A lambda 1059 library of Phaseolus vulgaris cv. ‘Tendergreen’ DNA was screened with a cloned lectin‐like cDNA. Among the phages selected was clone lambda B10 containing two complete lectin genes in the same orientation approximately 4 kb apart. The DNA sequences of the lectin genes and their flanking regions have been determined and their transcriptional initiation sites were located by S1 nuclease mapping. On the basis of the deduced amino acid sequences and compositions and the mol. wts. of their encoded glycoproteins, the genes, dlec1 and dlec2, are predicted to encode erythro‐ and leucoagglutinating phytohemagglutinins (PHA‐E and PHA‐L), respectively. The mRNA coding regions of dlec1 and dlec2 are 90% homologous, suggesting an origin involving duplication of an ancestral gene. Both lectin genes are intronless and have at least two ATG codons in a short (11‐14 bp) 5′‐untranslated region. Most of their 5′‐untranslated regions consist of alternating pyrimidines and purines (RY repeats). Upstream sequences are also highly conserved between dlec1 and dlec2, including stretches of nine or more alternating R and Y residues. RY repeats of such length are not found within the protein coding portion of dlec1, dlec2 or a Phaseolus lectinlike gene previously described. Overlapping double (dlec1) or triple (dlec2) polyadenylation addition signals are found and there is an unusually high degree of homology (84%) between their 3′‐untranslated regions.(ABSTRACT TRUNCATED AT 250 WORDS)
In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding β-phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal β-phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.
The nucleotide sequences of eight partial and five full-length phaseolin cDNA clones show that phaseolin polypeptides are encoded by two distinct gene families which differ in their coding regions by the presence or absence of two different size direct repeats. The alpha-type phaseolin polypeptides are encoded by genes containing direct repeats which encode 14 additional amino acids. Aside from these differences, the alpha-and beta-type phaseolin genes show a high degree of homology (98%) which is consistent with these genes being derived from a common ancestral gene. Much of the heterogeneity found in the phaseolin polypeptides appears to be due to post-translational processing. Nucleotide sequence analysis demonstrates that the alpha-type genes contain only a few amino acid replacement substitutions and that the beta-type genes appear to contain no amino acid replacement substitutions. S1 nuclease mapping shows a complex pattern for transcriptional initiation of phaseolin mRNA. Hydropathy analysis shows that phaseolin polypeptides are predominately hydrophilic, and that the two N-glycosyl recognition sites are located in different hydropathic environments.
The maize 15‐Kd zein structural gene was placed under the regulation of French bean β‐phaseolin gene flanking regions. Agrobacterium tumefaciens‐mediated transformation was used to insert the chimeric phaseolin–zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue‐specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin‐derived sequences, and was terminated within the phaseolin gene 3′ flanking region. Both zein‐ and phaseolin‐derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80‐fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20‐amino‐acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.
Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
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