Sequence and expression characteristics of three g-Box-binding factor cdnas from brassica napus

Waldmüller, Stephan; Link, Gerhard

doi:10.1007/bf00020981

Cited by 6 publications

(15 citation statements)

References 34 publications

(52 reference statements)

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“…For instance, the binding sites for bHLHs have been known for some members of the TF family. Both mouse c-myc [11] and Brassica bHLH [12] recognize CACGTG, which was initially known as the core of the G-box (−TCTTACACGTGGCAYY-) on the promoter of a small subunit of ribulose 1, 5-bisphosphate carboxylase gene [13]. Since the regulation of the anthocyanin pathway requires both MYB and bHLH (there is evidence for the bHLH’s binding to CACGTG [14]), this attribute of bHLH may serve as an anchor for obtaining the cis sites for MYB via our bioinformatic approaches.…”

Section: Introductionmentioning

confidence: 99%

A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

et al. 2013

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BackgroundTranscription factor (TF) binding sites (cis element) play a central role in gene regulation, and eukaryotic organisms frequently adapt a combinatorial regulation to render sophisticated local gene expression patterns. Knowing the precise cis element on a distal promoter is a prerequisite for studying a typical transcription process; however, identifications of cis elements have lagged behind those of their associated trans acting TFs due to technical difficulties. Consequently, gene regulations via combinatorial TFs, as widely observed across biological processes, have remained vague in many cases.ResultsWe present here a valid strategy for identifying cis elements in combinatorial TF regulations. It consists of bioinformatic searches of available databases to generate candidate cis elements and tests of the candidates using improved experimental assays. Taking the MYB and the bHLH that collaboratively regulate the anthocyanin pathway genes as examples, we demonstrate how candidate cis motifs for the TFs are found on multi-specific promoters of chalcone synthase (CHS) genes, and how to experimentally test the candidate sites by designing DNA fragments hosting the candidate motifs based on a known promoter (us1 allele of Ipomoea purpurea CHS-D in our case) and applying site-mutagenesis at the motifs. It was shown that TF-DNA interactions could be unambiguously analyzed by assays of electrophoretic mobility shift (EMSA) and dual-luciferase transient expressions, and the resulting evidence precisely delineated a cis element. The cis element for R2R3 MYBs including Ipomoea MYB1 and Magnolia MYB1, for instance, was found to be ANCNACC, and that for bHLHs (exemplified by Ipomoea bHLH2 and petunia AN1) was CACNNG. A re-analysis was conducted on previously reported promoter segments recognized by maize C1 and apple MYB10, which indicated that cis elements similar to ANCNACC were indeed present on these segments, and tested positive for their bindings to Ipomoea MYB1.ConclusionIdentification of cis elements in combinatorial regulation is now feasible with the strategy outlined. The working pipeline integrates the existing databases with experimental techniques, providing an open framework for precisely identifying cis elements. This strategy is widely applicable to various biological systems, and may enhance future analyses on gene regulation.

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Section: Introductionmentioning

confidence: 99%

A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

et al. 2013

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“…Recognition site screening [42] of a ZAPII cDNA library representing cotyledons of 4-day-old lightgrown Brassica napus seedlings has been described [43]. In a secondary screening, protein replica of plaque-purified clones were analyzed in duplicate with a trimer of the RbcS IV G-box (5'-GATCC[TCTGCCACGTGGCCTT]3 G-3') and a monomer of the RbcS III 'split' GS-box (GS-box: 5'-GATCCTTTCTACACCTTTCTTTAATCCTGTGGCA GTG-3', with the separated G-box half-elements underlined), respectively.…”

Section: Expression Library Screening Electrophoretic Mobility Shift mentioning

confidence: 99%

“…Total RNA was isolated from cotyledons of Brassica napus seedlings that were grown either in complete darkness or under constant white light (photon fiuence rate 250 #mol m -2 s-l; [43]). Cotyledon RNA (20 #g per lane) was separated on a 1.7% formaldehyde agarose gel and then transferred onto nitrocellulose membranes as described [17].…”

Section: Rna Isolation and Rna Gel Blot Analysismentioning

confidence: 99%

“…G-box elements of several light-inducible RbcS promoters have recently been used to clone and characterize G-box-binding proteins (GBFs) from Arabidopsis [35], Lycopersicum [23], Brassica [43], Zea [6] and Glycine [ 16]. Three proteins all belong to a group…”

Section: Introductionmentioning

confidence: 99%

“…This motif, which contains a 12-14 base pair spacer interrupting the CAC...GTG half-elements of the G-box core sequence was found in some but not all promoters of the RbcS gene family from Brassica napus [2,11,29,43]. For instance, in the light inducible Brassica RbcS IV promoter the GS box is located downstream of a continuous-type G box [11,43]. For instance, in the light inducible Brassica RbcS IV promoter the GS box is located downstream of a continuous-type G box [11,43].…”

Section: Introductionmentioning

confidence: 99%

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GSBF1, a seedling-specific bZIP DNA-binding protein with preference for a ?split? G-box-related element in Brassica napus RbcS promoters

Waldmüller¹,

Müller²,

Link³

1996

Plant Mol Biol

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Promoters of RbcS genes may contain a GS-box, which is a cis element with a core sequence related to the G-box, but split by a spacer of about 14 bp. Here we describe GSBF1, a DNA-binding protein that specifically interacts with a GS-box element located proximal to the G-box in the Brassica napus RbcS IV promoter. Sequence analysis of GSBF1 revealed a basic region/leucine zipper (bZIP) domain that displays structural features distinct from that of G-box binding factors (GBFs). Gel shift experiments showed that recombinant GSBF1 does not efficiently bind to the continuous G-box motif. RNA gel blot analysis indicated that GSBF1 transcripts are cotyledon-specific and accumulate to the highest levels during late seedling development in a ligh-dependent manner. During the same time period, RbcS IV transcript levels decreased simultaneously, suggesting that GSBF1 acts as a developmental, stage-specific, negative regulator of RbcS IV gene expression in rape seedlings.

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Isolation and Analysis of Gene Regulatory Sequences

Hehl¹,

Steffens²,

Wingender³

2004

Handbook of Plant Biotechnology

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Database‐assisted bioinformatic approaches facilitate the analysis of gene regulatory sequences and allow the identification of putative transcription factor binding sites or common regulatory motifs in similarly expressed genes. These approaches complement experiments that identify specific regulatory sites. They may be easily established if the respective transcription factors responsible for gene expression have already been cloned and annotated to the databases. In this chapter we summarise recent results on transcriptional regulation in plants and describe the most abundant transcription factor families of Arabidopsis thaliana . We summarise experimental approaches to delineate gene regulatory sequences that should precede a database‐assisted analysis. The plant cis‐regulatory DNA element database PLACE and the transcription factor databases PlantCARE and TRANSFAC® are described, including connected software tools. PlanCARE is a plant specific database and TRANSFAC® covers transcription factors of all eukaryotes. The content and use of the TRANSFAC® database is presented in detail. Furthermore, we give examples on the identification of putative transcription factor binding sites in previously identified regulatory sequences and compile other databases that are relevant for the identification of regulatory sequences.

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Sequence and expression characteristics of three g-Box-binding factor cdnas from brassica napus

Cited by 6 publications

References 34 publications

A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

GSBF1, a seedling-specific bZIP DNA-binding protein with preference for a ?split? G-box-related element in Brassica napus RbcS promoters

Isolation and Analysis of Gene Regulatory Sequences

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