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

Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters
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.
Cis-elements, Electrophoretic mobility shift assay, Nuclear binding factor, Pisum sativum sad gene, Proximal promoter, Stress-induced
0167-4781
231-244
Gittins, John R.
c4d269cc-aae0-4182-bc81-78dc724f7d95
Schuler, Mary A.
556972bc-02ec-4231-b328-2d67904b1bf5
Strid, Åke
5291be25-7846-4524-b9cb-d4f0fc2504cc
Gittins, John R.
c4d269cc-aae0-4182-bc81-78dc724f7d95
Schuler, Mary A.
556972bc-02ec-4231-b328-2d67904b1bf5
Strid, Åke
5291be25-7846-4524-b9cb-d4f0fc2504cc

Gittins, John R., Schuler, Mary A. and Strid, Åke (2002) Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1574 (3), 231-244. (doi:10.1016/S0167-4781(01)00366-9).

Record type: Article

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.

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More information

Published date: 12 April 2002
Keywords: Cis-elements, Electrophoretic mobility shift assay, Nuclear binding factor, Pisum sativum sad gene, Proximal promoter, Stress-induced
Organisations: Ocean and Earth Science

Identifiers

Local EPrints ID: 380345
URI: http://eprints.soton.ac.uk/id/eprint/380345
ISSN: 0167-4781
PURE UUID: 97fb4c8b-530d-45ca-abf4-f7c1466ecbb2

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Date deposited: 12 Aug 2015 15:22
Last modified: 08 Jan 2022 15:23

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Contributors

Author: John R. Gittins
Author: Mary A. Schuler
Author: Åke Strid

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