PHYSICAL MAPPING OF HUMAN CHROMOSOME 17Q23/Q24

这是第一次在MBRS计划下提交新的赠款提案 并扩大了目前基于聚合酶链式反应的试点项目 17q22-q23的YAC重叠群特征。长期目标是 对人类17号染色体q23-q24区域进行物理作图。 该区域包含多个遗传标记，如GHC、CsA、PL、 GAA、CSN4A和TK1。一些标记之间已显示出连锁。一个 很少有内部探针，如pC63、pTHH59、pTh17.12、128E1和pRMU1 在该地区可用。然而，极少数YAC(人工酵母 染色体)已从长弧的这一远端区域分离出来 这个项目的具体目标是开始 构建17q23-q24中YAC重叠的物理地图 区域。了解这一地区的物理地图是很重要的 导致不同疾病的几个基因的分子基础 这样的胎盘催乳素缺乏，生长激素缺乏，酸- 麦芽糖酶缺乏症、庞贝病与高钾性周期性麻痹 人类。此外，对某些基因组的表征 序列将引领未来发展早期分子诊断 对适当的障碍进行测试。例如，急性早幼粒细胞白血病 白血病可以通过检测重排和易位来诊断 将RARA基因(17q21)插入染色体15q22的MYL基因座。这也会有帮助的 建立检测MYL-RARA融合转录本的方法 (聚合酶链式反应)扩增。 构建重叠重叠群的起点将是 来自CSN4A标记物(高钾性周期性麻痹)的已知YAC或 通过分离已知标记的YAC(例如，GAA标记(Pompe 疾病、酸麦芽糖酶缺乏症)}。构建一种 17q23-24区域的物理图谱如下：染色体行走 将通过使用(I)‘载体’-聚合酶链式反应和/或反向聚合酶链式反应 利用载体聚合酶链式反应技术扩增小末端特异片段 合适的YAC克隆；(Ii)酶促循环测序 使用市场上可获得的自动或手动试剂盒的方法 DNA测序仪，(Iii)设计对 片段(STS)，并检测它们的人类和酵母总DNA 模板和(Iv)将引物提交给大学基因组中心。的 密歇根用于基于聚合酶链式反应的YAC筛选分离重叠克隆(S)。 在染色体行走的过程中，其他已知的遗传标记如 上面讨论的也将使用sSTSS来隔离重叠的YAC 17q23-24区域的克隆。猪瘟病毒的限制性内切酶片段分析 脉冲场凝胶电泳法(PFGE/Fige)重叠YAC 反转凝胶电泳法)将生成 将用来确定染色体行走的方向。 几个探针，如pBR322的左右片段，人COT-1 DNA 在这些分析中将使用内部标记物。

This is a first time new grant proposal submitted under the MBRS Program of NIH and an expansion of a current pilot project on PCR-based characterization of YAC contigs of 17q22-q23. The long term goal is to carry out physical mapping of q23-q24 region on human chromosome 17. This region consists of several genetic markers such as GHC, CSA, PL, GAA, CSN4A and TK1. A linkage has been shown between some markers. A few internal probes such as pC63, pTHH59, pTh17.12, 128E1 and pRMU1 are available in this region. However, very few YACs (yeast artificial chromosomes) have been isolate from this distal region of the long are of chromosome 17. The specific aims for this project are to begin construction of a physical map with overlapping YACs in the 17q23-q24 region. The physical mapping of this region is important to understand the molecular basis of several genes responsible for different disorders such a placental lactogen deficiency, growth hormone deficiency, acid- maltase deficiency, Pompe disease and hyperkalemic periodic paralysis in humans. In addition, the characterization of the certain genomic sequences will lead in the future to develop early molecular diagnostic tests for the appropriate disorders. For example, acute promyelocytic leukemia can be diagnosed by the detection of rearranged and translocate RARA gene (17q21) into myl locus of chromosome 15q22. It would also help to develop detection of myl-RARA fusion transcript by mRNA-PCR (polymerase chain reaction) amplification. The starting point in construction of overlapping contigs will be either from a known YAC for CSN4A marker (hyperkalemic periodic paralysis) or by isolating a YAC for a known marker {for example GAA marker (Pompe disease, acid-maltase deficiency)}. The strategies for building a physical map in the 17q23-24 region are as follows: chromosome walking will be done by employing (i) 'vectorette'-PCR and/or inverse PCR or Alu- vector PCR techniques to amplify small end-specific fragments in the appropriate YAC clones, (ii) cycle sequencing the fragment by enzymatic method using commercially available kits for either automated or manual DNA sequencer, (iii) designing PCR primers that are unique for the fragment (STS) and testing them for total human and yeast DNAs as template and (iv) submitting the primers to the Genome center, Univ. of Michigan for PCR-based YAC screening to isolate overlapping clone(s). In the course of chromosome walking, the other known genetic markers as discussed above will also be used a sSTSs to isolate overlapping YAC clones in the 17q23-24 region. Analyses of restriction fragments of overlapping YACs by PFGE (pulsed-field gel electrophoresis)/FIGE (field- inversion gel electrophoresis) would generate 'fingerprint' data which will be used in order to determine the direction of chromosome walking. Several probes such a left and right fragments of pBR322, human cot-1 DNA and internal marker will be used in these analyses.