Isolation of cDNA clones specific for collagen IV and laminin from mouse teratocarcinoma cells.
Isolation of cDNA clones specific for collagen IV and laminin from mouse teratocarcinoma cells.
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从小鼠畸胎癌细胞中分离出 IV 型胶原蛋白和层粘连蛋白特异的 cDNA 克隆。
DOI：
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发表时间：
1983
影响因子：
11.1
通讯作者：
L. Gudas
中科院分区：
文献类型：
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作者：
S. Wang;L. Gudas
经维甲酸和N6，O2‘-二丁酰cAMP(Bt2cAMP)处理后，F9小鼠畸胎瘤干细胞中层粘连蛋白和IV型胶原蛋白的合成被刺激约20倍。构建了维甲酸、Bt2cAMP和茶碱处理的F9细胞(F9-R+DBC细胞)的cDNA文库，以分离IV型胶原或层粘连蛋白的编码基因。通过与从F9干细胞和F9-R+DBC细胞中分离的Poly(A)+RNA合成的cDNA进行差异菌落杂交，筛选出重组质粒。然后以差异杂交质粒为探针，与RNA转移印迹杂交，确定其特异性mRNA的大小。只进一步分析了含有大分子量mRNA特异的c DNA序列的质粒。通过杂交筛选、体外翻译和免疫沉淀等研究表明，其中一个质粒克隆pc15含有与IV型胶原(α2)mRNA同源的c DNA，另一个质粒克隆pc 156含有与层粘连蛋白B m RNA同源的c DNA。经RNA印迹分析，编码IV型胶原(α2)的信使核糖核酸大小为7.6kb，层粘连蛋白信使核糖核酸大小为6.8kb。应用RNA印迹杂交技术，研究了维甲酸和Bt2cAMP作用于F9细胞后，编码IV型胶原(α2)和层粘连蛋白B的基因表达水平的时程变化。F9干细胞中存在IV型胶原(α2)和层粘连蛋白B的mRNAs。在加入维甲酸和Bt2cAMP后约12小时，IV型胶原(α2)和层粘连蛋白B的mRNA水平略有升高，在药物治疗后12至24小时间急剧增加。
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.