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.

为了确定负责人红细胞型丙酮酸激酶（R-PK）的红细胞特异性表达的调节基因序列，我们准备了两个人类PK Mini-Gene构建体，并确定了19行转基因小鼠的19行。我们已经表明，我们已经表明，含有4 Gata和2 Cacccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccced疾病，启动子不足以在PK转基因小鼠中体内转录。我们确定了人类PK LR基因上游的两个DNAsei高度敏感性位点（HS），并添加了近端HS（HS-I），使链接的PK传输能够在erythroid-shroid中表达HHS-erys-erys-erys-erys-erys-erys-erys-ery in yys-eyy s erys-人类pk lr-gene.HS-I包含一个GATA，一个CACCC和一个嘌呤富图案（Agggagaag），该组织类似于在大鼠pk lr-gene中鉴定出的红细胞特异性增强子，我们已经确定了三个PK Transgenic小鼠在人类R-PK sequul Suere the Multh besta bests bests sequul Serike in the Multh beitss中，该序列是Mun lim crike。将基因座链接到R-PK启动子和编码序列，用于显微注射。PK转基因小鼠的红细胞PK活性约为正常对照的2-3倍（83-122 IU/GHB（83-122 IU/GHB，正常范围41.6*5.8），在PK Zymogrim中表现出了pk Zymogriments。 LCR PK转基因可能受位置效应的影响，并充当红细胞特异性增强子。

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。

Kanno H, Fujii H, Wei DC, Chan LC, Hirono A, Tsukimoto I, Miwa S: "Frame shift mutation, exon skipping and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency." Blood. 89(11). 4213-4218 (1997)

Kanno H、Fujii H、Wei DC、Chan LC、Hirono A、Tsukimoto I、Miwa S：“剪接位点突变引起的移码突变、外显子跳跃和双密码子缺失是丙酮酸激酶缺陷的原因。”