PHYSICAL MAP OF HUMAN CHROMOSOME 13Q32

广泛的，长期的目标是使用体细胞和分子 用分子探针来定义和物理绘制区域的技术 染色体13 q的缺失似乎是细胞遗传学上负责 13 q综合征的症状这一区域因此被假定含有基因 对大脑眼睛和四肢的发育至关重要映射到这个的探针 该区域将在临床上用于检测患有以下疾病的患者中的缺失： 提示表型但核型正常。物理地图和相关的 克隆的DNA片段可用于定位重要的发育基因 在这个区域内。最终目标是了解几个人类 分子水平上的畸形我们会进行以下工作 具体目标，以便开始朝着这些长期目标取得进展。 1)使用来自许多患者的淋巴母细胞系（LCL） 在13号染色体的远端长臂中缺失，以产生一组 体细胞杂交体，每个体细胞杂交体含有缺失的13号染色体。一 将选择标记基因插入LCL的染色体中 使用逆转录病毒载体。微细胞介导的染色体转移将是 用于制造杂交种，并且标记将被选择用于。2)映射所有的 现有的远端染色体13 q探针对这组患者衍生 13号染色体缺失这是通过标准的南方 印迹3)制备含有未缺失的远端的杂交细胞系 13号染色体的一部分作为其唯一的人类组成部分。紫外线照射将 用于染色体片段化，位于13q上的UV修复基因， 选择为。4)使用基于脉冲场凝胶电泳的技术 分离来自关键13 q的大限制性片段， 地区人特异性Alu探针用于检测大片段， 在DNA的Southern转移中存在于正常DNA中，但不存在于缺失的DNA中 正常和缺失的染色体。识别这些条带的探针是 通过从这些条带中克隆序列而制成。5)分离探针 它通过随机作图来区分缺失组染色体 来自13q噬菌体文库的克隆针对缺失组。6)开始一个漫长的 由删除面板定义的区域的范围限制性图谱。这是 用正常DNA的PFGE完成，然后将其转移， 与一组定位的13 q探针杂交。

The broad, long term objectives are to use somatic cell and molecular techniques to define with molecular probes and to physically map a region of chromosome 13q whose deletion appears cytogenetically to be responsible for the 13q- syndrome. This region is thus assumed to contain genes critical for brain, eye and limb development. Probes which map to this region will be useful clinically for detecting deletions in patients with suggestive phenotype but normal karyotype. The physical map and associated cloned DNA segments can be used to locate important developmental genes within this region. The ultimate goal is to understand several human malformations at the molecular level. We will carry out the following specific aims in order to begin progress towards these long term goals. 1) Use lymphoblastoid cell lines (LCL's) derived from a number of patients with deletions in the distal long arm of chromosome 13 to create a panel of somatic cell hybrids each of which contains a deleted chromosome 13. A selectable marker gene will be inserted into the chromosomes of the LCL's using a retrovirus vector. Microcell mediated chromosome transfer will be used to make hybrids and the marker will be selected for. 2) Map all of the existing distal chromosome 13q probes to this panel of patient derived chromosome 13 deletions. This is accomplished with standard Southern blotting. 3) Make a hybrid cell line which contains the undeleted distal portion of chromosome 13 as its only human component. UV irradiation will be used to fragment the chromosome and a UV repair gene located on 13q is selected for. 4) Use a pulsed field gel electrophoresis based technique to isolate large restriction fragments which arise from the critical 13q region. A human specific Alu probe is used to detect large fragments which are present in normal but not in deleted DNA in Southern transfers of DNA from deleted and normal chromosomes. Probes which recognize these bands are made by cloning sequences from the bands themselves. 5) Isolate probes which distinguish between deletion panel chromosomes by mapping random clones from a 13q phage library against the deletion panel. 6) Begin a long range restriction map of the regions defined by the deletion panel. This is accomplished with PFGE of normal DNA which is then transferred and hybridized with a battery of mapped 13q probes.