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blot analysis using a cloned phaseolin gene as probe confirms that phaseolin is indeed encoded in a. multigene family (unpublished data).We have previously reported (7) the isolation and partial nucleotide sequence of the phaseolin genomic clone AG-APVPh 177.4 (A177.4) and of a cloned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...
The fraction of poly(A)-containing RNA isolated from ripening bean (Phaseolus vulgaris) cotyledons that sedimented at 16 S in linear logarithmic sucrose gradients was at least as active a messenger as viral RNA when added to a cell-free protein-synthesizing system from wheat germ. The major products synthesized in vitro were polypeptides of about 47,000 and 43,000 daltons, corresponding to two of the three subunits of G1 protein, the most abundant bean seed storage protein. No trace of the largest (53,000 daltons) subunit was found among the polypeptides synthesized in vitro. Proof that the 47,000- and 43,000-dalton polypeptides coded by the 16S RNA were indeed subunits of G1 protein was obtained by immunoprecipitation with monovalent antibody to G1 protein and by electrophoretic mapping of peptides on acrylamide gels after digestion of mixtures of authentic protein and radioactive translation products with protease V8, chymotrypsin, and trypsin. The subunits synthesized in vitro were slightly smaller than the native subunits, probably because they lacked the sugar residues present on the holoprotein.
Electrophoretic analysis of the major seed protein, GI globulin, from four strains of Phaseolus vulgaris L. revealed a three-banded pattern for two strains having a high methionine content (BBL 240 and PI 302,542). The other two strains (PI 207,227 and P1 229,815) known to have a lower seed methionine content, had a two-banded subunit pattern for the GI globulin. Analytical ultracentrifugation confirmed that globulin from the two-banded strains underwent pHdependent reversible dissociation similar to that previously found for a three-banded cultivar; additionally, the protomer molecular weight showed that three subunits of about 50,000 molecular weight each were present in the GI globulin of the two-banded strain. Gel patterns of Gl globulin from the two strains used as parents, BBL 240 and PI 229,815, showed differences in the largest subunit, which appeared as either a 53,000 molecular weight polypeptide known to be present in the three-banded strain, or While soybeans have the highest protein content and economic value, the oil present in these seeds is a hindrance to protein studies, and seeds of Phaseolus vulgaris L. were selected as the preferred material. Dry seeds of P. vulgaris contain 20 to 30% protein by weight, depending on the cultivar (5). Of this protein, 50 to 75 % is globulin (requiring appreciable salt for solubility) which can be separated into two fractions. In Osborne's studies on legumes he adopted the term legumin (first used by Braconnot in 1827) for the fraction requiring the highest level of salt for solubility, and coined the term vicilin for the fraction soluble in lower salt concentrations (9). Through the years, misuse of these terms has occurred; they have been applied to proteins from many genera despite the differing properties of protein in a given fraction from species to species. To avoid the resulting confusion, we designated the globulin fraction which first precipitates on addition of water to a saline solution of P. vulgaris protein as the Gl fraction, and the second fraction (obtained by extensive dialysis) as the G2 fraction (7).Despite the apparently simple separation of the two globulin fractions on the basis of solubility, great difficulty has been experienced in obtaining them free from traces of cross-contamination. We developed an acidic extraction procedure (7) which achieves this goal, permitting characterization of the Gl and G2 globulin fractions (15). Recent articles (12, 18) have described separation of globulins using zone precipitation, which performs the same function as our isoelectric separation, but is lengthier and limits the amount of material that can be handled.Although the G2 fraction gives a single boundary sedimenting at 6.6S in the analytical ultracentrifuge (16), it is not homogeneous, and contains several growth-inhibiting substances. This fraction has a high content of sugar-containing proteins, and a major component, glycoprotein I (10), possesses hemagglutinating properties, i.e., it is a lectin. Purification of lectin from Phas...
Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-I (GI) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 nillimeters long. The 7-to 9-millimeter stages represent a 2-day transition period over which genetic information for the GI protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that GI globulin was the major storage protein, representing some 50%o of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed GI polypeptides to be among the polysome-directed products.The genetic information for French bean (Phaseolus vulgaris L. cv. Tendergreen) storage proteins appears to be repressed in all tissues except for certain stages in the development of the cotyledon, when it is actively expressed. At maturity, a dry seed (450 mg) contains approximately 90 mg of protein (20%); about 50% of this is globulin. Because of the active synthesis and accumulation of relatively few molecular species over a short and well defined period, the bean cotyledon is an excellent tissue for studies toward understanding the molecular basis for regulation of specific gene expression.Aspects of the changes in biochemistry, morphology, and fine structure of P. vulgaris seeds during development have been studied extensively (2,6,24,31 Purified Gl and G2 proteins were prepared as described previously (29). For immunochemical studies, the protein samples were prepared by grinding 0
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