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DNA-protein interactions in an alpha-amylase gene transcription complex

Reference	BBS/E/C/00803300
Principal Investigator / Supervisor	Dr Alison Huttly
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	38,530
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

The hormone gibberellin (GA) controls many processes in plant growth and development. One of the best understood is the synthesis of hydrolytic enzymes by the aleurone layer of the cereal grain during endosperm mobilisation, where a direct link has been established between increased GA levels and increased transcription of specific genes such as those encoding the enzyme alpha-amylase. Application of abscisic acid (ABA) to aleurone cells prevents GA induced transcription of alpha-amylase genes. Our objective is to determine the mechanism by which GA and ABA control gene expression in aleurone cells. A structural analysis of a wheat alpha-amylase promoter was carried out by assaying the ability of mutant promoters to direct expression transiently in aleurone protoplasts, combined with in vitro DNase I foot printing of the same promoter. This indicated the presence of a number of binding sites for trans- acting factors, each essential for promoter function. We used this information to isolate cDNA clones for aleurone proteins capable of binding to the alpha- amylase promoter by screening an expression library, constructed from aleurone mRNA, with labelled double stranded oligonucleotides of the functional cis- elements. 2 Two clones which bound to one element were subjected to DNA sequence analysis and were found to encode two unrelated proteins with the exception of a conserved stretch of ca. 60 amino acids, present twice in ABF1 and once in ABF2, that contains a possible zinc-finger motif. ABF2 also contains a possible leucine zipper motif. The potential role of these proteins in controlling alpha-amylase gene transcription is being investigated by determination of the precise DNA-binding characteristics of heterologously expressed protein by gel retardation and DNAse I foot printing and relating this to functional analysis of mutated alpha- amylase promoters by transient expression in aleurone protoplasts. In addition the role of these proteins will be investigated. by an antisense approach or expression of dominate negative mutants in aleurone protoplasts. Expression of the wheat Em gene family in aleurone cells is also under investigation. Objectives 1996 Continue investigation on the binding specificity of ABF1 and ABF2. Construct mutated forms of the proteins and assess their ability for DNA-binding activity by gel retardation, and for ability to bind zinc or other co-factors following expression in E. coli. 1997 Complete and submit to Plant Molecular Biology final publication on DNase1 footprinting of wild oat will-amylase promoter. Continue investigation into the roles of ABF1,2 and 3 in aleurone cells and determine their optimal binding sites. Look for interacting partners using the yeast two hybrid system and through gel retardation and super-shifts with the oat GA-myb homologue.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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