Poltorak A, Smirnova I, He X, Liu M Y, Van Huffel C, McNally O, Birdwell D, Alejos E, Silva M, Du X, Thompson P, Chan E K, Ledesma J, Roe B, Clifton S, Vogel S N, Beutler B
Howard Hughes Medical Institute, Dallas, TX 75235-9050, USA.
Blood Cells Mol Dis. 1998 Sep;24(3):340-55. doi: 10.1006/bcmd.1998.0201.
On the basis of 2093 meioses analyzed in two separate intraspecific backcrosses, the location of the mouse Lpsd mutation was circumscribed to a genetic interval 0.9 cM in size. A total of 19 genetic markers that lie in close proximity to the mutation were examined in mapping. Most of these were previously unpublished polymorphic microsatellites, identified by fragmentation of YAC and BAC clones spanning the region of interest. Lpsd was found to be inseparable from the microsatellite marker D4MIT178, and from three novel polymorphic microsatellites identified near D4MIT178. The mutation was confined between two novel microsatellite markers, herein designated "B" and "83.3." B lies centromeric to the mutation, and was separated by four crossovers in a panel of 1600 mice; 83.3 lies distal to the mutation and was separated by three crossovers in a panel of 493 mice. 66 BAC clones and one YAC clone were assembled to cover > 95% of the critical region. Estimates based on pulsed field gel electrophoresis and fluorescence in situ hybridization indicate that the The B-->83.3 interval is about 3.2 Mb in length. A minimal area of zero recombinational distance from Lpsd was also assigned, and found to occupy approximately 1.2 Mb of physical size. To identify gene candidates, nearly 40,000 sequencing runs were performed across the critical region. Selective hybridization and exon trapping were also employed to identify genes throughout the "zero" region. Only a single intact gene was identified within the entire critical region. This gene encodes the Toll-4 receptor, a member of the IL-1 receptor family.
基于在两次独立的种内回交中分析的2093次减数分裂，小鼠Lpsd突变的位置被限定在一个大小为0.9厘摩的遗传区间内。在定位过程中检查了总共19个与该突变紧密相邻的遗传标记。其中大多数是以前未发表的多态微卫星，通过跨越感兴趣区域的YAC和BAC克隆的片段化鉴定出来。发现Lpsd与微卫星标记D4MIT178以及在D4MIT178附近鉴定出的三个新的多态微卫星不可分离。该突变被限制在两个新的微卫星标记之间，在此命名为“B”和“83.3”。B位于突变的着丝粒侧，在一组1600只小鼠中通过四次交换与之分离；83.3位于突变的远端，在一组493只小鼠中通过三次交换与之分离。组装了66个BAC克隆和一个YAC克隆以覆盖关键区域的>95%。基于脉冲场凝胶电泳和荧光原位杂交的估计表明，B到83.3区间的长度约为3.2兆碱基。还确定了与Lpsd零重组距离的最小区域，发现其物理大小约为1.2兆碱基。为了鉴定候选基因，在关键区域进行了近40000次测序运行。还采用了选择性杂交和外显子捕获来鉴定整个“零”区域的基因。在整个关键区域内仅鉴定出一个完整基因。该基因编码Toll-4受体，它是IL-1受体家族的成员。
