Molecular cloning of gene sequences transcriptionally regulated by retinoic acid and dibutyryl cyclic AMP in cultured mouse teratocarcinoma cells.
Molecular cloning of gene sequences transcriptionally regulated by retinoic acid and dibutyryl cyclic AMP in cultured mouse teratocarcinoma cells.
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DOI：
10.1016/0012-1606(85)90377-x
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发表时间：
1985
影响因子：
2.7
通讯作者：
S. Wang;G. Larosa;L. Gudas
中科院分区：
文献类型：
--
作者：
S. Wang;G. Larosa;L. Gudas
F9小鼠畸胎癌干细胞在维甲酸（RA）诱导下分化为原始内胚层细胞。从RA和二丁酰环AMP处理的F9畸胎瘤细胞合成的cDNA文库，已筛选出RA调控的基因序列。通过杂交筛选和体外翻译，pcJ 6、pcJ 31和pcF 117同源mRNA分别编码40，000、35，000 - 37，000和24，000 Da的多肽。pcI 56和pcI 5同源mRNA编码层粘连蛋白B和胶原IV（S-Y. Wang和L. J. Gudas，1983，Proc. Natl. Acad. Sci. USA80，5880-5884）。在RA添加之前，这些基因序列对应于低丰度mRNA（小于总细胞mRNA的0.05%）。在向F9细胞中加入RA（5 × 10− 7 M）后24小时内，这些序列的表达急剧增加，在药物加入后72小时，这些基因的诱导可达到5- 30倍。添加较低浓度的RA导致这些基因表达的较小增加。如果用RA（5 × 10− 7 M）和双丁酰环腺苷酸（2cAMP）处理F9细胞，这5种可诱导基因的特异性mRNA水平比干细胞中的mRNA水平进一步增加约30- 110倍;但2cAMP单独不能诱导这些基因的表达。在P19畸胎瘤干细胞系中，J6和J31的表达受RA调控，而I56（层粘连蛋白）和I5（IV型胶原）则不受RA调控。P19畸胎瘤干细胞系在对RA的反应中分化为成纤维细胞类型。体外转录实验表明，层粘连蛋白和IV型胶原受RA的转录调控;但2cAMP也增强了F9细胞中这些基因的转录速率。
F9 mouse teratocarcinoma stem cells differentiate into primitive endoderm cells in response to retinoic acid (RA). A cDNA library, synthesized from RA and dibutyryl cyclic AMP-treated F9 teratocarcinoma cells, has been screened for gene sequences regulated by RA. By hybridization-selection andin vitrotranslation, the pcJ6, pcJ31, and pcF117 homologous mRNAs encode polypeptides of 40,000; 35,000–37,000; and 24,000 Da respectively. The pcI56 and pcI5 homologous mRNAs encode laminin B and collagen IV (S-Y. Wang and L. J. Gudas, 1983,Proc. Natl. Acad. Sci. USA80,5880–5884). Prior to RA addition, these gene sequences correspond to low-abundance mRNAs (less than 0.05% of total cellular mRNAs.). Within 24 hr after the addition of RA (5 × 10−7M) to F9 cells, the expression of these sequences increases dramatically, and at 72 hr after drug addition, a 5- to 30-fold induction of these genes can be attained. Addition of lower concentrations of RA results in a smaller increase in the expression of these genes. If the F9 cells are treated with both RA (5 × 10−7M) and dibutyryl cyclic AMP (but2cAMP), the levels of mRNA specific for these five inducible genes are further increased by approximately 30- to 110-fold over those in the stem cells; but2cAMP alone does not induce the expression of these genes. The expression of J6 and J31, but not I56 (laminin) and I5 (collagen IV), is also regulated by RA in the P19 teratocarcinoma stem cell line, which differentiates into a fibroblastic cell type in response to RA.In vitrotranscription experiments demonstrate that laminin and collagen IV are transcriptionally regulated by RA; but2cAMP also enhances the rate of transcription of these genes in F9 cells.