Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters

John R. Gittins, Mary A. Schuler, Åke Strid

Research output: Contribution to journalArticle

Abstract

DNA fragments containing the 5′ promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.

Original languageEnglish (US)
Pages (from-to)231-244
Number of pages14
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1574
Issue number3
DOIs
StatePublished - Apr 12 2002

Keywords

  • Cis-elements
  • Electrophoretic mobility shift assay
  • Nuclear binding factor
  • Pisum sativum sad gene
  • Proximal promoter
  • Stress-induced

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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