HUMAN DISTAL XQ DNA--ANALYSIS WITH YACS

长期目标是制造酵母人工染色体 基于YAC的Xq 24-q28人类DNA图谱（50 Mb;约占基因组的1.5%）。 一开始，几乎所有的YAC/探针图都是重叠的， 该地区，和绘图现在将继续更完善的结构 功能水平。 结构分析将集中在位于末端的序列， 染色体，包括Xq 28端粒、脆性X染色体和常见染色体 Xq27.2-27.3中的脆性位点，将对其进行详细分析。 在 此外，定义的序列元素的分布将被确定， 在YAC和YAC重叠群中跨细胞遗传学条带。 了实验 设计用于测试关于总体GC分布的示意图 含量;高度重复序列（Alu，LI）;选择的基因座 中度重复序列pTR 5;和一些简单重复序列 [包括聚（dGdC）。（dA-dT）和脆性X染色体上的（CCG）n基序 网站]。 函数分析将比较几种映射技术 6-磷酸葡萄糖脱氢酶附近的转录单位 （G6 PD）;在Xq 26上的8 Mb重叠群的部分中;以及在搜索 一种基因，其中病变负责一个特定的X连锁 疾病 将转染含有G6 PD的YAC，以确定其本身 或与适当的选择标记相匹配，它们可以在 基因组中的同源位点。 类似的实验将测试 脆性X区域中的脆性可以纯粹定位于（CCG）n 重复序列或需要其他结构特征。 最后，一种方法将结构和功能研究扩展到一个 进化的观点。 人类G6 PD基因已被测序。 现在将尝试确定基因的重要区域， 通过与相应序列的比较，观察其演化特征， 通过PCR从其他灵长类动物扩增的部分，以及完整的 克隆小鼠基因的序列。 保留的假定面积 功能的重要性将因此推断;最后，YAC拟合 将在这些区域中修改选择性标记， 将被转染以测试预测。

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.