Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters.
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.
Gittins
John R
JR
Biokemi och Biofysik, Institutionen för Kemi, Göteborgs Universitet, P.O. Box 462, S-405 30 Göteborg, Sweden.
Schuler
Mary A
MA
Strid
Ake
A
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D018532:Pisum sativum / Q000235:genetics* / Q000528:radiation effects; D010940:Plant Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D014466:Ultraviolet Rays; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010940:Plant Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D000426:Alcohol Dehydrogenase