Sandilands Aileen, Wang Xin, Hutcheson Aileen M, James John, Prescott Alan R, Wegener Alfred, Pekny Milos, Gong Xiahou, Quinlan Roy A
Department of Molecular and Cellular Pathology, University of Dundee, Dundee DD1 5EH Scotland, UK.
Exp Eye Res. 2004 Apr;78(4):875-89. doi: 10.1016/j.exer.2003.09.028.
Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.
在此，我们报道了小鼠Bfsp2基因的首个自然突变。对129X1/SvJ小鼠的Bfsp2基因进行特征分析时发现了一个突变，该突变缺失了外显子2的剪接受体位点。这导致外显子1错误地与外显子3拼接，从而在阅读框中引起移码，并在Bfsp2转录本的外显子3的第2位引入了一个终止密码子。对从129S1/SvImJ、129S2/SvPas和129S4/SvJae品系分离的晶状体RNA进行的逆转录聚合酶链反应（RT-PCR）研究证实，在这些不同的129品系中存在这种突变，并且在CBA和101品系中也发现了类似的突变，但在C3H或C57BL/6J小鼠品系中未发现。预计这种突变会导致产生一种严重截短的蛋白质产物，称为CP49，其基本上仅包含外显子1，但针对CP49的多克隆抗体未能在从129S1/SvImJ或129S4/SvJae制备的提取物中检测到全长或CP49片段，这表明这些129品系缺乏CP49蛋白。与最近报道的Bfsp2基因敲除情况类似，与C57BL/6J相比，129S1/SvImJ和129S4/SvJae品系中的丝状肌动蛋白水平及其蛋白水解加工也发生了改变。对129S2/SvPas晶状体细胞骨架进行电子显微镜检查发现，与CP49基因敲除相比，细胞骨架的形态变化相似，珠状和中间丝明显被定义不清的丝状物质所取代。免疫电子显微镜以及CP49/波形蛋白双敲除小鼠的产生表明波形蛋白是这种残留物质的关键成分。本报告中关于129及其他小鼠品系中Bfsp2自然突变的研究，对于在基因敲除策略中使用129X1/SvJ品系的晶状体研究也具有重要意义。
