Boehmler Wendy, Petko Jessica, Canfield Victor A, Levenson Robert
Department of Biological Sciences, York College of Pennsylvania, York, PA, USA.
Methods Mol Biol. 2013;964:201-14. doi: 10.1007/978-1-62703-251-3_12.
In this chapter, we describe the identification and cloning of D2-like dopamine receptor (DR) genes in zebrafish, a vertebrate model genetic organism. To identify DR genes, we performed searches of the zebrafish genomic sequence database that yielded contig segments of several D2-like DR genes. From these sequences, we amplified full-length cDNAs encoding three D2, one D3, and three D4 DR receptor subtypes via RT-PCR. The predicted proteins displayed 57-72% amino acid identity when compared to their human DR counterparts. To validate the identity of zebrafish DR genes, each of the genes was mapped by using the T51 radiation hybrid panel. With the exception of drd2b and drd4b, each of the zebrafish DR genes mapped to chromosomal positions that were syntenic with regions of human chromosomes containing orthologs of the zebrafish DR genes. To further validate the identity of the D2-like DR genes in zebrafish, we conducted phylogenetic analysis which supported the predicted identities of the cloned DR receptor cDNAs.
在本章中，我们描述了斑马鱼（一种脊椎动物模式生物）中D2样多巴胺受体（DR）基因的鉴定和克隆。为了鉴定DR基因，我们对斑马鱼基因组序列数据库进行了搜索，得到了几个D2样DR基因的重叠群片段。从这些序列中，我们通过逆转录聚合酶链反应（RT-PCR）扩增出了编码三种D2、一种D3和三种D4 DR受体亚型的全长cDNA。与人类DR对应物相比，预测的蛋白质显示出57%-72%的氨基酸同一性。为了验证斑马鱼DR基因的身份，我们使用T51辐射杂种细胞系对每个基因进行了定位。除了drd2b和drd4b外，斑马鱼的每个DR基因都定位到了与包含斑马鱼DR基因直系同源物的人类染色体区域同线的染色体位置。为了进一步验证斑马鱼中D2样DR基因的身份，我们进行了系统发育分析，该分析支持了克隆得到的DR受体cDNA的预测身份。
