5?-upstream cis-elements and binding factor(s) potentially involved in light-regulated expression of a Brassica napus rbcS gene

One of the essential issues regarding evolution of polyploid species is how duplicate genes are expressed. Most studies on gene expression in polyploids have been based on isozyme analyses; RNA analysis has not been widely used partially due to difficulties in distinguishing homologous transcripts which usually have the same length and similar or almost identical sequences. In this study, a method combining RT-PCR with RFLP was used to analyze transcripts of homologous genes in natural and synthetic Brassica amphidiploids. Sequences coding for several known genes were selected and used to synthesize gene-specific primers. Total RNAs were used as templates for RT-PCR to amplify homologous transcripts in three diploid parental species, three cultivated amphidiploid species and six synthetic amphidiploids. For each gene, initial PCR products amplified in all species had identical length; however, homologous transcripts in the diploid and amphidiploid species could be distinguished after digesting the PCR products with restriction enzymes. Preliminary results based on three genes indicated that both transcripts from the diploid parents were expressed in the synthetic and natural amphidiploids. This study represents the first application of RT-PCR and RFLP analysis to investigate expression of homologous genes in higher plants. The technique is a sensitive, simple and efficient method for distinguishing homologous transcripts in a mixed RNA population and can be applied to many types of studies on expression of homologous genes.

Section: Introductionmentioning

confidence: 94%

A method for examining expression of homologous genes in plant polyploids

Song

Osborn

1994

“…In contrast, a 200-fold molar excess of the GS-box oligonucleotide did not interfere with the G-box-binding of the recombinant GB Fs. Moreover, no binding could be observed when employing radiolabelled GS-box oligonucleotides in the binding reactions, indicating that the three BnGBFs are not related to the GS-box-binding activity previously identified in whole cell extracts of Brassica napus [ 11 ].…”

Section: Cloning Of Three Different Gbf Cdnas From Brassica Napusmentioning

confidence: 99%

“…We have previously analysed the 5'-upstream regions of members of the rbcS family from Brassica napus for eis elements, which include the TATA-box, G-box, GS-box and I-box [ 1,10,11 ]. As a first step towards characterization of cognate trans factors, we isolated GBF cDNAs by recognition site screening [31 ].…”

Section: Cloning Of Three Different Gbf Cdnas From Brassica Napusmentioning

confidence: 99%

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

Waldmüller¹,

Link²

1995

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G-box-binding factors (GBFs) are bZIP proteins that have been implicated in the transcriptional control of a number of plant genes including the family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Using rbcS promoter regions as recognition site probes, we have cloned three Brassica GBFs designated as BnGBF1a, 1b and 2a. RNA gel blot analyses showed that all three BnGBF sequences give transcripts of the same size (1.3 kb) but in different amounts at a constant ratio in various tissues and developmental stages (1a > 2a > 1b). Transcript pools were largest in photosynthetically active organs such as leaves and cotyledons. Pool sizes correlated with those of total rbcS transcripts.

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

Self Cite

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.