The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

Abstract

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal a-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal a-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal a-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein.Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle a-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal (-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal a-actin genes.The actins represent a multigene family of highly conserved proteins found in all eucaryotes. Differences in amino acid sequence among the various actins have shown that at least six different isoforms are expressed in vertebrates (52, 53). Two striated muscle isoforms, skeletal a and cardiac a (52), and two smooth muscle isoforms (53) are found in the contractile apparatus of muscle fibers, whereas two cytoskeletal isoforms, p and y, are present in the cytoskeleton of all cells (51). These actin proteins are extremely conserved in amino acid sequence.Actin gene expression is tissue specific and developmentally regulated (27,29,32,35). By studying the structural organization of the actin gene family, one can begin to look for the controlling elements which modulate the expression of these genes during development. Here we present the complete nucleotide sequence of the single genomic copy of the mouse skeletal a-actin gene. The coding region of this gene is interrupted by five introns which are located in the same positions as introns previously identified in other vertebrate skeletal a-actin genes (13, 54). A comparison of the nucleotide sequences of several vertebrate skeletal aactin genes reveals several blocks of highly conserved sequences in the 5'-flanking region and in both the 5'-and 3'-untranslated regions. Interestingly, the conserved sequences in the 5'-flanking region and within the first untranslated exon can potentially form several hairpin loops by base pairing between adjacent inverted complementary sequences. These regions do not correspond to the potential hairpin structure in the corresponding portion of the rat cytoskeletal ,B-actin gene (33). Furthermore, it is possible to form long hairpin loops within the first intron and one stem loop in the 3'-untranslated region upstream from the putative...

Expression of Gap-Junctional Protein (Connexin 43 or .ALPHA.1 Gap Junction) is Down-Regulated at the Transcriptional Level during Adipocyte Differentiation of H-1/A Marrow Stromal Cells.

“…The GPD probe was a 0.9 kb EcoRI fragment of pL10.9 (28). The actin probe was a 6.8 kb EcoRI fragment ofpSP62-PL (15). With the use of this a-actin DNA, /3-actin mRNA was detected by RNAblot analysis to check the total amount of RNAloaded.…”

“…RNAwas prepared from cultured cells by homogenization in guanidinium isothiocyanate, followed by centrifugation over a cesium chloride cushion (5). The RNAwas then electrophoresed in a 1.0% agarose gel, transferred to a nylon filter (Du Pont Company NENProducts), and hybridized with CDNAinserts labeled with 32P-dCTP by the random-primer method (9) at 65°C for [14][15][16] hours in buffer containing 5xSSPE…”

Cross-binding of factors to functionally different promoter elements in c-fos and skeletal actin genes.

“…The MAPF footprint occurs within a dyad symmetry element that is conserved in the actin genes (22,32,33,47). These sequences are found in muscle and nonmuscle genes, and they have been collectively referred to as "CArG boxes" (29).…”

A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

“…Comparison of nucleotide sequences within these regulatory regions revealed stretches of several conserved motifs shared by many of these myogenic cell-specified genes (25,34,44,50). However, no common sequence has been identified that coordinates the transcriptional activation of all myogenic cell-regulated genes.…”