TRANSCRIPTION MAPPING THROUGH THE 11P13 ANIRIDIA LOCUS

这项提案要求根据以下规定界定无光区的界限 三个关键患者的易位断裂点，并进行测序 12个现有克隆的片段(以及其他来自具有良好特征的 如有必要，显微切割文库)，该文库定位于该区域。底漆 将从这些序列中设计和合成，并将 用于分离整个无虹膜区域的YAC克隆。YAC将会是 通过荧光原位杂交定位回11p13，并 其特点是其内容为单拷贝片段和重叠 其中，通过比较它们之间的Alu扩增产物。这些内部ALU 数据段将克隆到PUC 18中(或者，YAC将 直接亚克隆和单拷贝序列鉴定 与人类总DNA的探针杂交)和单拷贝亚克隆 将被用作针对动物园斑点、Northerns和cDNA文库的探针。 将特别注意任何检测到记录的克隆 胎儿大脑，因为我们相信参与眼睛发育的基因将是 在胎儿大脑中表达。YAC将被放置在远程物理上 通过映射单副本子克隆和现有父代来映射两者 克隆，并通过比较稀有切割酶在 根据现有的图谱，将YAC定位到基因组DNA中的位置。脱氧核糖核酸 克隆的序列及其映射信息将是 作为STS输入GenBank，YAC映射信息将为 储存在索尔克人类基因组研究所的数据库中。 这些克隆的DNA资源将被用来检测病毒的无虹膜区域 其他有眼部缺陷的患者，如缺陷症，调查 这些疾病之间有遗传关系的可能性。

This proposal calls for defining the limits of the anirdia region according to the translocation breakpoints of three key patients, and sequencing segments of twelve existing clones (and others from a well-characterized microdissection library if necessary) which map in that region. Primers for PCR will be designed and synthesized from these sequences, and will be used to isolate YAC clones throughout the aniridia region. YACs will be mapped back to 11p13 by fluorescent in-situ hybridization, and characterized for their content of single-copy segments and for overlaps among them, by comparison of their inter-Alu PCR products. These inter-Alu segments will be cloned into pUC 18, (alternatively, the YACs will be subcloned directly and single-copy sequences identified by absence of hybridization to a probe of total human DNA), and the single copy subclones will be used as probes against Zoo blots, Northerns and cDNA libraries. Special attention will be given to any clones detecting transcripts in fetal brain, since we believe genes involved in eye development will be expressed in fetal brains. YACs will be placed on the long-range physical map both by mapping the single copy subclones and the existing parental clones, and by comparing the restriction sites for rare-cutting enzymes in the YACs to the sites in genomic DNA, according to the existing map. DNA sequences of the clones, along with their mapping information, will be entered as STS's into GENBANK, and the YAC mapping information will be stored in the database at the Salk Institute for Human Genome Research. These cloned DNA resources will be used to examine the aniridia region of other patients with eye defects, such as coloboma, to investigate the possibility of a genetic relationship between these disorders.