Regulation of human pyruvate kinase gene in red cells

红细胞中人丙酮酸激酶基因的调控

• 批准号: 09670165
• 负责人: KANNO Hitoshi
• 金额: $ 2.3万
• 依托单位: Okinaka Memorial Institute for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670165/
• 关键词: Glycolysis; Genetic disease; Metabolic disorder; Transgenic mice; Gene therapy; Transcriptional regulation; Hemolytic anemia; Erythroenzymopathies; 細胞特異的発現; 遺伝病; 代謝異常

To identify regulatory gene sequence(s) responsible for the erythroid-specific expression of human erythroid-type pyruvate kinase (R-PK), we prepared two human PK mini-gene constructs and established 19 lines of transgenic mice.Although we have shown that the human R-PK promoter containing 4 GATA and 2 CACCC motifs had prominent transcription activity in both K562 and MEL cells, the promoter was not sufficient for in vivo transcription in the PK transgenic mice.We identified two DNaseI hypersensitive sites (HS) upstream of the human PK LR-gene, and an addition of the proximal HS (HS-I) enabled the linked PK transgene to express in an erythroid-specific manner, suggesting that the HS-I is an erythroid-specific enhancer of the human PK LR-gene.The HS-I contained one GATA, one CACCC and one purine rich motif (AGGGAGAAG), and the organization was similar to the erythroid-specific enhancer which had been identified in rat PK LR-gene.We have established three PK transgenic mice lines which overexpress human R-PK in red cells.The mu' LCR, the regulatory sequence of human beta-like globin locus was linked to the R-PK promoter and coding sequences, and used for microinjection.Red cell PK activities of the PK transgenic mice were about 2-3 fold of normal controls (83-122 IU/gHb, normal range 41.6*5.8), and human R-PK activities were demonstrated in the PK zymograms.Since the mu' LCR did not confer the position-independent, copy number-dependent expression of the PK transgene, the LCR might be influenced by position effect, and functioned as an erythroid-specific enhancer.

为了鉴定人红系特异性表达人红细胞型丙酮酸激酶(R-PK)的调控基因序列(S)，我们制备了两个人PK微型基因构建体，建立了19个转基因小鼠系。虽然含有4个GATA基序和2个CACCC基序的人R-PK启动子在K562和MEL细胞中都具有显著的转录活性，但在PK转基因小鼠中，启动子不足以在体内转录。我们鉴定了人PK LR-基因上游的两个DNaseI高敏感位点(HS)。Hs-I是人PK受体基因的红系特异性增强子。HS-I含有一个GATA、一个CACCC和一个富嘌呤基序(AGGGAAG)，其组织结构与已在大鼠PK LR基因中发现的红系特异性增强子相似。我们建立了三个在红细胞中高表达人R-PK的PK转基因小鼠。人β样珠蛋白基因的调控序列Mu‘LCR与R-PK启动子和编码序列相连，PK转基因小鼠的红细胞PK活性约为正常对照小鼠的2-3倍(83-122IU/gHb，正常范围41.6*5.8)，并且在PK酶谱中证实了人的R-PK活性。由于Mu‘LCR不提供PK转基因基因的位置依赖、拷贝数依赖的表达，LCR可能受位置效应的影响，并作为红系特异性增强子发挥作用。

项目成果

Tsujino K, Kano H, Hashimoto K, Fujii H, Jippo T, Morii E, Lee Y-M, Asai H, Miwa S, Kitamura Y.: "Delayed onset of hemolytic anemia in CBA-Pk-1^<slc>/Pk-1^<slc> mice with a point mutation of the gene encoding red blood cell type pyruvate kinase" Blood. 91

Tsujino K、Kano H、Hashimoto K、Fujii H、Jippo T、Morii E、Lee Y-M、Asai H、Miwa S、Kitamura Y.：“CBA-Pk-1^<slc>/Pk- 中溶血性贫血延迟发作

Hitoshi Kanno: "Expression and enzymatic characterization of human qlucose phosphate isomerase (GPI) variants accounting for GPI deficiency" Blood Cells Mol Dis. 24. 54-61 (1998)

Hitoshi Kanno：“导致 GPI 缺陷的人葡萄糖磷酸异构酶 (GPI) 变体的表达和酶学特征”Blood Cells Mol Dis。

Yoshiaki Kajiyama: "p53 gene mutation in 150 dissected lymph nodes in a patient with esophageal cancer" Dis Esophagus. 11. 279-283 (1998)

Yoshiaki Kajiyama：“食管癌患者 150 个解剖淋巴结中的 p53 基因突变”Dis Esophagus。

Hitoshi Kanno: "Expression and enzymatic characterization of human glucose phosphate isomerase(GPI)variants accounting for GPI deficiency" Blood Cells Mol Dis. 24. 54-61 (1998)

Hitoshi Kanno：“导致 GPI 缺乏的人葡萄糖磷酸异构酶 (GPI) 变体的表达和酶学特征”Blood Cells Mol Dis。

Murakami K, Kanno H, Miwa S, Piomelli S.: "Human HK_R isozyme : the organization of the hexokinase-I gene, the erythroid-specific promoter and transcription initiation site." Mol Genet Metab. (in press). (1999)

Murakami K、Kanno H、Miwa S、Piomelli S.：“人类 HK_R 同工酶：己糖激酶-I 基因、红细胞特异性启动子和转录起始位点的组织。”