The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.

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.

We have isolated a genomic clone, related in sequence to the skeletal-actin gene sub-family. It is expressed in the skeletal muscle of embryos from the neurula stage onwards and in tadpoles, but not in adults. The equivalent Xenopus Zuevis gene is expressed as a major transcript in adult muscle, as well as at earlier stages. The intron/exon structure is typical of vertebrate skeletal-actin genes, as is the possession of multiple copies of three serum-response elements in the promoter of this gene. TheXenopus actin and fl-globin genes were fused in their second introns. This construct, which contained 2.4 kb of upstream sequence, was injected into fertilized eggs at the two-cell stage. It showed the normal pattern of tissue-specific transcription. Thus all of the information necessary for appropriate expression of this actin gene in the embryo is contained in the region that extends from a point 2.4 kb upstream of transcription initiation to the centre of the second exon. A series of enhancer constructs were made in which upstream regions of the actin gene were placed upstream of a X. laevis p-globin gene. The region immediately adjacent to the promoter, containing the three serum-response elements, was able to drive muscle-specific expression, and there was also a general enhancement of transcription by regions further upstream.