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

我们之前开发了恶性转化细胞，命名为FRTL- tc，具有促甲状腺激素（TSH）受体，来源于FRTL细胞。为了进一步表征这些FRTL- tc细胞，我们克隆了与FRTL细胞相比FRTL- tc细胞中表达增加的mRNA物种对应的cdna。利用FRTL-Tc细胞的聚腺苷化mRNA构建lambda gt10文库。然后利用FRTL和FRTL- tc细胞制备的mRNA合成的单链cDNA探针，通过差异斑块过滤杂交对文库进行筛选。在筛选的25,000个克隆中，发现3个克隆（Cl3， C17和C140）代表FRTL-TC细胞中mRNA水平较高的物种。FRTL- tc细胞中C13、C17和C140对应mrna的密度定量分别为2.84、1.33和5.86，FRTL细胞中的水平为1.0。C13、C17和C140的大小分别为1.0、0.8 kb。Northern blot分析显示，这些克隆对应的mrna大小分别为1.0、2.0和0.8 kb。在Eisher大鼠的所有组织中均发现C13和C140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与已知的核苷酸序列无同源性，但C13与人类核糖体蛋白Po有90%的同源性。电镜观察显示，FRTL- tc细胞比FRTL细胞存在更多的游离核糖体。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。

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.