Complete nucleotide sequence of a French bean storage protein gene: Phaseolin

Abstract

The complete nucleotide sequences of the gene and the mRNA coding for a specific phaseolin type French bean major storage protein have been determined. Comparison of these sequences reveals a phaseolin gene structure consisting of 80 base pairs (bp) of 5' untranslated DNA, 1,263 bp of protein-encoding DNA which is interrupted by five intervening sequences (IVSI,72 bp; IVS2, 88 bp; IVS3, 124 bp; IVS4, 128 bp; and IVS5, 103 bp), and 135 bp of 3' untranslated DNA. Sequences characteristic of eukaryotic promoters "CCAAT" and "TATA" are present in the 5' flanking DNA, and the eukaryotic poly(A) addition signal A-A-T-A-A-A occurs 16 bp before the first nucleotide of poly(A). The derived amino acid sequence yields an amino acid composition and a molecular weight compatible with those found for the a-type phaseolin protein. Two regions that probably serve as carbohydrate-peptide linkage recognition sites have been identified. A region of highly hydrophobic amino acids at the NH2 terminus of the protein suggests the presence of a signal peptide in the newly synthesized phaseolin protein."Phaseolin" is the name of a group of polypeptides which make up the major storage glycoprotein in the seeds of French bean (Phaseolus vulgaris L.), representing about 50% of the total protein in mature seeds (1). One-dimensional NaDodSO4/ polyacrylamide gel electrophoresis of phaseolin isolated from cotyledons of the cultivar Tendergreen resolves three polypeptide bands-a, ,B, and y, of [51][52][53][47][48], and 43-46 kilodaltons (kDa), respectively (2, 3). All three polypeptides are encoded in 16S mRNA species, and these proteins accumulate rapidly in the developing seed cotyledon, beginning when the cotyledons are about 7 mm in length and continuing until the cotyledons reach 17-19 mm in length (4). Two-dimensional gel electrophoretic separation of phaseolin resolves five polypeptides, indicating charge and molecular weight heterogeneity in the phaseolin protein pool (2). Peptide mapping of these phaseolin proteins after proteolytic and chemical cleavages shows that all of these proteins are highly homologous (5, 6), suggesting that they may be encoded in a multigene family. Genomic blot analysis using a cloned phaseolin gene as probe confirms that phaseolin is indeed encoded in a. multigene family (unpublished data).We have previously reported (7) the isolation and partial nucleotide sequence of the phaseolin genomic clone AG-APVPh 177.4 (A177.4) and of a cloned phaseolin cDNA, AG-cpPVPhl (cDNA1) which contains about 40% of a phaseolin mRNA transcript (7).In this paper we report the complete nucleotide sequence determination of the phaseolin genomic clone A177. A177.4 and its subclone AGpPVPh7.2 (p7.2) have been described (7). Additional pBR322 subclones from A177.4 (used to facilitate DNA sequence determinations) are the 3.0-kilobase pair (kbp) EcoRI-BamHI subelone AG-pPVPh3.0 and the 3.8-kbp Bgl II-BamHI subclone AG-pPVPh3.8 (see Fig. 1, clone 177.4 for location of these subcloned regions). These subclones were construc...

A Small Phospholipase A2-α from Castor Catalyzes the Removal of Hydroxy Fatty Acids from Phosphatidylcholine in Transgenic Arabidopsis Seeds

“…A common motif (-LHKP) is found at the C terminus of both the rice and castor sPLA 2 a sequences, constituting a putative ER retention signal (Singh et al, 2012). RcsPLA 2 a and RcsPLA 2 b were cloned from a castor endosperm complementary DNA (cDNA) library, introduced into the expression vector pGate-DsRed-Phas (Lu et al, 2006), and expressed under the control of the seed-specific phaseolin promoter (Slightom et al, 1983) in the Arabidopsis CL37 line. CL37 is an Arabidopsis line expressing RcFAH12 and accumulating 17% HFAs, including ricinoleic acid and densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid [18:2-OH]; Lu et al, 2006).…”

Phaseolin: Structure and Evolution

“…Three TATA box sequences are located upstream from the mRNA cap at positions -28, -37 and -39. Slightom et al [25] postulated that the sequence at -28 was likely to be the most important in driving high expression from the phas promoter; this was later confirmed by Grace et al [40] who used an in vitro transcription system that revealed that the sequence and spacing of the TATA box elements are critical for accurate initiation from the -phaseolin promoter. The A+T content of the introns is similar to that in soybean proteins, but is considerably higher than those of non-plant species.…”

“…Zein, the major storage prolamin storage protein of maize [42] is encoded by a family of highly similar genes bearing a TATA motif at -32, similar to that of most eukaryotes. In contrast, the zein CCAT sequence is located at -112 bp [25]. The hexanucleotide poly (A) addition signal sequence AATAAA is located 16 bp 5' to the first nucleotide of poly (A).…”

“…The complete nucleotide sequence of phaseolin [25] revealed a gene structure that includes a total of 1990 bp distributed as: 80 bp of 5' untranslated region, 1263 bp of protein-encoding DNA interrupted by five introns (a total of 515 bp: IVS1, 72bp; IVS2, 88bp; IVS3, 124 bp; IVS4 128 bp and IVS5, 103 bp) and 135 bp of 3' untranslated DNA. Thus, the original mRNA transcript of 1990 bp must be processed by five or more RNA splicing events to result in a 1475 bp mRNA molecule.…”

“…Although most cDNA clones were not full-length, sequence comparison with that of thephaseolin gene resulted in the first demonstration of introns in a plant gene [24]. Subsequently, full-length cDNA and genomic clones were isolated and characterized [24,25]. The availability of the complete nucleotide sequence of phaseolin was important in revealing its amino acid sequence, an essential step towards the twin goals of improving digestibility by eliminating the asparagine residues at which the Nglycans were linked and improving the nutritional value of the protein by the insertion of additional methionine codons into the -phaseolin gene [26].…”