Positional cloning of mouse morphological mutants

小鼠形态突变体的定位克隆

• 批准号: 09440257
• 负责人: SHIROISHI Toshihiko
• 金额: $ 8.7万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09440257/
• 关键词: Tail-short (Ts); morphological anomalies; homeotic transformation; positional cloning; cDNA selection; ribosomal protein; Rpl38; transgenesis; リボソーム蛋白; Rpl38遺伝子; トランスジェネシス; Rim4変異、Hx変異; マイクロサテライト; BACクローン; shh遺伝子; 形態形成; マウス; 突然変異; 連鎖解析 /

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

小鼠突变尾部短（TS）表现出缩短的扭结尾巴和许多骨骼异常，包括沿轴向骨骼的同型前后图案问题。 TS基因被映射到11号染色体的三层区域。为了阐明TS基因在小鼠胚胎发生中的功能，我们打算通过位置克隆来克隆基因。首先，我们基于突变库存TSJ/ LE-TS/ +和日本野生小鼠衍生的MSM菌株之间的大规模间反向交叉进行了对该基因的精细映射。 TS基因映射到两个微卫星标记物D1IMIT128和DLIMIT256之间的0.16厘米区域。我们筛选了小鼠YAC和BAC文库，并具有与TS基因座紧密相关的微卫星标记，并获得了YAC和BAC克隆。与孤立的克隆一起行走的进一步的染色体使我们能够构建一个完整的BAC重叠群，覆盖了TS人口式基因，而TS的临界区域则在两个新的STS之间狭窄，DI IRIN56和D…更多的Linigi7。该重叠群由3个BAC克隆组成，跨越了250KB DNA片段。我们通过使用相应的BAC定向cDNA选择，将几个cDNA克隆从临界区域分离为TS候选基因。我们已经获得了源自小鼠胚胎cDNA库的几个克隆。在这些候选基因中，编码核糖体蛋白亚基之一的RP138基因的表达水平在TS突变小鼠中降低。对TS突变体的基因组结构的分析表明，它具有含有RPL38的18KB长缺失。据报道，其他两个突变，分别是Rabo Torcido（RBT）和尾部 - 短shionogi（TSS），其表型与TS相似，并且要映射到11号染色体中的同一区域。我们发现RBT和TSS分别具有框架移位突变和插入性突变。为了确认RPL38是TS的真正基因，我们试图用RPL38的野生型基因通过转基因来挽救TS突变的致命表型。在包含RPL38和RPL38 cDNA的BAC表达上，都恢复了TS突变体在一定遗传背景下的生存能力，并完全恢复了形态表型。这似乎是TS突变体的致死性和形态异常，包括其同型转化，是由RPL38突变引起的。较少的

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