Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle
Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle
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DOI：
10.1089/104454999315655
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发表时间：
1999-01-01
影响因子：
3.1
通讯作者：
Gong, ZY
中科院分区：
文献类型：
--
作者：
Xu, YF;He, JY;Gong, ZY
斑马鱼肌球蛋白轻链2 cDNA克隆的分离和表征。克隆的序列分析显示与编码快速骨骼肌同种型MLC 2f的哺乳动物和禽类基因具有高度同源性。原位杂交和北方印迹杂交分析表明，斑马鱼MLC 2f mRNA只在快速骨骼肌中表达。个体发育学上，MLC 2f mRNA出现在受精后16小时左右（hpf）的前几个发育良好的前体节中。在后期阶段，MLC 2f mRNA也可以在鳍芽、眼肌和颌肌中检测到。为了开发一个有用的模型系统，用于分析肌肉基因调控，斑马鱼MLC 2f基因的启动子分离和连接到氯霉素乙酰转移酶（CAT）报告基因。MLC 2f/CAT嵌合构建体通过直接注射到斑马鱼骨骼肌中进行分析，并观察到显著的CAT活性;相反，从类似注射的催乳素基因启动子/CAT基因构建体产生很少或没有CAT活性。在MLC 2f启动子区域的1 kb内，鉴定了几个MEF 2结合位点和E盒，表明MLC 2f可以由肌肉转录因子MEF 2和肌源性bHLH蛋白调节。5'缺失分析表明，距转录起始位点的近端79个核苷酸（其含有单个MEF 2结合位点）足以驱动注射肌肉中的高水平CAT活性。在-79-bp构建体中MEF 2元件的内部缺失导致CAT活性降低80%，而在-1044-bp构建体中相同MEF 2元件的内部缺失对诱导的CAT活性没有影响。这些观察结果表明，MEF 2元件是重要的激活肌细胞中的MLC 2f基因，和近端MEF 2元件的损失的影响可以补偿的上游MEF 2：元件的存在。本研究还表明，直接注射DNA到骨骼肌中是一种有效的和有价值的方法来分析肌肉基因启动子在斑马鱼。
A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the zebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. Ontogenetically, the MLC2f mRNA appears around 16 hours postfertilization (hpf) in the first few well-formed anterior somites. At later stages, the MLC2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the promoter of the zebrafish MLC2f gene was isolated and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constructs were analyzed by direct injection into the zebrafish skeletal muscle, and significant CAT activity was observed; in contrast, little or no CAT activity was generated from a similarly injected prolactin gene promoter/CAT gene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-binding sites and E-boxes were identified, suggesting that MLC2f can be regulated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the transcription start site, which contains a single MEF2-binding site, is sufficient to drive a high level of CAT activity in injected muscle. Internal deletion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044-bp construct had no effect on induced CAT activity. These observations suggest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensated for by the presence of the upstream MEF2: elements. This study also demonstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.