Daubas P, Klarsfeld A, Garner I, Pinset C, Cox R, Buckingham M
Départment de Biologie Moléculaire, Institut Pasteur, Paris, France.
Nucleic Acids Res. 1988 Feb 25;16(4):1251-71. doi: 10.1093/nar/16.4.1251.
Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.
将编码MLC1F和MLC3F的小鼠肌球蛋白碱性轻链基因、小鼠α-心肌肌动蛋白基因以及鸡乙酰胆碱受体α亚基基因的近端上游侧翼序列与细菌氯霉素乙酰转移酶（CAT）基因相连，并转染到源自小鼠骨骼肌的原代培养物或成肌细胞系中。我们证明小鼠MLC1F/MLC3F基因有两个功能性启动子。在原代肌肉培养物中，外显子1（MLC1F）侧翼的1200 bp序列和外显子2（MLC3F）侧翼的438 bp序列可在肌管中指导CAT活性，但在成肌细胞或非肌源性3T6和CV1细胞中则不能。在原代培养系统中，α-心肌肌动蛋白（320 bp）和乙酰胆碱受体α亚基（850 bp）上游序列也呈现出发育调控表达。用成肌细胞系进行的转染实验显示，给定的启动子构建体有不同结果，这反映了不同细胞系之间调控因子水平可能存在差异。不同的肌肉基因启动子在给定的细胞系中表现不同，表明这些启动子对调控因子的需求不同。
