小鼠新基因Ayu17-449的克隆及表达分析

在利用PU8捕获载体从小鼠ES细胞中寻找有关对发育起重要作用基因时，一阳性ES克隆编号为Ayu17-449被捕获，经过Southern blotting法证实捕获载体单一整合在Ayu17-449号ES细胞的基因组中。通过用5＇RACE法得到所捕获基因的一小段cDNA，在EST数据库中比对，得到一5523bp cDNA序列，在Celera数据库中它包含于两个相邻基因，根据这两个基因的mRNA设立了一系列的引物进行RT-PCR和测序，用这两个基因的不同片段分别作探针进行Northern blotting分析，确定这是一个RNA约9kb并编码1920个氨基酸的新基因（定名为Ayu17-449基因，其cDNA序列和编码蛋白序列发表在NCBI数据库，编号为DQ079067）。Northern blotting揭示Ayu17-449基因高度表达在小鼠的脑、肾脏、心脏、肺、肌肉和胃等组织。PU8捕获载体具有X-gal报告基因，能从蛋白表达水平揭示它所捕获的基因的表达模式。X-gal染色结果显示，Ayu17-449蛋白高度表达在小鼠的脑、肾脏、心脏等组织，与Northern blotting法的结果高度一致。X-gal染色切片结果进一步证明Ayu17-449蛋白主要表达在脑的神经细胞和肾脏近曲小管细胞中。Ayu17-449基因的编码蛋白在数据库（Scansite，http：//scansite．mit．edu/）做功能基团分析后，揭示其编码蛋白的N末端含有Granin基团，大量文献证实Granin基团具有参与激素的分泌的功能，显示Ayu17-449基因可能与激素的分泌有关。

Cloning and Expression Analysis of a Murine Novel Gene,Ayu17-449

We used gene trapping vector PU8 to search some interesting genes which play important roles in mouse development from murine ES cells. One positive ES colony termed Ayu17-449 was trapped. Its partial cDNA was obtained by using 5′ RACE method. It is homologous to a 5 523 bp cDNA fragment (GI: 20879412) in EST database. Further analysis of the 5 523 bp cDNA sequence in Celera mouse gene database showed that it overlaps two genes. We designed serials of DNA primers according to the mRNAs of these two genes for RT-PCR and Northern blotting analysis, and identified a novel RNA about 9 kb (we named it as Ayu17-449) encoding 1 920 aa. This gene is expressed highly in the brain, kidney, heart, lung, muscle and stomach. The expressed protein contains a Granin motif on its N-terminus, showing that this gene may be involved in hormone secretion.