Cis -Regulatory elements conserved in the proximal promoter region of an ascidian embryonic muscle myosin heavy-chain gene

Abstract

The B-line muscle cells of the ascidian embryo are specified autonomously depending on determinants prelocalized in the myoplasm of unfertilized eggs. Expression of muscle-specific actin and myosin heavy-chain genes commences in the B-line presumptive muscle cells as early as the 32-cell stage. To explore the intrinsic genetic program for this differentiation, we analysed cis-regulatory elements of the Halocynthia roretzi muscle myosin heavy-chain gene (HrMHC1). Comparison of the entire amino acid sequence of HrMHC1 with those of other invertebrates and vertebrates indicated that HrMHC1 resembles myosin heavy-chain of vertebrate skeletal and cardiac muscles. A fusion gene was constructed consisting of 132 bp upstream the 5'-end of HrMHC1 gene fused to a bacterial lacZ reporter. When the fusion gene was microinjected into fertilized eggs, the reporter gene was eventually expressed only in muscle cells of tailbud embryos. It has been reported that 103 bp of sequence 5' of the transcription start site of the ascidian embryonic muscle actin gene (HrMA4) contains information sufficient for muscle-specific expression (Hikosaka et al. 1994). Comparison of the 132 bp of sequence 5' of the HrMHC1 gene with the 103 bp of sequence 5' of the HrMA4 gene revealed several common motifs shared by the two genes (E-box, GATA box and Boxes A, B, T1 and T2). Point mutations inserted into these motifs suggested that the Box T1/T2 (TTTTTTCTTTCA) is critical for the promoter activity of the HrMHC1 gene.