Lin X, State M W, Vaccarino F M, Greally J, Hass M, Leckman J F
Child Study Center, Yale University School of Medicine, New Haven, Connecticut, 06520-7900, USA.
Genomics. 1999 Aug 15;60(1):96-104. doi: 10.1006/geno.1999.5882.
Homeodomain (HD) genes are helix-turn-helix transcription factors that play key roles in the specification of cell fates. In the central nervous system (CNS), HD genes not only position cells along an axis, but also specify cell migration patterns and may influence axonal connectivity. In an effort to identify novel HD genes involved in the development of the human CNS, we have cloned, characterized, and mapped the human homologue of the murine HD gene Orthopedia (Otp), whose product is found in multiple cell groups within the mouse hypothalamus, amygdala, and brain stem. Human cDNA and genomic libraries were screened with probes derived from mouse Otp sequences to find the human homologue, OTP. The deduced amino acid sequence of the open reading frame of the human cDNA is 99% homologous to mouse Otp and demonstrates a high degree of conservation when compared to sea urchin and Drosophila. OTP was mapped to human chromosome 5q13.3 using radiation hybrid panel mapping and fluorescence in situ hybridization. Flanking markers were identified from YAC clones containing OTP. A single putative OTP gene product was found in 17-week human fetal brain tissue by Western blot analysis using a novel polyclonal antibody raised against a conserved 13-amino-acid sequence at the C-terminus of the OTP protein. Expression in the developing human hypothalamus was confirmed by immunohistochemistry.
同源结构域（HD）基因是螺旋-转角-螺旋转录因子，在细胞命运的特化过程中发挥关键作用。在中枢神经系统（CNS）中，HD基因不仅沿轴定位细胞，还确定细胞迁移模式，并可能影响轴突连接。为了鉴定参与人类中枢神经系统发育的新型HD基因，我们克隆、表征并定位了小鼠HD基因正位基因（Otp）的人类同源物，其产物存在于小鼠下丘脑、杏仁核和脑干的多个细胞群中。用人cDNA和基因组文库，以源自小鼠Otp序列的探针进行筛选，以寻找人类同源物OTP。人cDNA开放阅读框推导的氨基酸序列与小鼠Otp有99%的同源性，与海胆和果蝇相比显示出高度保守性。使用辐射杂种板定位和荧光原位杂交将OTP定位到人类染色体5q13.3。从含有OTP的酵母人工染色体（YAC）克隆中鉴定侧翼标记。通过使用针对OTP蛋白C末端保守的13个氨基酸序列产生的新型多克隆抗体进行蛋白质印迹分析，在17周龄的人类胎儿脑组织中发现了单一推定的OTP基因产物。免疫组织化学证实了其在发育中的人类下丘脑中的表达。
