TRANSCRIPTION MAPPING THROUGH THE 11P13 ANIRIDIA LOCUS

该提案要求根据以下内容界定无核武器区的界限： 三个关键病人的易位断裂点， 十二个现有的克隆片段（和其他来自一个良好的特征 显微切割文库，如果需要的话），其在该区域中映射。 引物 将从这些序列设计和合成，并将 用于分离整个无虹膜区域的YAC克隆。 YAC将是 通过荧光原位杂交定位回11p13， 其特征在于它们的单拷贝段的内容和重叠 其中，通过比较它们之间的Alu PCR产物。 这些铝间 片段将被克隆到pUC 18中，（或者，YAC将被 直接亚克隆，单拷贝序列通过不存在 与总人DNA的探针杂交）和单拷贝亚克隆 将用作针对Zoo印迹、Northerns和cDNA文库的探针。 将特别注意任何克隆检测转录本， 胎儿的大脑，因为我们相信与眼睛发育有关的基因， 在胎儿大脑中的表达。 YAC将被放置在远程物理 通过映射单拷贝亚克隆和现有亲本 克隆，并通过比较稀有切割酶的限制性位点， 根据现有的地图，将YAC定位到基因组DNA中的位点。 DNA 克隆的序列，沿着它们的映射信息，将被 作为STS输入GENBANK，YAC映射信息将 存储在索尔克人类基因组研究所的数据库中。 这些克隆的DNA资源将用于研究无虹膜区域的 其他眼缺损患者，如缺损，以调查 这些疾病之间存在遗传关系的可能性。

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.