Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif

Abstract

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.Seed storage proteins serve as a nitrogen and sulfur source for the developing plant seedling during the period of early germination. Storage proteins from cultivated plant species also constitute a major animal and human protein source. Since storage proteins are only expressed in embryonic and/ or endosperm tissues during seed development, they are presently in the focus for studies on developmentally regulated gene expression in plants. Napin is one of the dominating storage proteins of Brassica napus (rape) and variant forms are found among the Brassicaceae, e. g. Raphanus sativus (radish) [l], Sinapis alba (white mustard) [2], and Arabidopsis thaliana [3]. In addition, homologous storage proteins have been characterised in Bertholletia exelsa (brazil nut) [4], Hellianthus annus (sun flower) [5], Triticum aestivum (wheat) [6], and Ricinus communis (castor bean) [7]. The presence of a related protein in the spores of a fern (Matteucia struthiopteris) has also been reported [8, 91. Napin is encoded by a gene family [lo, 111 and several isoforms of napin exist [12]. The sequences of three napin genes, napA [lo], pGNA [ll], and BngNAPl [13], have so far been published. Radke et al. [14] have shown that 300 bp of the 5' flanking sequence in front of the translation initiation codon are sufficient to give a developmentally faithful expression of a chimeric gene construct in transgenic B. napus plants. However, it has not been ascertained whether all sequence motives regulating the napin gene expression are included in this segment of the upstream region.With the underlying assumption that regulatory promoter elements will prove to be conserved between the different Correspondence to

High Affinity Amino Acid Transporters Specifically Expressed in Xylem Parenchyma and Developing Seeds of Arabidopsis

“…Two potential TATA boxes were identified 106 and 164 bases upstream of the ATG (Fig 2B). A motif (napin motif TACACAT) responsible for specific binding of a protein extracted from developing Brassica napus seeds was found at position -218 (40). Repeated CAT elements were found around -480 bp, and a SEF1 element (Soybean embryo factor 1) is located around -790 bp.…”

A cotyledon regulatory region is responsible for the different spatial expression patterns of Arabidopsis 2S albumin genes

“…In the promoter region of Brassica napus 2S albumin genes (napins), sequence elements interacting with nuclear proteins have been mapped (Ericson et al, 1991 ;Gustavsson et al, 1991 ;St.~lberg et al, 1993). A search for homologies in the analogous regions of the Arabidopsis and napin 2S albumin genes made it possible to design a series of hybrid at2S1/at2S2 promoters in which conserved sequences were exchanged.…”

Expression in Escherichia coli of Sin a 1, the Major Allergen from Mustard

“…based on the primary structure of the natural protein obtained from mustard seeds [15]. Two DNA fragments were produced after amplification (data not shown), one of them approximately 440 bp in size, in good agreement with the size expected from the amino acid sequence of Sin a 1 plus the described IPFs from homologous 2 s proteins [17,[22][23][24][25][26][27]. This fragment was isolated from agarose gels and cloned into pUC18.…”

“…It was expected that the product of such a construction should be conformationally very similar to the natural protein, because the folding of this molecule in vivo must occur before the proteolytic processing of the IPF [36]. Given that napins are encoded by genes without introns [17,[22][23][24][25][26][27], it was possible to use a genomic sequence for the expression of Sin a 1 allergen, which, in conjunction with the use of PCR, avoids the tedious screening of cDNA libraries.…”

“…The primers used encode the N-terminal and C-terminal amino acid sequences of the Sin a 1 allergen [IS]. The codon usage was selected on the basis of the nucleotide sequences of homologous proteins [17,[22][23][24][25][26][27]. BamHI and EcoRI restriction sites were incorporated at the 5' ends of each primer to facilitate the ligation of the amplified DNA into vectors [28].…”