Isolation of a new gene by a PCR-cloning method.

通过 PCR 克隆方法分离新基因。

基本信息

批准号：
05671009
负责人：
KIMURA Shigeru
金额：
$ 1.41万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671009/
关键词：
PCR differential display cloning cancer cell lines クローニング degenerate primer Ca結合蛋白

项目摘要

A lot of oncogenes and tumor suppressor genes were discovered in past several years, but these genes are not enough to explain the mechanism of carcinogenesis. There are a number of important genes that regulate cell proliferation and differentiation, and certain genetic changes of such genes cause a malignant transformation. Most of them have not been discovered yet. In this research, we tried to isolate a new gene concerning cell differentiation or proliferation.We used a differential display technique to identify several cDNA sequences. This method is rapid to detect differences of expression of mRNA between one material and the other. We prepared cancer cell lines treated with or without PMA (Phorbol Myristate Acetate), isolated mRNA from each of them, and synthesized cDNA.Consequence of DNA sequencing, we obtained some cDNA fragments not recorded in the genebank. We examined their expression patterns using Northern blotting in several cancer cell lines, and tried to isolate the complete cDNA of these. Finally, we obtained three attracted cDNA fragments of unknown genes.The clone9 which was isolated from LNCaP,derived from prostatic cancer cell line, was expressed strongly in all examined cell lines including leukemia, brest, colon, prostate and cervix of uterus. The size of mRNA is estimated about 1.7kb, and its amino acid sequence demonstrated partial homology with tyrosine-phosphatase.The clone16-5 was also isolated from LNCaP which demonstrated remarkable expression broadly in our cell lines. The size of mRNA is estimated about 3.7kb.The clone15-1 showed interesting expression pattern. Its expression was only observed in LNCaP treated with PMA,the size of mRNA was predicted 3.8kb.This gene may correlate with a cell differentiation or proliferation.We will try to isolate complete cDNA of these genes, and characterize of their functions.

在过去的几年中，发现了许多癌基因和肿瘤抑制基因，但是这些基因不足以解释癌变的机理。有许多重要的基因调节细胞增殖和分化，并且这种基因的某些遗传变化会导致恶性转化。他们中的大多数尚未发现。在这项研究中，我们试图隔离一个有关细胞分化或增殖的新基因。我们使用差分显示技术来识别多个cDNA序列。该方法迅速检测一种材料与另一种材料之间mRNA表达的差异。我们制备了用或不含PMA处理的癌细胞系（佛罗威尔肉豆蔻酸酯乙酸肉豆蔻酸酯），从它们中分离出mRNA并合成cDNA。DNA测序的产生，我们获得了Genebank中未记录的一些cDNA片段。我们使用几种癌细胞系中的Northern印迹检查了它们的表达模式，并试图分离它们的完整cDNA。最后，我们获得了三个未知基因的吸引cDNA片段。从前列腺癌细胞系中分离出的clone9在所有检查的细胞系中都强烈表达，包括白血病，布雷斯特，结肠癌，前列腺和子宫宫颈。估计mRNA的大小约为1.7kb，其氨基酸序列与酪氨酸 - 磷酸酶的部分同源性。克隆16-5也从LNCAP中分离出来，这在我们的细胞系中广泛表现出显着的表达。 mRNA的大小估计约为3.7kb。克隆15-1显示出有趣的表达模式。它的表达仅在用PMA处理的LNCAP中观察到，预测mRNA的大小为3.8KB。该基因可能与细胞分化或增殖相关。我们将尝试隔离这些基因的完整cDNA，并表征其功能。