Molecular cloning of cDNAs corresponding to mRNAs whchi increase in malignantly transformer FRTL cells.

对应于在恶性转化FRTL细胞中增加的mRNA的cDNA的分子克隆。

基本信息

批准号：
02807223
负责人：
ENDO Toyoshi
金额：
$ 0.9万
依托单位：
Yamanashi Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807223/
关键词：
thyroid cancer molecular biology ribosome heat shock protein Po protein GRP78 protein thyrotropin 甲状腺癌腫瘍マ-カ-甲状腺乳頭癌

项目摘要

We previously developed malignantly transformed cells designated as FRTL-Tc with thyroid stimulating hormone (TSH) receptors which were derived from FRTL cells. To characterize these FRTL-Tc cells further, we cloned cDNAs corresponding to mRNA species showing increased expression in FRTL-Tc cells compared to FRTL cells. A lambda gt10 library was constructed using polyadenylated mRNA from FRTL-Tc cells. The library was then screened for the cDNAs by differential plaque filter hybridization using single stranded cDNA probes synthesized from mRNA prepared from FRTL and FRTL-Tc cells. Of 25, 000 clones screened, 3 clones (Cl3, C17 and C140) were found to represent mRNA species whose levels were higher in FRTL-TC cells.Densitometric quantification of the mRNAs corresponding to C13, C17 and C140 in FRTL-TC cells gave values of 2.84, 1.33 and 5.86, respectively, when the levels in FRTL cells were taken as 1.0. The sizes of C13, C17 and C140 were 1.0, 0.8 kb, respectively. Northern blot analysis showed that the sizes of the corresponding mRNAs to these clones were 1.0, 2.0 and 0.8 kb, respectively. The mRNAs corresponding to C13 and C140 were found in all tissues investigated from Eisher rats, but the expression of C17 MRNA was extremely high in the thyroid only. Determination of the partial nucleotide sequences of these three clones showed they were neither genes for thyroglobulin nor thyroid peroxidase. C17 and C140 bore no homology to any known nucleotide sequences, but C13 was 90% homologous with the human ribosomal protein, Po. Electron microscopy showed that more free ribosomes existed in FRTL-Tc cells than in FRTL cells. Characterization of these genes might provide insights into the novel opportunity to study the nature of thyroid cancer as well as the function of normal thyroid epithelial cells.

我们之前开发了具有源自 FRTL 细胞的促甲状腺激素 (TSH) 受体的恶性转化细胞，命名为 FRTL-Tc。为了进一步表征这些 FRTL-Tc 细胞，我们克隆了与 mRNA 种类相对应的 cDNA，与 FRTL 细胞相比，FRTL-Tc 细胞中的表达增加。使用来自 FRTL-Tc 细胞的聚腺苷酸化 mRNA 构建 lambda gt10 文库。然后使用从FRTL和FRTL-Tc细胞制备的mRNA合成的单链cDNA探针，通过差异噬菌斑过滤杂交来筛选文库中的cDNA。在筛选的 25, 000 个克隆中，发现 3 个克隆（C13、C17 和 C140）代表 FRTL-TC 细胞中水平较高的 mRNA 种类。对 FRTL-TC 细胞中对应于 C13、C17 和 C140 的 mRNA 进行光密度定量，得出当 FRTL 细胞中的水平取为时，值分别为 2.84、1.33 和 5.86 1.0。 C13、C17和C140的大小分别为1.0、0.8 kb。 Northern blot分析显示这些克隆相应的mRNA大小分别为1.0、2.0和0.8 kb。在 Eisher 大鼠研究的所有组织中都发现了与 C13 和 C140 相对应的 mRNA，但 C17 mRNA 仅在甲状腺中表达极高。对这三个克隆的部分核苷酸序列的测定表明它们既不是甲状腺球蛋白基因也不是甲状腺过氧化物酶基因。 C17 和 C140 与任何已知的核苷酸序列没有同源性，但 C13 与人类核糖体蛋白 Po 有 90% 同源性。电镜显示FRTL-Tc细胞中存在比FRTL细胞更多的游离核糖体。这些基因的表征可能为研究甲状腺癌的性质以及正常甲状腺上皮细胞的功能提供新的机会。