Two cis-regulatory elements are essential for the muscle-specific expression of an actin gene in the ascidian embryo.

Muscle cells in the anterior and middle part of the ascidian larval tail differentiate in a highly autonomous manner. Because this autonomy is due to so-called muscle determinants in the myoplasm of fertilized eggs, there is a genetic cascade for muscle cell differentiation in ascidian embryos, which begins with muscle determinants and ends with the expression of muscle-specific structural genes. In this genetic cascade, an important research subject is the disclosure of the molecular nature of muscle determinants. We approached this issue by going upstream of the cascade by analyzing transcriptional control mechanisms of muscle-specific structural genes. The HrMA4a actin gene, a muscle-specific structural gene, has been analyzed for this purpose. It has been demonstrated that a short upstream sequence up to -103 bp from the transcription start site is sufficient for the muscle-specific expression of HrMA4a. In the present study, we performed a more detailed analysis using the β-galactosidase gene as a reporter. We demonstrated here that, within the proximal region (-103 to -66) of the HrMA4a gene, there are two short sequences essential to the muscle-specificity of HrMA4a promoter, one is 9 bp long (5'-TCGCACTTC-3') and the other is 13 bp long (5'-GTGATAACAACTG-3').