Dubin R A, Wawrousek E F, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1989 Mar;9(3):1083-91. doi: 10.1128/mcb.9.3.1083-1091.1989.
The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.
克隆了小鼠αB-晶状体蛋白基因并检测了其表达。在小鼠中,不仅在晶状体中检测到显著水平的αB-晶状体蛋白RNA,在心脏、骨骼肌、肾脏和肺中也检测到;在脑和脾中分别检测到低水平和痕量水平。肺、脑和脾中的RNA种类比其他组织中的RNA种类大400至500个碱基。晶状体、心脏、骨骼肌、肾脏和脑中的转录起始于同一位置。构建了一个小鼠αB-晶状体蛋白微型基因并将其导入小鼠种系,结果表明其表达与内源基因的表达平行。转基因RNA总是在晶状体、心脏和骨骼肌中被检测到,而在肾脏和肺中的表达则是可变的;脑和脾中是否存在转基因表达仍不确定。这些结果表明,控制αB-晶状体蛋白基因表达的调控序列位于转录起始位点上游666个碱基对与聚腺苷酸(poly(A))添加位点下游2.4千碱基对之间,且不位于内含子内。对一系列αB-晶状体蛋白微型基因缺失突变体进行的转染研究表明,-222至-167位之间的序列是在原代胚胎鸡晶状体细胞中高效表达所必需的;在这个体外系统中,聚腺苷酸添加信号下游的序列对于表达是可有可无的。