Positional cloning of mouse morphological mutants

突变的小鼠短尾巴(Ts)表现出短而扭曲的尾巴和许多骨骼异常，包括沿着轴向骨骼的同源前后图案问题。为了阐明ts基因在小鼠胚胎发育中的作用，本研究拟通过定位克隆的方法克隆ts基因。首先，我们基于突变株Tsj/Le-ts/和日本野鼠来源的MSM菌株之间的大规模亚种间回交，对该基因进行了精细的定位。将TS基因定位于D1IMit128和DlIMit256两个微卫星标记之间的0.16 cM区域。我们筛选了与ts基因座紧密连锁的微卫星标记的小鼠YAC和BAC文库，获得了YAC和BAC克隆。与分离克隆的进一步染色体行走使我们能够构建一个覆盖ts致病基因的完整的BAC重叠群，并且ts的临界区在两个新的STS DI IRin56和D…之间变窄更多的插图。该重叠群由3个BAC克隆组成，跨越250kb的DNA片段。我们通过使用相应的BAC进行定向选择，从关键区中分离到了几个作为ts候选基因的cDNA克隆。我们已经从小鼠胚胎cDNA文库中获得了几个克隆。在这些候选基因中，编码核糖体蛋白亚基之一的Rp138基因在ts突变小鼠中的表达水平降低。对ts突变体的基因组结构分析表明，该突变体含有18kb的Rpl38缺失。另外两个突变，Rabo torcido(RBT)和TSS(TSS)，被报道具有与ts相似的表型，并被定位到11号染色体的同一区域。我们发现RBT和TSS分别存在移码突变和插入突变。为了确定Rpl38是TS的致病基因，我们尝试用Rpl38野生型基因来挽救ts突变的致死表型。含有Rpl38的BAC和Rpl38的过表达都恢复了ts突变体在一定遗传背景下的生存能力，并完全挽救了其形态表型。因此，看来ts突变体的致死性和形态异常，包括它们的同源异型转化，都是由Rpl38突变引起的。较少

A mouse mutation Tail-short (Ts) exhibits shortened kinky tail and numerous skeletal abnormalities including homeotic anteroposterior patterning problem along the axial skeleton. Ts gene was mapped to the teromeric region of the chromosome 11. To elucidate the function of the Ts gene in mouse embryogenesis, we intended to clone the gene by means of positional cloning. First, we employed a fine mapping of this gene based on a large scale intersubspecific backcross between the mutant stock TsJ/Le-Ts/ + and Japanese wild mouse-derived MSM strains. Ts gene was mapped to a 0.16cM region between two microsatellite markers, D1IMit128 and DlIMit256. We screened mouse YAC and BAC libraries with microsatellite markers tightly linked to the Ts locus and have obtained YAC and BAC clones. Further chromosome walking with the isolated clones allowed us to construct a complete BAC contig covering the Ts causative gene and the critical region of the Ts was narrowed between two new STSs, DI IRin56 and D … More lINigI7. This contig consists of 3 BAC clones, spanning a 250kb DNA fragment. We have isolated several cDNA clones as Ts candidate gene from the critical region by directed cDNA selection using the corresponding BAC. We have obtained several clones derived from mouse embryonic cDNA library. Among these candidate genes, the expression level of Rp138 gene, which encodes one of ribosomal protein subunits, was reduced in Ts mutant mouse. Analysis of the genomic structure of the Ts mutant revealed that it had an 18kb long deletion containing Rpl38. Other two mutations, Rabo torcido (Rbt) and Tail-short shionogi (Tss), were reported to have phenotype similar to Ts and to be mapped to the same region In the chromosome 11. We found that Rbt and Tss have a frame shift mutation and an insertional mutation, respectively. In order to confirm that Rpl38 is the causative gene for Ts, we tried to rescue the Ts mutant lethal phenotype by transgenesis with the wild-type gene of Rpl38. Over expression both of the BAC containing Rpl38 and the Rpl38 cDNA restored the viability of the Ts mutant in a certain genetic background and completely rescued the morphological phenotype. Thus, it appeared that the lethality and the morphological anomalies of Ts mutants, including their homeotic transformation, are caused by the Rpl38 mutation. Less