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>Molecular Cloning and Functional Analysis of the Promoter of Rat Skeletal Muscle Voltage-Sensitive Sodium Channel Subtype 2 (rSkM2): Evidence for Muscle-Specific Nuclear Protein Binding to the Core Promoter
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Molecular Cloning and Functional Analysis of the Promoter of Rat Skeletal Muscle Voltage-Sensitive Sodium Channel Subtype 2 (rSkM2): Evidence for Muscle-Specific Nuclear Protein Binding to the Core Promoter
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机译:Molecular Cloning and Functional Analysis of the Promoter of Rat Skeletal Muscle Voltage-Sensitive Sodium Channel Subtype 2 (rSkM2): Evidence for Muscle-Specific Nuclear Protein Binding to the Core Promoter
ABSTRACTrSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt −102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5′ upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additionalciselement (−645/−506). The positive and negativecis-elements together drive transcription of the chloramphenicol acetyltransterase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (−44/−29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (−44/−29) are competed by unlabeled −44/−29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides −40 to −38 and −34 to −32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-spec
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