The molecular regulation mechanism in the cardiac calcium signaling proteins

为了检测和鉴定负责RYR 2和受磷蛋白的转录调节的序列，我们分离了基因的5 ′-侧翼区，并且为了鉴定基因的5 ′-侧翼区中的潜在调节元件，将基因的5 ′-侧翼区中的一系列5 ′-缺失构建体与荧光素酶基因融合，RYR 2基因的启动子活性测定结果表明，在RYR 2基因的5 '端侧翼区存在一个含有正调控元件的区域。对GC盒和调控区的置换突变分析表明，除了GC盒位于-56至-51处外，还有两个调控元件（RYR 2 P1和RYR 2 P2）对启动子活性至关重要。这些结果表明，Sp1和转录因子结合RYR 2 P1和RYR 2 P2协同增强RYR 2基因的表达。在瞬时转染实验中， 关于我们 通过对骨骼肌细胞启动子-荧光素酶基因构建体的研究，我们发现在-209 ~-90之间存在一个抑制RYR 2基因表达的负调控区，而在phopholamban基因上游-2080 bp处，5'端逐渐缺失至-96 bp处的荧光素酶基因构建体显示出较高的荧光素酶活性。相反，从-78到-4 bp的进一步缺失导致荧光素酶活性显著降低。结果表明，受磷蛋白基因的-96 ~-78区域是受磷蛋白基因的主要正调控元件，序列分析表明，在受磷蛋白基因转录起始位点的-93位点有一个典型的CCAAT序列，在-10位点有一个TATA盒。在受磷蛋白基因的5 ′侧翼区产生内部缺失突变体。CAAT盒的去除将荧光素酶活性降低至对照的40%。CAAT盒和位于-113位点的C/EBP β元件的内部缺失使活性降低至对照的5%。凝胶位移分析表明，来自心脏的核蛋白与CAAT盒结合。这些结果表明，CAAT盒与C/EBP β元件一起对受磷蛋白基因的转录活性具有关键作用。少

In order to examine and identify sequenses responsible for transcriptional regulation of the RYR2 and phospholamban, we have isolated the 5'-flanking region of the genes, and to identify potential regulatory elements in the 5'-flanking region of the genes, a series of 5'-deletion constructs in the 5'-flanking region of the genes were fused to the luciferase gene, and then their promoter activity in rat neonatal cardiac myocytes was determined.As to the RYR2 gene, the results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyzes of substitutional mutations introduced into the GC boxes and the regulatory region indicated that besides the GC box located at-56 to-51, two regulatory elements (RYR2P1 and RYR2P2) and essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2P1 and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment inv … More olving the promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between-209 and-90 which represses the expression of the RYR2 gene in skeletal muscle cells.In another hand, luciferase gene constucts including-2080 bp of the upstream region of phopholamban gene and progressive 5' deletions up to-96 bp showed a high level of luciferase activity. In contrust, further deletions from -78 to -4 bp resulted in remarkable decrease of luciferase activity. These results indicated that the region from-96 to-78 of the phospholambn gene contains the major positive regulatory element.Sequence analysis revealed a canonial CCAAT sequence at position-93 from the tentative transcription initiation site and a TATA box at-10.To confirm the functional significance of CAAT box for the transcription of phospholamban gene, internal deletion mutants were generated in the 5' flanking region of the phospholamban gene. Removal of the CAAT box reduced the luciferase activity to 40% that of the control. Internal deletion of both CAAT box and C/EBP beta element, which is located at-113, reduced the activty to 5% of the control. Gel shift assay indicated that nuclear proteins from heart bind to the CAAT box. These results suggested that the CAAT box together with the C/EBP beta element has critical role for the transcriptional activity of the phospholamban gene. Less