Nakazawa Asako, Inaba Yoko, Kamijima Atsuko, Kondo Noriaki, Ito Michihiko, Shiba Tadayoshi, Takamatsu Nobuhiko
Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan.
Gene. 2004 Mar 31;329:71-9. doi: 10.1016/j.gene.2003.12.010.
The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.
花栗鼠冬眠特异性蛋白HP - 55是一个140 kDa复合物的组成部分，其水平在冬眠期间的血液中急剧下降。它与α1 -抗胰蛋白酶（AT）高度同源。在花栗鼠中，除了HP - 55（或CM55 - ML）之外，还有几个α1 - AT样基因在肝脏中表达，并且在冬眠期间具有不同的调控模式：在冬眠的花栗鼠中，CM55 - ML基因的表达水平大幅降低，CM55 - MS基因的表达水平略有增加，而CM55 - MM基因的表达几乎不受影响。作为理解这些花栗鼠α1 - AT样基因与冬眠相关基因调控的第一步，我们分离了CM55 - ML、CM55 - MM和CM55 - MS基因的基因组克隆，并分析了它们的启动子活性。这些α1 - AT样基因由五个外显子组成，并且显示出与人α(1)-AT基因相似的基因结构，这表明它们是由一个祖先α1 - AT基因的复制产生的。使用HepG2和COS - 7细胞进行的瞬时转染研究表明，对于所有三个α1 - AT样基因，大约150 bp的5'侧翼序列足以实现肝脏特异性启动子活性，并且HNF - 1与启动子区域的结合可以反式激活转录。此外，对由CM55 - ML和CM55 - MS基因序列组成的嵌合启动子活性的分析表明，CM55 - MS基因中缺乏TATA盒样序列是其启动子活性较弱的原因。
