Wang S Y, Gudas L J
Proc Natl Acad Sci U S A. 1983 Oct;80(19):5880-4. doi: 10.1073/pnas.80.19.5880.
The synthesis of the proteins laminin and collagen IV is stimulated approximately equal to 20-fold in F9 mouse teratocarcinoma stem cells after treatment of the cells with retinoic acid and N6, O2'-dibutyryl-cAMP (Bt2cAMP). A cDNA library from F9 cells treated with retinoic acid, Bt2cAMP, and theophylline (F9-R + DBC cells) was constructed to isolate cDNA coding for collagen IV or laminin. The recombinant plasmids were screened by differential colony hybridization to cDNA synthesized from poly(A)+ RNA isolated from F9 stem and F9-R + DBC cells. Differentially hybridizing plasmids were then used as probes to hybridize to RNA transfer blots to determine the size of their specific mRNA. Only plasmids containing cDNA sequences specific for high molecular weight mRNA were further analyzed. Studies by hybridization-selection, in vitro translation, and immunoprecipitation showed that a plasmid clone, pc15, contains cDNA homologous to collagen IV (alpha 2) mRNA, and another plasmid clone, pc156, contains cDNA homologous to laminin B mRNA. By RNA blot analyses, the size of mRNA coding for collagen IV (alpha 2) is 7.6 kilobases; the size of mRNA for laminin is 6.8 kilobases. Using the technique of RNA blot hybridization, we studied the time course of the increase in mRNA coding for collagen IV (alpha 2) and laminin B in F9 cells after retinoic acid and Bt2cAMP treatment. Both collagen IV (alpha 2) and laminin B mRNAs are present in F9 stem cells. Collagen IV (alpha 2) mRNA and laminin B mRNA levels increase slightly at approximately 12 hr after retinoic acid and Bt2cAMP addition, with a dramatic increase between 12 and 24 hr after drug treatment.
用视黄酸和N6，O2'-二丁酰环磷腺苷（Bt2cAMP）处理F9小鼠畸胎瘤干细胞后，层粘连蛋白和IV型胶原蛋白的合成受到刺激，增加幅度约为20倍。构建了一个来自用视黄酸、Bt2cAMP和茶碱处理过的F9细胞（F9-R + DBC细胞）的cDNA文库，以分离编码IV型胶原蛋白或层粘连蛋白的cDNA。通过差异菌落杂交筛选重组质粒，以与从F9干细胞和F9-R + DBC细胞分离的聚腺苷酸加尾RNA（poly(A)+ RNA）合成的cDNA杂交。然后将差异杂交的质粒用作探针与RNA转移印迹杂交，以确定其特异性mRNA的大小。只有包含高分子量mRNA特异性cDNA序列的质粒才会进一步分析。通过杂交选择、体外翻译和免疫沉淀研究表明，一个质粒克隆pc15包含与IV型胶原蛋白（α2）mRNA同源的cDNA，另一个质粒克隆pc156包含与层粘连蛋白B mRNA同源的cDNA。通过RNA印迹分析，编码IV型胶原蛋白（α2）的mRNA大小为7.6千碱基；层粘连蛋白的mRNA大小为6.8千碱基。利用RNA印迹杂交技术，我们研究了视黄酸和Bt2cAMP处理后F9细胞中编码IV型胶原蛋白（α2）和层粘连蛋白B的mRNA增加的时间进程。IV型胶原蛋白（α2）和层粘连蛋白B的mRNA在F9干细胞中均有表达。添加视黄酸和Bt2cAMP后约12小时，IV型胶原蛋白（α2）mRNA和层粘连蛋白B mRNA水平略有增加，药物处理后12至24小时之间急剧增加。
