Transgenic approach to clarify regulatory mechanisms of odorant receptor gene expression

最近，我们成功地用YAC转基因系统表达了小鼠气味受体（OR）基因M0R28（神经科学学报，367,2000）。基因组分析显示，小鼠mrr28基因上游68 kb的1.8 kb DNA区域与人类同源物hr28上游32 kb的序列高度同源（gene 292,73,2002）。这个同源区域可能包含一个顺式作用的调控元件，这是我们用转基因小鼠进行的缺失研究表明的表达morr28簇所必需的。为了更好地理解嗅觉感觉图谱是如何组织的，我们分析了单个肾小球中通过嗅觉神经元轴突运输的OR mRNA物种。通过对嗅球切片进行激光显微解剖，我们发现从肾小球中分离出的ormrna分子并不总是具有高度的序列相似性。嗅上皮（OE）切片的原位杂交显示，在相邻肾小球中检测到mrna的OR基因倾向于在4区的同一限制区域内表达，即使其OR序列不相似（Tsuboi等人提交）。为了研究OE区域结构的功能意义，我们使用差异显示方法寻找区域特异性表达的基因。在从大鼠OE分离的克隆中，我们发现了一种新的嗅觉蛋白，称为0-MACS，它与其他中链酰基辅酶a合成酶家族蛋白不同，在OE中特异性表达。这项研究表明，这种新蛋白可能在以特定区域的方式处理气味或在发育过程中OE的区域模式中发挥重要作用(Eur。J. Bichem. 270, 1995,2003)。

Recently, we succeeded the expression of a murine odorant receptor (OR) gene, M0R28 with the YAC transgenic system (Nat. Neurosci. 3, 687, 2000). Genome analysis has revealed that a 1.8 kb DNA region 68 kb upstream of the mouse M0R28 gene is highly homologous to the sequence 32 kb upstream of the human ortholog H0R28 (Gene 292, 73, 2002). This homologous region may contain a cis-acting regulatory element necessary for the expression of the M0R28 cluster indicated by our deletion studies with the transgenic mice.To better understand how the olfactory sensory, map is organized, we analyzed individual glomeruli for OR mRNA species transported through the axons of olfactory neurons. Using the laser micro-dissection method against olfactory bulb sections, we have found that the OR mRNA molecules, isolated from glomeruli adjacent to the projection sites for the zone 4 OR, MOR28, do not always share high sequence similarities. In situ hybridization of olfactory epithelium (OE) sections revealed that the OR genes whose mRNAs are detected in the adjacent glomeruli tend to be expressed in the same restricted area within zone 4, even when the OR sequences are not similar (Tsuboi, et al. submitted).To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein, termed 0-MACS, which is specifically expressed in the OE unlike other medium-chain acyl-CoA synthetase family proteins. This study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development (Eur. J. Bichem. 270,1995, 2003).