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...
We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-4-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-HA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor.However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-levelinducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.Inducible promoter systems are very useful for controlling the levels of gene expression of transfected genes for basic studies of the cell biological effects of a given gene product and, in some cases, for practical genetic engineering applications. We and others have previously described the use, in mammalian cells, of the lac operator-repressor system for this purpose (6,9,11,15,16,25). It was shown that insertion of the lac operator (lacO) into an otherwise constitutive eucaryotic promoter provided lacI-dependent expression when the genes were transfected into eucaryotic cells. Furthermore, treatment of the lacI+ cells with isopropyl-,-D-thiogalactoside (IPTG) resulted in significant derepression. Thus, the lac operator-repressor system was shown to be potentially useful for the design of inducible gene expression systems for transfected genes. This system may be particularly attractive for use with eucaryotic cells and organisms because the inducer, IPTG, does not occur in tissue culture media or in body fluids.However, the levels of induction by IPTG observed by us for a chimeric Moloney sarcoma virus simian virus 40 (SV40) enha...
The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-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 alpha-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 alpha-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 beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.
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