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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

We identified cis elements in the 5′-flanking region of rat Na,K-ATPase α2 subunit gene (Atp1 a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5′-deletion mutation analysis, the region between nucleotide positions –175 and –108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides –144 to –139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides –123 to –118) and the GGGAGG sequence (nucleotides –114 to –109) act as positive regulatory elements. Gel-retardation analysis revealed that binding factors are an E-box-binding protein and Sp1. DNase I foot-printing and methylation-interference analyses revealed that Sp1 binds to the region from nucleotides –122 to –101 and the E-box-binding protein to the region from nucleotides –144 to –136. T4 DNA polymerase footprinting revealed that there are three Sp1-binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.

Abbreviations
Atp1a2

Na,K-ATPase α2 subunit gene

CAT

chloramphenicol acetyltransferase

Enzymes
 

Na,K-ATPase (EC 3.6.1.3)

 

T4 DNA polymerase (EC 2.7.7.7)

 

restriction endonucleases (EC 3.1.21.4)

 

Klenow fragment (Escherichia coli DNA polymerase I) (EC 2.7.7.7)

 

chloramphenicol acetyltransferase (EC 2.3.1.28)

 

T4 polynucleotide kinase (EC 2.7.1.78)

 

DNase I (EC 3.1.21.1)

REFERENCES

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  2. Abstract
  3. REFERENCES
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