测试有关总体GC分布的猜想 内容；高度重复的序列(Alu，Li)；为 适度重复的序列pTR5；以及一些简单的序列重复 [包括聚(DGdC).(da-dT)和脆性X的(CCG)n基序 站点]。 功能分析将比较几种映射技术 葡萄糖6-磷酸脱氢酶附近的转录单位 (G6PD)；在Xq26上的8Mb重叠群的部分中；以及在搜索 一种基因，其中的损伤负责一种特定的X-连锁 疾病。 含有G6PD的YAC将被转染，以确定是否如此 或配备适当的选择标记，它们可以在 基因组中的同源位置。可比较的实验将测试 脆性X区域的脆性可以仅局限于(CCG)n 重复序列或需要其他结构特征。 最后，一种方法将把结构和功能研究扩展到 进化论观点。人类G6PD基因已被测序。 现在将尝试确定基因的重要区域和 通过比较序列和对应序列来观察ITS进化的特征 从其他灵长类动物身上扩增出的部分，也包括完整的 克隆的小鼠基因的序列。推定的保护区 因此，功能重要性将被推断；最后，YAC适合 带有选择标记的基因将在这些区域被修改，YAC 将被转化来测试预测。

The long-range objective is to make a yeast artificial chromosome (YAC)-based map of Xq24-q28 human DNA (50 Mb; about 1.5% of the genome). A start has been made with an overlapping YAC/probe map of nearly all of the region, and mapping will now be continued to more refined structural and functional levels. Structural analysis will focus on sequences placed at the ends of chromosomes, including the Xq28 telomere, and the Fragile X and common fragile site in Xq27.2-27.3, which will be analyzed in detail. In addition, the distribution of defined sequence elements win be determined in YACs and YAC contigs across cytogenetic bands. The experiments are designed to test conjectures about the distribution of overall GC content; highly repetitive sequences (Alu, LI); selected loci for the moderately repetitive sequence pTR5; and some simple sequence repeats [including poly(dGdC).(dA-dT), and the (CCG)n motif at the Fragile X site]. Functional analyses will compare several techniques for mapping transcription units in the vicinity of glucose 6-phosphate dehydrogenase (G6PD); in portions of an 8 Mb contig across Xq26; and in the search for a gene in which lesions are responsible for a particular X-linked disease. G6PD-containing YACs will be transfected to determine if, either as such or fitted with appropriate selective markers, they can recombine at homologous sites in the genome. Comparable experiments will test whether fragility in the Fragile X region can be localized purely to the (CCG)n repeat sequence or requires other structural features. Finally, one approach will extend structural and functional studies to an evolutionary perspective. The human G6PD gene has been sequenced. Attempts will now be made to identify important regions of the gene and observe features of its evolution by comparing sequences to corresponding portions amplified by PCR from other primates, and also to the complete sequence of the cloned mouse gene. Conserved areas of putative functional importance will thus be inferred; and finally, YACs fitted with selective markers will be modified in those regions, and the YACs will be transfected to test predictions.