title: Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo.
abstract: Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.
authors: State Key Laboratory of the Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, 361102, Fujian, China.
authors: Jun
authors: J
authors: Feng
authors: Guang
authors: G
authors: Li
authors: Xin
authors: X
authors: Liu
authors: Jing
authors: J
authors: Wang
authors: Yi-Quan
authors: YQ
authors: Wang
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D004625:Embryo, Nonmammalian; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D064175:Lancelets / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D024363:Transcription Initiation Site
chemical_list: D000199:Actins