Determination of the sequence requirements for the expression of a Xenopus borealis embryonic/larval skeletal actin gene

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Abstract
In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealisα3B embryonic/Iarval skeletal actin gene, reside in a 156-nucleotide fragment of the promoter that spans nucleotides -197 to -42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box1) exhibits two binding activities using band-shift analysis. One of these binding activities contains components antigenically related to a serumresponse factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility-shift analysis. Although the shifted complex coelectrophoreses with the CArG box1/serum-response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides -197 and -167. In the actin promoter, a downstream region can apparently fulfil this function.