Kamada S, Miwa T
Department of Oncogene Research, Osaka University, Japan.
Gene. 1992 Oct 1;119(2):229-36. doi: 10.1016/0378-1119(92)90276-u.
A CArG box motif [CC(A+T-rich)6GG] is one of the DNA elements required for muscle-specific gene transcription. Nuclear factors in mouse C2 myogenic cells strongly bind to the CArG box in the first intron of the gene (Sm alpha-A) encoding human smooth muscle alpha-actin. To clone cDNAs of the CArG box-binding factor (CBF), lambda gt11 cDNA expression libraries from C2 cells were screened for in situ DNA binding specific for this CArG box sequence. The 1.6-kb cDNA (CBF-A) encoding 285 amino acids (aa) was obtained, and a beta-galactosidase fusion protein, bacterially produced from the cDNA, bound to DNA fragments containing several CArG boxes. When the expression level of CBF-A in C2 cells increased by transfection of CBF-A expression plasmids, Sm alpha-A transcription was repressed. The deduced aa sequence of CBF-A is similar to some single-stranded (ss) nucleic acid-binding proteins. The fusion protein could bind to ssDNA, whereas CBF in C2 cell nuclear extracts could not. From these results, CBF-A is a novel CArG box-, ssDNA- and RNA-binding protein, as well as a repressive transcriptional factor.
一个CArG框基序[CC(A+T富含区)6GG]是肌肉特异性基因转录所需的DNA元件之一。小鼠C2成肌细胞中的核因子与编码人平滑肌α-肌动蛋白的基因（Smα-A）第一个内含子中的CArG框强烈结合。为了克隆CArG框结合因子（CBF）的cDNA，对来自C2细胞的λgt11 cDNA表达文库进行筛选，以寻找对该CArG框序列具有原位DNA结合特异性的克隆。获得了编码285个氨基酸（aa）的1.6kb cDNA（CBF-A），并且由该cDNA在细菌中产生的β-半乳糖苷酶融合蛋白与含有几个CArG框的DNA片段结合。当通过转染CBF-A表达质粒使C2细胞中CBF-A的表达水平增加时，Smα-A转录受到抑制。CBF-A推导的氨基酸序列与一些单链（ss）核酸结合蛋白相似。融合蛋白可以与ssDNA结合，而C2细胞核提取物中的CBF则不能。根据这些结果，CBF-A是一种新型的CArG框、ssDNA和RNA结合蛋白，也是一种抑制性转录因子。
