Postic C, Niswender K D, Decaux J F, Parsa R, Shelton K D, Gouhot B, Pettepher C C, Granner D K, Girard J, Magnuson M A
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Genomics. 1995 Oct 10;29(3):740-50. doi: 10.1006/geno.1995.9943.
We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1 beta and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from approximately -9.2 to approximately +15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from -335 to +736 bp (1071 bp), relative to the transcription start site in beta cells. These sequences revealed that mouse GK is > 94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-specific expression of GK to be performed.
我们克隆并鉴定了一段包含完整葡萄糖激酶（GK）基因的83 kb小鼠基因组DNA片段。该基因的11个外显子总跨度为49 kb，外显子1β和1L被35 kb的间隔分开。共测定了25266 bp的DNA序列信息：相对于肝细胞转录起始位点，从大约-9.2 kb到大约+15 kb（24195 bp）；相对于β细胞中的转录起始位点，从-335 bp到+736 bp（1071 bp）。这些序列显示小鼠GK与大鼠和人类GK的同源性>94%。小鼠肝脏GK mRNA受禁食和再喂食的调节，这在大鼠中也有发生。对小鼠、大鼠和人类基因的上游和下游启动子区域进行比对，发现了几个可能有助于转录调控的进化保守区域。然而，转基因小鼠中的融合基因研究表明，肝细胞转录起始位点附近的保守区域本身不足以在肝脏中实现不依赖位置的表达。使用DNA酶I对小鼠基因48 kb区域的染色质结构进行分析，发现了8个肝脏特异性超敏位点，其位置范围在肝脏转录起始位点上游0.1至36 kb之间。包含完整小鼠GK基因的单一连续DNA片段的获得，应有助于进一步开展GK细胞特异性表达的研究。
