PHYSICAL MAP OF HUMAN CHROMOSOME 13Q32

广泛而长期的目标是利用体细胞和分子 用分子探针定义和物理定位区域的技术 染色体13q的缺失似乎是细胞遗传学上的原因 对于13q综合征。因此，这个区域被认为含有基因。 对大脑、眼睛和肢体发育至关重要。映射到此的探测器 区域将在临床上用于检测慢性支气管炎患者的缺失 提示表型，但正常核型。物理地图和关联的 克隆的DNA片段可用于定位重要的发育基因 在这一区域内。最终目标是理解几个人 分子水平上的畸形。我们将进行以下工作 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) 通过在13号染色体的远端长臂上的缺失来创建一组 体细胞杂交种，每种都含有缺失的13号染色体。 可选择标记基因将被插入到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 用来制造杂交种，该标记将被选为。2)映射所有 现有的远端染色体13q探针到这组患者衍生 13号染色体缺失。这是通过标准的Southern实现的 吸墨水。3)建立含有未缺失末端的杂交细胞系 13号染色体的一部分作为其唯一的人类成分。紫外线照射将会 被用来对染色体进行分段，位于13q上的紫外线修复基因是 选择用于。4)使用基于脉冲场凝胶电泳的技术 分离产生于关键13q的大的限制性片段 区域。人类特有的Alu探针用于检测大片段， 在DNA Southern转移中存在于正常DNA中而不存在于缺失DNA中 从缺失的和正常的染色体中。识别这些波段的探测器是 这是通过克隆乐队本身的序列而产生的。5)隔离探头 它们通过随机映射来区分缺失面板染色体 对照删除面板从13q噬菌体文库中克隆。6)开始一段漫长的旅程 由删除面板定义的区域的范围限制图。这是 用正常DNA的PFGE完成，然后转移和 与一组测绘的13q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.