Isolation and characterization of cDNA clones of mRNAs expressed selectively in the immature cells of developing brain.

在发育中的大脑的未成熟细胞中选择性表达的 mRNA 的 cDNA 克隆的分离和表征。

• 批准号: 06454691
• 负责人: USUI Hiroshi
• 金额: $ 4.54万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454691/
• 关键词: brain development; gene expression; cDNA cloning; differential screening; subtractive cDNA cloning; brain; development

We first established several improved procedures to isolate cDNA clones of mRNAs expressed selectively in the immature cells of developing brain. A modified differential screening procedure, which utilized two vector(pT7T3D and pBluescript) -system, showed clear signals with low background levels. Southern blot analysis of amplified cDNA library DNAs were shown to be able to replace the Northern blot analysis. These procedures were reliable especially for the analysis of cDNA clones whose corresponding mRNAs were relatively abundantly expressed. An improved subtractive cDNA cloning procedure, named Directional tag PCR subtraction, were demonstrated to be efficient to isolate differentially expressed clones of relatively rarely expressed mRNAs. These three procedures require no additional RNA for the screening after construction of the cDNA libraries, enabling the analysis of tiny brain regions of particular interest.By use of the procedures described above, we then isolated rat fetal brain-enriched (FBE) clones whose corresponding mRNAs were expressed preferentially in the prenatal stages of brain development. We successfully identified 22 distinct FBE clones whose mRNAswere expressed at least 5-fold more in the fetal brain than in the adult brain. The nucleotide sequence analysis of the 22FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (alpha tubulin M alpha 1, beta tubulin M beta 5, thymosin beta 10, stathmin, beta tubulin M beta 2, alpha-internexin, ferritin Lg subunit, neuronatin, and amphoterin). In situ hybridization analysis showed that mRNAs corresponding tothe FBE clones decreased during braindevelopment with various expression patterns. The mRNA of a newly isolated FBE clone was shown to be expressed selectively in the immature cells in theexternal germinal layr of the cerebellum and the subventricular zone of developing brain.

我们首先建立了几个改进的程序，以分离出在发育中的未成熟细胞中有选择性地表达的mRNA的cDNA克隆。经过修改的差分筛选程序，利用两个向量（PT7T3D和PBLUESCRIPT）系统显示出较低背景水平的明显信号。放大cDNA库DNA的Southern印迹分析可替代北印迹分析。这些程序是可靠的，特别是对于分析相应mRNA相对表达的cDNA克隆的分析。证明了改进的减法cDNA克隆程序，称为方向性TAG PCR减法，可以有效地分离出相对较少表达的mRNA的差异表达的克隆。这三个程序在构建cDNA文库后不需要其他RNA进行筛查，从而可以分析特定感兴趣的微小大脑区域。通过使用上述过程，我们隔离了大鼠胎儿脑增强（FBE）克隆（FBE）的克隆，其相应的mRNA在大脑发育的产物中优先表达。我们成功地确定了22个不同的FBE克隆，其mRNASWERE在胎儿大脑中的表达至少要比成人大脑高5倍。 The nucleotide sequence analysis of the 22FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (alpha tubulin M alpha 1, beta tubulin M beta 5, thymosin beta 10, stathmin, beta tubulin M beta 2, alpha-internexin, ferritin Lg subunit, Neuronatin和Amphoterin）。原位杂交分析表明，在脑开发过程中，与各种表达模式相对的mRNA相应的TOTHE FBE克隆降低。新分离的FBE克隆的mRNA被证明在小脑的外部生发层中的未成熟细胞和发育中的大脑的室内室内区域中有选择地表达。

项目成果

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Falk,J.D.：“人类染色体 9q32-34 上转录序列的鉴定和表征”J. Mol。

Falk, J.D.: "Identification and characterization of transcribed sequences on human chromosome 9q32-34" Journal of Molecular Neuroscience. 5(3). 165-179 (1994)

Falk, J.D.：“人类染色体 9q32-34 上转录序列的识别和表征”分子神经科学杂志。

熊西敏郎: "廣川タンパク質化学 第9巻 脳神経タンパク質、接着タンパク質" グリア線維性酸性タンパク質(glial fibrillary acidic protein; GFAP), 5 (1995)

Toshiro Kumanishi：“广川蛋白质化学第9卷：脑神经蛋白质，粘附蛋白质”胶质纤维酸性蛋白（GFAP），5（1995）

薄井 宏: "新しいcDNAサブトラクション法(Directional tag PCR subtraction法)を用いた“線状体特異的"cDNAクローンの単離" Neuropathology. 14(supple). 204- (1994)

Hiroshi Usui：“使用新的 cDNA 消减法（定向标签 PCR 消减法）分离‘纹状体特异性’cDNA 克隆”《神经病理学》14（补充）。

Kumanishi, T.: "Glial fibrillary acidic protein ; GFAP.(Japanese)" In Yajima, H.et al.(eds).Protein Chemistry-Molecular Structures and Functions-14 volumes.Tokyo : Hirokawa Publishing Company. 3-8 (1995)

Kumanishi, T.：“胶质原纤维酸性蛋白；GFAP。（日语）”载于 Yajima, H.等人（编）。蛋白质化学-分子结构和功能-14 卷。东京：广川出版公司。