Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and characterization of its 5'-flanking region

Ichiro Manabe, Masahiko Kurabayashi, Yukio Shimomura, Makoto Kuro-o, Noboru Watanabe, Masafumi Watanabe, Masanori Aikawa, Toru Suzuki, Yoshio Yazaki, Ryozo Nagai

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Abstract

To examine the molecular mechanisms that regulate the expression of the SMemb/NMHC-B gene, a nonmuscle myosin heavy chain isoform predominantly expressed in fetal aorta, we have isolated and characterized the 5'-flanking region of the rabbit SMemb/NMHC-B gene. Transient transfection experiments demonstrated that 105 base pairs of 5'-flanking sequence was necessary to direct high level transcription in C2/2 cells, vascular smooth muscle cells derived from rabbit aorta. An essential cis-regulatory element was localized between -100 and -91 base pairs from the transcription start site based on the results that replacement mutagenesis within this region significantly reduced promoter activity. Sequence of this region is completely conserved between mouse and rabbit and fits no known DNA binding consensus. Gel mobility shift assays revealed that a specific DNA-protein complex was formed at this site with nuclear extracts from C2/2 cells, which can be competed by H-2Kb CCAAT box but not by Hsp70 CCAAT box or other CCAAT-containing sequences. We conclude that expression of the SMemb/NMHC-B gene is regulated through an interaction between a sequence element located at -100 and a distinct member of CCAAT-binding proteins.

Original languageEnglish (US)
Pages (from-to)598-605
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume239
Issue number2
DOIs
StatePublished - Oct 20 1997

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Keywords

  • Factor
  • Myosin heavy chain
  • Smooth muscle
  • Transcription

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Manabe, I., Kurabayashi, M., Shimomura, Y., Kuro-o, M., Watanabe, N., Watanabe, M., Aikawa, M., Suzuki, T., Yazaki, Y., & Nagai, R. (1997). Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and characterization of its 5'-flanking region. Biochemical and Biophysical Research Communications, 239(2), 598-605. https://doi.org/10.1006/bbrc.1997.7512