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Endoplasmic Reticulum Targeting and Glycosylation of Hybrid Proteins in Transgenic Tobacco
Abstract
The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide.
... 3b, d, f, 4b, d, f). SA and SE GUS proteins were glycosylated with N-glycosylation through Golgi apparatus because sporamin sorting sequences were introduced in SA and SE constructs (Iturriaga et al. 1989;Matsuoka and Nakamura 1991). The decrease of GUS activities with N-glycosylation through the Golgi apparatus has been discussed in previous studies (Iturriaga et al. 1989;Firek et al. 1994;Honma and Yamakawa 2015). ...
... SA and SE GUS proteins were glycosylated with N-glycosylation through Golgi apparatus because sporamin sorting sequences were introduced in SA and SE constructs (Iturriaga et al. 1989;Matsuoka and Nakamura 1991). The decrease of GUS activities with N-glycosylation through the Golgi apparatus has been discussed in previous studies (Iturriaga et al. 1989;Firek et al. 1994;Honma and Yamakawa 2015). Sporamin proteins are sorted to vacuoles and are accumulated in vacuole (Matsuoka and Nakamura 1991;Shewry 2003;Yang et al. 2005). ...
... In leaves, stems, white roots and red roots, there was not always a consistent sucrose concentration-dependent increase in GUS activity (Fig. 3c, e), as was found in an earlier study on transgenic N. plumbaginifolia (Honma and Yamakawa 2015). The decrease of GUS activities of Sa and SE in leaves, stems, white roots, and red roots of after sucrose treatment is probably due to the N-glycosylation of GUS according to the previous reports (Iturriaga et al. 1989;Matsuoka and Nakamura 1991). However, GUS activities in leaves, stems, white roots, and red roots of SU, Sa, and SE were increased by sucrose treatment, compared with no sucrose treatment (Figs. 3a,c,e,4a,c,e). ...
Key message
We developed transgenic sweet potato with Spomin (sucrose-inducible minimal promoter)-GUS gene-fused constructs. Induced GUS activities by Spomin were higher than those by CaMV 35S promoter.
Abstract
We developed transgenic sweet potato (Ipomoea batatas L. Lam. cv. Kokei no. 14) plants with Spomin (sucrose-inducible minimal promoter)-GUS gene-fused constructs with signal peptides for sorting to cytosol, apoplast and ER, and we analyzed the GUS expression pattern of cut tissue after sucrose treatment. Induced GUS activities by Spomin were several hundred times higher than those by the CaMV 35S promoter. Also, GUS activities in storage roots induced with a Spomin–cytosol-GUS construct were higher than those with either Spomin–apoplast or –ER-GUS constructs. The induced GUS activities by Spomin were higher in storage roots without sucrose treatment than those with sucrose treatment. Chilling (4 °C) storage roots with Spomin constructs for 4 weeks produced higher GUS activities than in storage roots stored at 25 °C for 4 weeks. The calculated maximum GUS content in the storage roots was up to about 224.2 μg/g fresh weight. The chilling treatment increased the free sucrose content in the storage roots, and this increase in endogenous sugar levels induced increased GUS activities in the storage roots. Therefore, Spomin appears to be a useful promoter to develop protein production systems using sweet potato variety Kokei no. 14 storage roots by postharvest treatment.
... However, the synthesis of heterologous proteins still imposes major challenges in fungal expression hosts [11]. One reason is the occurrence of atypical post-translational modifications during conventional secretion via the endomembrane system [12]. Furthermore, secreted fungal proteases are often destructive to the exported products [9,13]. ...
Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.
... It is used with different types of promotors including constitutive or tissue-specific promoters, for instance, Cauliflower Mosaic Virus (CaMV) 35S which is considered to be a very strong constitutive and the mostly used promoter in plant biotechnology. E. coli GUSB can be glycosylated in plant in the ER when the GUSB gene is fused to Patatin signal peptide that leads to considerable reduction of enzyme activity (Iturriaga et al., 1989). To overcome this glycosylation, E. coli GUS gene has been modified by sitedirected mutagenesis which can be especially used in protein targeting (Farrell and Beachy, 1990). ...
... We aimed at developing a stable expression system that allows the production and secretion of a fungal laccase by transgenic plant cells. Recombinant proteins bearing an N-terminal signal in their primary sequence are capable to enter the cell secretory pathway via the endoplasmic reticulum and then be secreted in the apoplast (Iturriaga et al. 1989;Lund et al. 1989;Denecke et al. 1990;De Loose et al. 1991;Ponstein et al. 1996;Benchabane et al. 2008). Calreticulins are calcium binding proteins highly conserved and ubiquitously expressed in plants (Mariani et al. 2003). ...
Some plant species colonize mining-contaminated soils. They are adapted to harsh growth conditions and show high native phytoremediation abilities. For environmental cleanup, these species can be improved by genetic manipulation. Laccases are ligninolytic enzymes that oxidise a broad range of substrates by a radical-catalysed reaction mechanism using oxygen as the electron acceptor. Fungi of the genus Pycnoporus produce laccases with important biotechnological, industrial and environmental applications. Here we describe a successful attempt of Agrobacterium rhizogenes mediated stable transformation of Nicotiana glauca, a species naturally resistant to metal-contaminated soils. The coding region of a Pycnoporus sanguineus laccase gene was fused with the Nicotiana plumbaginifolia calreticulin apoplast targeting signal and driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter (2X35S). The obtained transgenic N. glauca callus cells secreted a recombinant fungal laccase into the growth medium and were capable to degrade an anthraquinone dye, Remazol Brilliant Blue R. Under phenol and cadmium stresses, the transgenic calli not only maintained their growth capacity but their relative growth rate was greater compared to callus transformed with empty vector. This work shows that stable expression of a fungal laccase in a plant species resistant to heavy metals presents a successful strategy that potentially can be used to combat combined organic and inorganic contamination.
... Upon traversal of the endomembrane system during conventional secretion in eukaryotes, this enzyme is artificially N-glycosylated at a single site, leading to a strong reduction in enzyme activity [50]. Therefore, bacterial Gus cannot be secreted in an active state using the classical secretory machinery [52]. On the other hand, translational fusions of Gus to the N-terminus of Cts1 (Gus-Cts1) show activity in the culture supernatant, indicating that the ER is circumvented during this export route [50]. ...
Protein export in eukaryotes can either occur via the classical pathway traversing the endomembrane system or exploit alternative routes summarized as unconventional secretion. Besides multiple examples in higher eukaryotes, unconventional secretion has also been described for fungal proteins with diverse functions in important processes such as development or virulence. Accumulating molecular insights into the different export pathways suggest that unconventional secretion in fungal microorganisms does not follow a common scheme but has evolved multiple times independently. In this study, we review the most prominent examples with a focus on the chitinase Cts1 from the corn smut Ustilago maydis. Cts1 participates in cell separation during budding growth. Recent evidence indicates that the enzyme might be actively translocated into the fragmentation zone connecting dividing mother and daughter cells, where it supports cell division by the degradation of remnant chitin. Importantly, a functional fragmentation zone is prerequisite for Cts1 release. We summarize in detail what is currently known about this potential lock-type mechanism of Cts1 secretion and its connection to the complex regulation of fragmentation zone assembly and cell separation.
... GLB1 sequences encoding the mature β-gal were also cloned into a pBC-SK(−) vector with an in-house patatin optimized signal peptide (PoSP) using Phusion High-Fidelity DNA polymerase (Finnzymes) and In-Fusion® (Clontech) seamless cloning. PoSP is based on a plant signal peptide derived from potato patatin tuber storage protein [45] with the following modification: Ala-Ser-Ser downstream of the initiation codon [46], a "Kozak" sequence for plants [47], nucleotide modifications to enhance RNA stability, and a cleavage site to yield Val as first amino acid based on the plant N-end rule [48]. Together all these modifications have improved the expression of different recombinant proteins in our system (Condori, unpublished results). ...
... However, in rare cases, such as when a protein of bacterial origin has an inadvertent glycosylation site in a particularly strategic position like the catalytic site, glycosylation can cause inactivation of the protein. The popular marker protein, GUS, is inactivated by glycosylation (Iturriaga et al. 1989;Farrell and Beachy 1990), thereby limiting the native protein's use as marker, when targeted to intracellular sites that glycosylate the protein. Thus, proteins targeted for expression should be scanned for potential sites of glycosylation. ...
Recombinant DNA technology has been used to add new functionalities to maize seed. Some of these added functionalities, such as the production of pharmaceuticals or the introduction of enzyme pathways to increase nutritional value, rely on the efficient expression of heterologous proteins. Maize is an exemplary system for the production of recombinant proteins, particularly those that are used for human and veterinary health, because it is a safe and abundant crop. This chapter reviews some of the traits that make maize a popular choice for recombinant protein production, assesses the various factors that contribute to the high-level expression of heterologous proteins, and analyzes examples of successful approaches. It also considers strategies to minimize the inadvertent mixing of recombinant plants with food and feed streams, and the unintended escape of genetically engineered germplasm.
... Furthermore , the presence of a C-tenninal tetrapeptide sequence, -Lys-Asp-Glu-Leu (- KDEL), previously reported for other proteins to be a retention signal for the ER, represents further evidence for the retention of Zm-ERabp 1 in the lumen of the ER. The signal K/HDEL is well recognized in eucaryotic organisms to he responsible for retrieval of proteins from a post-ER salvage compartment by the active participation of a receptor system, and has an apparently similar function in plants (Iturriaga et aI., 1989; Pelham, 1991). Although the primary sequences of the proteins encoded by this gene fami Iy do not fit the structural requirements of 'animal' receptor proteins, ...
Plants modify gene expression and metabolism in response to a large variety of exogenous and endogenous signals. Among the various signals sensed by plants the phytohormones e.g. auxins, cytokinins, ethylene, abscisic acid and gibberellins, have received particular attention. It has been argued that, similar to hormone action in `vertebrates’ the first step in phytohormone action is the interaction of a ligand with binding sites, most likely proteins, located either at the plasma membrane or at various other intracellular locations. It is thought that binding of phytohormones to such proteins should be specific, reversible, of high affinity and saturable and result in a defined biological response.
... Construction of the cassette containing the double enhanced 35S constitutive plant promoter 40 , a translational enhancer from Tobacco Etch Virus (TEV) 41 , the plant signal peptide derived from the potato patatin tuber storage protein (pat) 42 , and the RTB sequence was described previously (Reidy, 2006, etd-12202006-220049). For the fusion constructs, the RTB sequence was PCR-amplified using Pfu DNA polymerase to generate the RTB coding fragment. ...
Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in "hard-to-treat" organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis, and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human α-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts, and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues.
... In SU leaves, the high GUS activities that were observed ( Figure 3A) may have been caused by lack of N-glycosylation of GUS protein. GUS protein (accession number, S69414) has one site which is glycosylated with N-glycosylation, and GUS activity has been decreased by N-glycosylation (Iturriaga et al. 1989). Sporamin protein was transported to vacuoles by way of Golgi apparatus (Yang et al. 2005) and was glycosylated during transportation (Shimizu et al. 2005). ...
We produced transgenic Nicotiana plumbaginifolia plants which contained Spomin (sporamin minimal promoter)-GUS fused chimeric gene constructs with 5 types of signal sequences, such as cytosol, apoplast, ER, vacuole and plastid, and analyzed the GUS expression patterns after sucrose treatment. Spomin induced extremely high GUS activities after 6% and 10% sucrose treatment, especially in leaves. The high GUS activities were observed in leaves of the Spomin- ER-GUS construct treated with 6% or 10% sucrose. These were over 200 times higher than those in leaves with the 35S promoter-ER-GUS construct. The 10% sucrose treatment significantly altered GUS activities in all Spomin and 35S promoter constructs compared with those in the 6% sucrose treatment; some increased and some decreased. GUS activities in 2 months old plants were almost the same as 8 months old plants, indicating that GUS expression driven by Spomin was stably maintained. Also, even when sucrose treatment was stopped, GUS gene expression by Spomin continued for 10 days. © 2015 The Japanese Society for Plant Cell and Molecular Biology.
... For expression of RTB and mIL-12 fusions in transgenic plants, constructs that fused RTB to either the N-terminus (RTB:IL-12) or C-terminus (IL-12:RTB) of IL-12 were generated by utilizing RTB from plasmid R6-2 (Medina ) and a single chain form of mIL-12 ( Lieske et al., 1997). Constructs utilized a strong constitutive promoter de35S (the dual-enhanced 35S promoter, Lam et al., 1989), the tobacco etch virus (TEV) translational enhancer ( Carrington et al., 1990), and sequences encoding the signal peptide provided either by the patatin signal peptide (pat, Iturriaga et al., 1989) upstream of RTB ( Fig.Ⅳ.2), or the mIL-12 sequences (p40, endogenous) depending on orientation. These two constructs were then inserted into pBIB-Kan (Becker, 1990) for transformation of the plant cell via Agrobacterium tumefaciens-mediated transformation. ...
... The region of the patatin gene encoding the signal peptide (Iturriaga et al., 1989) was amplified from 100 ng of potato (Solonum tuberosum L.) genomic DNA using primers to create flanking restriction sites (underlined below), a 5' KpnI site, and a 3' XbuI site. The upstream oligonucleotide was 5'-GCGGGTACCAATGGCAACTACTAAATCT-3' and the downstream oligonucleotide was 5'-GGG= AGACGTAGCACATGTTGAACT-3'. ...
Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.
... Proteins secreted in cytosol are often degraded by proteases and till date it is not possible to establish protease free plant line. On the other hand glycosylation and disulfide bridge formation to recombinant protein is only possible in the endoplasmic reticulum (ER) and golgi apparatus of plant cells [71][72][73] which makes it mandatory for the glycoproteins to be transported to ER or golgi apparatus after translation. Targeting proteins to intracellular organelles (ER, chloroplast, and vacuole) [74] or secretory pathway (like ER or golgi apparatus) will facilitate proper folding of proteins which is essential for their correct functioning and increased expression level [75,76]. ...
Plant genetic engineering is conventionally used for transferring beneficial traits to plants. However in recent years the application of this technology for the production of commercially valuable simple or complex therapeutic proteins and industrial enzymes makes it more important and emerging field of study. The whole plant or plant parts or plant cell culture have been used for the production of biopharmaceuticals like cytokines, blood proteins, milk proteins, hormones, antibodies or antibody fragments, metabolic enzymes, hormones, antigens or vaccine epitopes and many more biological molecules used in animal, specially human health care. Safe, cost effective and considerable high level of recombinant protein production with the ability to properly fold and assemble eukaryotic proteins makes it an alternative competitive system with microbial and mammalian hosts. The increase in recombinant protein production varies with varying host plant species, transformation strategies, location of protein accumulation and the expression of foreign gene which can be optimized. Some of the strategies used till date to optimize the process with their success stories, have been discussed in this review. Although the increases in protein accumulation and their safe downstream processing is vital factor, but the acceptance of this technology also depends on human acceptance which also seems to be promising.
... The recombinant proteins that targeted either to the ER or secretory pathway showed to be properly folded, thereby increasing functional expression of recombinant protein in plants [46,52]. Transgenic plants expressing B6scFv with ER retention showed a high level of expression due to the effect of the ER targeting which is essential for the glycosylation and disulfide bridge formation [53,54]. The expression of normally secreted proteins, in the cytosol of plant cells is very low [55,56], but we observed a detectable amount of B6scFv antibodies in plants expressing the protein in cytosol (B6scFv1001) that may be due to the presence of Kozak sequences, double enhancer and non-codon biasness addition to the gene cassette. ...
Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.
... Recombinant proteins that could not be expressed because their prior toxicity to the cell were produced at high levels when targeted to specific organelles (Hood et al., 1997). Targeting to endoplasmic reticulum is essential for glycosylation (Ituriaga et al., 1989) and disulfide bridge formation (Bruyns et al., 1996;Shani et al., 1994;Vitale et al., 1993). In all instances, the target sequence must be properly cleaved, otherwise incomplete processing will affect the ultimate quality of the recombinant protein. ...
This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng56: 473–484, 1997.
... Recently, a predominant localization of diX-indigo crystals inside the endoplasmic reticulum and in the nuclear envelope was also reported and interpreted as resulting from a still unknown signal peptide within the GUS protein (Craig, 1992). However, I$-glucuronidase is glycosylated in the reticulum and should not display any activity in this compartment (Iturriaga et al., 1989). ...
For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of -glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified -glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl--d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.
We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS) gene–fused constructs with signal sequences for sorting to cytosol, apoplast, and ER, and we analyzed the GUS activities of hairy roots after sucrose treatment. Induced GUS activities by Spomin were about 10 times higher than those by the CaMV 35S promoter. GUS activities in hairy roots induced with a Spomin-UTR-GUS construct after 6% sucrose treatment were higher than those with either Spomin-UTR-GUS construct after 10% sucrose treatment, Spomin-apoplast or ER-GUS constructs after sucrose treatment. High GUS activities in hairy roots with Spomin constructs were induced during 20 wk by culturing hairy roots in LS liquid medium containing 6% sucrose in 300 ml conical flasks. Hairy roots in Linsmaier and Skoog (LS) liquid medium showed the increase of fresh weight (FW) for 16 wk. Total yield (μg) of GUS by the hairy roots with Spomin-UTR-GUS construct using 5 l culture container was calculated to about 6742 μg after 8-wk culture. Productive efficiencies of GUS were over 3.0% of total soluble protein after 1–2 wk 6% sucrose treatment. Productive efficiencies of GUS in transgenic N. plumbaginifolia hairy roots were higher than that in transgenic N. plumbaginifolia leaves, stems, and roots. And growth speeds of transgenic N. plumbaginifolia hairy roots were faster than transgenic N. plumbaginifolia plants. These results showed that N. plumbaginifolia hairy roots with Spomin-heterologous construct systems and sucrose treatment would be useful tools to develop protein production system.
The sections in this article are
Introduction
Endoplasmic Reticulum
G olgi Apparatus
Vacuoles
Secreted Proteins from A rabidopsis
Membrane Proteins
The Mechanism of Membrane Trafficking in Cells
Future Directions
PHOSPHORUS is a critical nutrient for all living organisms as a component ofmembrane phospholipids, nucleic acids, ATP, and many other biological molecules. A recent editorial addressing a potential crisis in phosphorus availability highlighted a variety of areas in which loss of this resource is a common concern [1]. Phosphorus availability plays a key role in issues of soil fertility and crop production, animal health and nutrition, as well as waste management and water quality. While many areas of the world lack sufficient phosphorus to sustain good crop yields, areas of intensive animal production experience an accumulation of excess phosphorus in soil as a result of repeated applications of phosphorus-rich manure. Runoff from pastures and croplands with elevated soil phosphorus levels can contaminate surface water and lead to environmental phosphorus pollution and eutrophication.
Gene transfer technology underpins both crop plant genetic engineering, where it is an obvious prerequisite, and the plant sciences generally, where it is becoming an essential tool for tackling fundamental problems in physiology, biochemistry, developmental biology, molecular biology and genetics. In particular, the use of transgenic plant technology will undoubtedly lead to the development of genetic-based strategies for gene cloning, enabling the linkage of genotype to phenotype, and therefore will provide revolutionary new approaches for identifying genes useful in pure and applied research.
Unconventional secretion of proteins in eukaryotes is characterized by the circumvention of the Endoplasmic Reticulum (ER). As a consequence proteins exported by unconventional pathways lack N -glycosylation, a post-transcriptional modification that is initiated in the ER during classical secretion. We are exploiting the well-established enzyme β-glucuronidase (GUS) to assay unconventional protein secretion (UPS). This bacterial protein is perfectly suited for this purpose because it carries a eukaryotic N -glycosylation motif. Modification of this residue by attachment of sugar moieties during the passage of the ER apparently causes a very strong reduction in GUS activity. Hence, this enzyme can only be secreted in an active state, if the export mechanism does not involve ER passage. Here, we describe a reporter system applied in the corn smut fungus Ustilago maydis that is based on this observation and can be used to test if candidate proteins are secreted to the culture supernatant via alternative pathways avoiding N -glycosylation. Importantly, this system is the basis for the establishment of genetic screens providing mechanistic insights into unknown UPS pathways in the future.
The seed, as a means of plant propagation, utilizes sophisticated and versatile storage mechanisms. Seeds store lipids or carbohydrates as a carbon source, protein for carbon and nitrogen, and some inorganics such as phosphate, which is sequestered in phytic acid. The seed is capable of storing these products for extended periods of time often under extreme conditions of cold, heat and water stress. The deposition and storage of proteins occurs in a variety of different tissues (e.g. cotyledons, perisperm, endosperm) within seeds. In addition, the subcellular localization of the stored proteins varies from species to species.
In many respects this chapter can be seen as a statement of the obvious — mankind has been obtaining tons of important protein products from plants for industrial uses for generations. What genetic engineering can do is to add a new source of variation to the natural variation that is already exploited in agriculture. In this review we will outline the recent progress made in the understanding of gene expression, gene product targetting, gene isolation and tissue culture that will be important for the development of plants and plant tissue culture as sources of novel proteins.
In higher plants, as in higher animals, fertilization marks the beginning of a new generation. A set of closely regulated developmental processes ensures the programmed differentiation of embryonic tissues that will form the new shoot and root system while vigorous accumulation of reserve materials synthesized by the parental organism provides an inheritance to be expended during the early stages of new growth. Reserve proteins must be capable of rapid assembly and accumulation in forms that avoid generating high osmotic pressures, they must be stable during overwintering or other unfavorable times and yet be readily available for controlled release of amino acids providing carbon and nitrogen sources for the fast growing seedling. These factors place similar constraints on the nature of the capital to be stored in monocotyledonous and dicotyledonous spermatophytes. In this article, these constraints will be examined in light of current molecular and cellular knowledge of phaseolin, the major storage protein of the common bean, Phaseolus vulgaris .
Genes encoding lytic proteins, attacin E (att E) and T4 lysozyme (T4 L), were cloned into 6 different plasmid binary vectors for use in Agrobacterium-mediated transformation of apple. All 6 constructs containing att E, T4 L, or both were successfully transferred to Galaxy by Agrobacterium-mediated transformation. Apple transgenics have been recovered with all six constructs. NPTII ELISA and transgene PCR have confirmed transgene transformation with att E and T4 L. Transformation rate varied among different plasmid constructs. The transformation rate of pCa2AMVAtt plasmid vector was lower than with other plasmids. All att E transgenic lines expressed att E. However, the protein expression level was different among transgenic lines. pCa2AMVSPAtt transgenic lines had low expression of att E compared to other att E transgenic lines. In pCa2AMVSPAtt transgenic lines, att E was detected in total apple protein but not in intercellular fluid. Inoculation of transgenic lines indicated that some lines have increased resistance to fire blight. Fusion att E was produced using the pRSET E. coli expression vector and purified. The purified fusion att E and T4 L were used successfully to obtain polyclonal antibodies for protein analysis.
Protein pharmaceuticals constitute a heavily increasing market. Thus, there is an urgent need to efficiently produce the molecules of interest. In a quest to develop a novel protein production system, the corn smut fungus Ustilago maydis was recently established as an expression platform. Inspired by the discovery of an unconventional secretion pathway used by the fungus to secrete the endochitinase Cts1, this eukaryotic system was employed to export heterologous proteins using Cts1 as a carrier. U. maydis displays several features that indicate a great potential for its utilization in biotechnology. However, as seen in other fungi, some bottlenecks were also identified such as limiting transcriptional levels and proteolytic degradation. This initially led to low amounts of secreted proteins. Therefore, the expression system was optimized by improving expression levels and reducing proteolytic degradation using a single-chain antibody as a proof-of-principle. Hence, optimized U. maydis strains are now on a promising way to be exploited for different biotechnological applications.
IntroductionTransfer of DNA into Plant Cells and Regeneration of Intact and Fertile PlantsControlled Expression of Foreign Genes in Transgenic PlantsImproving Plants with Respect to Agronomic TraitsEngineering of Other TraitsTransgenic Plants as a Means of Manipulating Protein and Carbohydrate Composition-Plants as Bioreactors
Plants respond to environmental stress, and the transduced signals cause expression of numerous genes associated with stress tolerance. A number of genes have been described that respond to water stress such as induced by drought and salinity in plants (Ingram and Bartels 1996; Bray 1997; Shinozaki and Yamaguchi-Shinozaki 1997, 1999, 2000; Hasegawa et al. 2000). More than 60 independent cDNAs for dehydration-inducible genes have been reported in Arabidopsis (Shinozaki and Yamaguchi-Shinozaki 1997, 1999, 2000). Functions of their gene products have been predicted from sequence homology with known proteins (Fig. 4.1). Genes induced during dehydration stress conditions are thought to function not only in protecting cells from dehydration by the production of important metabolic proteins (functional proteins) but also in the regulation of genes for signal transduction in the dehydration stress response (regulatory proteins). Northern analysis of dehydration-inducible genes revealed that there appear to be at least four independent signal-transduction pathways between the initial dehydration signal and gene expression (Fig. 4.2). Most of the dehydration-responsive genes are induced by the plant hormone abscisic acid (ABA), but others are not.
From its origins in the Andes, the cultivated potato has spread to most parts of the world in the last 400 years. It now is a major source of nutrition for people from a wide variety of cultural and ethnic backgrounds. In the developing countries of Asia and Africa, the rate of increase in potato production has been higher than that of most other crops, and presently potato ranks fourth in terms of total global food production (Bajaj and Sopory, 1986). When grown under optimal conditions, the yield of tubers can be as high as 100 tonnes/ha, and as such is second only to soybean in production of protein on a per acre basis (Johnson and Lay, 1974). Nutritionally, the tuber is a good source of protein, minerals and vitamin C but is an inefficient supplier of energy compared with grain crops such as rice. Commercial cultivars of potato are highly heterozygous tetraploids that are self-incompatible. These factors contribute to a very slow breeding cycle, and today the European and US markets are dominated by a few varieties that have existed for many years, each of which has substantial weaknesses. However, the ability to transfer single defined genes into potato using Agrobacterium vectors promises to have a disproportionately large effect on the improvement of potatoes.
Vaccination is the safest and most economical way to prevent diseases. On a global scenario, the developing countries are often the target of several infectious diseases. Prevention of these diseases through vaccination has proceeded rapidly in the last couple of decades with the developments in recombinant DNA technology. The major obstacle in developing effective vaccines for mass immunization is the expense of recombinant subunit vaccines and their inability to induce mucosal immunity. Plant based edible vaccines may serve as an attractive alternative to develop cost effective, safe and simple to administer. The oral delivery of edible vaccines induces mucosal immune response. Recent progress made in these areas and the prospects for future is reviewed briefly in this article.
A new reporter system for studying gene expression in plants is proposed that meets many requirements for such systems. When expressed in plant cells, a bacterial licB gene for thermostable lichenase produced the enzyme that retained its activity and was not subjected to any modifications in the heterologous host. Lichenase activity could be detected quantitatively and qualitatively by simple and sensitive methods. Light and wounding induced lichenase synthesis when the licB gene was put under the control of the appropriate inducible promoters, which made it possible to use the system in kinetic studies of gene expression. Efficient secretion of the bacterial enzyme into the plant apoplast indicates the possibility of using lichenase in the studies of protein secretion, transport, and targeting in plant tissues.
Although the advent of molecular biology and recombinant DNA technologies has caused tremendous excitement and enthusiasm among many scientists and policy-makers whose goal is the improvement of agriculture through research, the realities of the time scale over which these technologies must be applied, and the extent to which they are currently inadequate is now being appreciated. The main reason for these inadequacies is simply our overwhelming ignorance of the biology that underlies agriculture. Traditional crop manipulation, as typified by plant breeding, is an empirical craft that is not generally based on a firm understanding of the processes that govern the characters being manipulated. Although the methodology used in an advanced plant breeding program can be quite sophisticated, it is fundamentally limited by the need to visualize and select for traits from existing variation. The complexity of agriculture and the urgency of its success or failure has generally required this empirical approach to its manipulation, while the power of modern molecular biology lies in the acquisition of an understanding of the processes by experimental manipulation.
Bacterial glucanase with a carrot extensin leader peptide was shown to be effectively secreted into the intercellular space in tobacco transgenic plants. A comparison of the glucanase activities in two samples of transgenic plants indicated that the heterologous enzyme was more stable in the intercellular space than within the plant cells. To study the secretion of plant proteins and the role of β-1,3;1,4-glucanase in the plant defense response, a combination of the rbcS plant promoter, the sequence for the carrot extensin leader peptide, and the sequence for thermostable bacterial glucanase with a high specific activity was used.
The specificity of protein targeting processes is the basis of maintaining structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles. Studies in plants have progressed markedly in the last 5 years, and many of the specific signals involved in the transport and targeting of proteins to the nucleus, chloroplast, mitochondrion and microbody, and to organelles along the secretory pathway (endoplasmic reticulum [ER], Golgi complex, and vacuole) have been characterized. Exciting prospects include the identification of receptors involved in the recognition of protein targeting signals, mechanisms of vesicle targeting, and the role of mRNA targeting. Although important exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality — among plants, animals, and eukaryotic microorganisms — and even between prokaryotes and eukaryotes. More information is required about the structural features of proteins that allow for their stable accumulation in a particular subcellular compartment, of particular interest to the plant genetic engineer. Our understanding of the rules that govern protein folding and oligomer assembly and how these processes relate to a protein's ultimate stability in the cell is limited.
A b s t r a c t Hepatitis B is one of the alarming diseases globally. There are about 350 million chronic carriers in the world and it is estimated that 75-100 million of them will die of liver cirrhosis and/or hepatocellular carcinoma. Treatment is expensive and cure rates are not impressive. The advent of recombinant DNA technology resulted in the development of rDNA vaccine using yeast as an expression system. The expense of this vaccine prevented its inclusion in mass immunization programmes, especially in the developing countries. Plant based production of vaccines, preferably in edible parts like fruits, may enable the development of cost effective and more acceptable delicious vaccines. In order to develop edible vaccine for Hepatitis B, researchers world wide, have endeavored to express the surface antigen (HBsAg) of this virus in plants and they could successfully demonstrate the proper folding and immunogenicty of plant derived HBsAg. In our laboratory, Hepatitis B virus 's' gene coding for surface antigen was cloned into plant expression vectors, with and without endoplasmic reticulum retention signal. Transformed NT-I cell lines of tobacco and banana plants were obtained and analyzed for the integration of the transgene by PCR and expression levels were assayed by ELISA. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed tobacco cells. HBsAg was expressed both as intracellular and secreted forms in transformed NT-I cells. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium and expression in banana plants.
This chapter describes some of the critical factors in the design of transgenic plant expression systems and discusses the features of commercial production systems currently being developed. It also examines the range of applications of the technology and the commercial opportunities that exist and reviews the preliminary data from the large-scale recovery of recombinant proteins from plants, along with strategies for simplifying downstream purification. Economic analysis of preliminary data from recombinant protein production in high-yielding crop species and process simulations of downstream recovery operations have provided support for the predicted cost advantage of plant systems. Therefore, it seems likely that plants will be increasingly considered as the primary production vehicle, particularly for recombinant proteins required in large volumes or where high production costs now limit their application.
The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzymeß-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.
To determine whether expression of commonly used reporter genes can alter plant gene expression we used the RNA fingerprinting technique, cDNA-AFLP analysis. We analysed changes in gene expression in response to transient expression (agro-infiltration) of two derivatives of the green fluorescent protein (GFP) reporter gene. Sixty plant cDNA-AFLP fragments showed altered expression profiles in response to an endoplasmic reticulum (ER)-targeted GFP (GFP5) when compared with a non-targeted form (GFP4) or to controls. Two plant genes showed altered expression profiles in response to GFP4. One plant gene showed an altered expression profile in response to both GFP4 and GFP5. No changes in gene expression were observed in response to the widely used β-glucuronidase (GUS) reporter gene. However, altered expression profiles were observed when GUS was targeted to the ER (Cht-GUS) suggesting that reporter proteins can alter plant gene expression especially when ER-targeted. The GFP5-induced genes were also up-regulated in transgenic plants constitutively expressing a GFP5 transgene but only after agro-inoculation of these plants with GFP5. Many of the induced genes showed homology to genes involved in plant defence, suggesting that the plant response to ER-targeted reporter proteins can mimic the response to attack by pathogens.
L'emploi chez l'homme de protéines recombinantes d'intérêt thérapeutique, comme l'hémoglobine, nécessite que l'hôte transgénique utilisé permette d'obtenir de manière économique, des protéines présentant une activité biologique non altérée, en garantissant une sécurité maximale vis-à-vis du risque de contamination par des agents pathogènes. L'utilisation des plantes transgéniques répondrait à ces exigences. En particulier, parmi tous les sytèmes transgéniques, la plante serait l'un des systèmes qui permettrait de réduire considérablement les coûts de production, l'obtention de la biomasse par la culture en champs étant très économique.
Plant pathogens of the family Ustilaginaceae parasitise mainly on grasses and cause smut disease. Among the best characterised members of this family are the covered smut fungus Ustilago hordei colonising barley and oat as well as the head smut Sporisorium reilianum and the corn smut Ustilago maydis, both infecting maize. Over the past years, U. maydis in particular has matured into a model system for diverse topics like plant-pathogen interaction, cellular transport processes or DNA repair. Consequently, a broad set of genetic, molecular and system biological methods has been established. This set currently serves as a strong foundation to improve existing and establish novel biotechnological applications. Here, we review four promising aspects covering different fields of applied science: (1) synthesis of secondary metabolites produced at fermenter level. (2) Lipases and other hydrolytic enzymes with potential roles in biocatalytic processes. (3) Degradation of ligno-cellulosic plant materials for biomass conversion. (4) Protein expression based on unconventional secretion, a novel approach inspired by basic research on mRNA transport. Thus, plant pathogenic Ustilaginaceae offer a great potential for future biotechnological applications by combining basic research and applied science.
We have transformed embryo-derived cultures of two rice cultivars using several different Agrobacterium-mediated gene transfer systems. Mature embryos of cv. Nipponbare inoculated with the wide host range (WHR) supervirulent strain A281(pTiBo542) formed tumorigenic callus tissue that grew on hormone-free medium. The transformed status of this tissue was confirmed by DNA hybridization analysis that showed transferred DNA (T-DNA) present in the rice genome. Embryos of another variety, cv. Fujisaka 5, gave a hypersensitive response when inoculated with strain A281 but exhibited extensive root proliferation following inoculation with the limited host range (LHR) strain A856. These roots grew on hormone-free medium and produced octopine. Fujisaka 5 embryos subsequently inoculated with a disarmed WHR strain conferring kanamycin resistance and -D-glucuronidase (GUS) activity produced callus that grew on selective levels of kanamycin and this tissue fluoresced upon incubation with GUS substrate. GUS expression in the rice tissues was confirmed by Western blotting. We conclude that T-DNA has been transferred to, integrated and then expressed in rice cells.
IntroductionTransfer of DNA into Plant Cells and Regeneration of Intact and Fertile PlantsControlled Expression of Foreign Genes in Transgenic PlantsImproving Plants with Respect to Agronomic TraitsEngineering of Other TraitsTransgenic Plants as a Means of Manipulating Protein and Carbohydrate Composition-Plants as Bioreactors
The bacterial gene of the thermostable endo--1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.
β-glucuronidase (GUS) can be assayed in the spent media of plant tissues transformed with some GUS gene fusions (Jefferson,
1988). This approach is based on the presence of GUS in the media of transformed plant tissues expressing the gene and can
be used to monitor the progress of transformation without destruction of the tissue under study.
Although the application of filamentous fungi, such asAspergillus niger for the production of extracellular proteins is well established for several decades, hardly any information is available about the molecular mechanisms of the process of protein secretion in these organisms.
Two lines of research initiated towards a systematic analysis of the mechanism of protein targeting and secretion are presented in this paper.
1 — To study routing and targeting of proteins in filamentous fungi the availability of a versatile reporter/carrier protein will be of considerable importance. Experiments towards the identification of such a protein are presented.
2 — In analogy to the situation inSaccharomyces cerevisiae, the availability of defined (conditional) mutations in the secretion pathway will provide very important information about the organisation of the pathway. Therefore, based on results obtained inS. cerevisiae, the cloning of several fungal ‘secretion’ genes was started. The results of the cloning and characterisation of one of these genes is presented.
The storage proteins and lectins that accumulate in the protein bodies
of developing legume cotyledons undergo a number of processing steps
along the transport pathway from their site of synthesis to their site
of deposition. The polypeptides are synthesized on polysomes attached to
the endoplasmic reticulum. Synthesis of the polypeptides is always
accompanied by the co-translational removal of a signal peptide. Those
proteins that are glycoproteins in their mature form are
co-translationally glycosylated with high-mannose oligosaccharide side
chains. Co-translational sequestration into the lumen of the endoplasmic
reticulum is followed by the formation of oligomers. Transport of these
oligomers to the Golgi complex may occur via tubular connections between
the endoplasmic reticulum and the Golgi. In the Golgi complex some of
the high-mannose side chains are modified by the removal of five to six
mannosyl residues, and the addition of fucosyl and terminal
N-acetylglucosaminyl residues. This phenomenon has so far been observed
only for phytohaemagglutinin, the lectin of Phaseolus vulgaris. From the
Golgi complex the storage proteins and lectins are transported to the
protein bodies. This transport is mediated by small electron-dense
vesicles. In the protein bodies two types of processing occur:
proteolytic processing resulting in the formation of smaller
polypeptides, and glycolytic processing resulting in the removal of the
terminal N-acetylglucosaminyl residues from the modified carbohydrate
side chains. All storage proteins and lectins undergo some of these
processing steps, and specific examples are discussed in this paper.
The translocation of secretory proteins across the endoplasmic reticulum involves the recognition and cleavage of an amino-terminal extension called the signal sequence1. The structure of signal peptides appears to be ubiquitous in having a very hydrophobic central core2, so that the signal sequence in secretory proteins from one organism could possibly be recognized by the processing and transport apparatus of another. We therefore wished to investigate whether a protein, α-amylase, one of several hydrolytic enzymes secreted from the aleurone of wheat into the endosperm during germination, could be processed and secreted in an active form from the yeast Saccharomyces cerevisiae, secretion being dependent upon the plant signal sequence. Here, synthesis of α-amylase was by inserting a cDNA clone coding for the entire α-amylase structural gene3 into a yeast expression vector4. The α-amylase protein coded for by this gene fusion has the signal sequence located internally, not at the N-terminal end of the polypeptide. Nevertheless, it is processed and the processed form is secreted into the medium in an active form. There are potential industrial applications for yeast that secrete a functional α-amylase.
In this report, we describe the efficient expression and glycosylation, in insect cells, of -phaseolin polypeptides (M
r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M
r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 g/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (GC) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.
A simple and efficient method for direct in. vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from
a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts
were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wfieat germ cell-free protein synthesizing systems.
Genes coding for the high-Mr ['high-molecular-weight' (HMW)] glutenin subunit 12 and for a gamma-gliadin from wheat (Triticum aestivum, cv. Chinese Spring) were subcloned into transcription-translation vectors. In each case transcription in vitro yielded a RNA transcript which when added to a rabbit reticulocyte cell-free translation system directed the synthesis of a polypeptide of appropriate Mr by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). When dog pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. When translation and translocation of the gamma-gliadin was carried out under conditions favouring the formation of disulphide bonds, a polypeptide was synthesized which had a faster mobility on SDS/PAGE carried out under non-reducing conditions than under reducing conditions. This suggests that the processed and translocated gamma-gliadin forms an intramolecular disulphide bond or bonds during synthesis in vitro.
We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease.
A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame
to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this
vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like
endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies
showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene.
The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information
for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads
the way for experiments to identify the cell-type-specific regulatory elements.
To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.
We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.
Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.
We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence
and the mature exported protein is described. The predictive accuracy is estimated to be around 75–80% for both prokaryotic
and eukaryotic proteins.
Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.
Plant transformation has formed the basis for a renewed interest in plant development and genetics as well as stimulating exciting developments in agricultural biotechnology. The cause of this interest is the soil bacterium Agrobacterium tumefaciens and its complex relationship with plant cells. In this review we will describe both the interaction of Agrobacterium with plant cells which leads to DNA transfer and integration into the nuclear genome, and how an understanding of this process has led to the design of vectors for plant transformation.
Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.
We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5' upstream and 1600 bp 3' downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration
method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had
been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then
transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were
confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines
gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable
to plant species that can be infected by Agrobacterium and regenerated from leaf explants.
The particles of cultures of cucumber mosaic virus (CMV) contain the three genomic species of single-stranded RNA and a sub-genomic species that acts as messenger RNA for CMV particle protein. Some cultures also contain a single-stranded linear RNA molecule that is typically ~335 nucleotides long1,2. This extra molecule, termed satellite RNA, does not share appreciable nucleotide sequence with CMV genomic RNA3 but replicates only in plants1 or protoplasts4 that are infected with CMV. CMV isolates that do not contain satellite RNA can be cultured repeatedly in plants in a satellite-free state, but when satellite RNA is added to such cultures it is synthesized and persists as a component of the virus isolate5. The effect of satellite RNA on CMV infections depends on the strain of satellite: in many cases the usual symptoms of CMV are suppressed and as a result the infected plants show few symptoms of infection6–8. However, the presence of other strains of satellite RNA leads to the production of severe symptoms that are quite distinct from those of CMV8. Here we describe the transformation of tobacco plants with DNA copies of CMV satellite RNA, the production of satellite RNA transcripts by such plants and the acquisition of satellite RNA by CMV cultures grown in them. These results suggest a means of protecting plants against the effects of CMV and also suggest a method by which satellite RNAs may have evolved.
The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown-gall tumours after infection of wounded dicotyledonous plants. Large plasmids (Ti-plasmids) are responsible for the oncogenicity of the bacterium1-3. Crown-gall tumours contain a DNA segment, called the T-DNA, which is homologous with a defined part of the Ti-plasmid present in the tumour-inducing bacterium, and is stably integrated into the plant genome4-7. Apart from the T-DNA another region of the Ti-plasmid-called the vir-region, is essential for tumour induction8-11. We report here the interaction of two compatible plasmids, one containing the vir-region, the other carrying the T-DNA on a wide host-range replicon. An A. tumefaciens strain harbouring both plasmids has a normal tumour-inducing capacity, although neither plasmid is functional alone. With this approach, the T-DNA on one plasmid can, because of its size, be easily genetically manipulated using Escherichia coli as a host. Transfer of this plasmid into an A. tumefaciens strain harbouring the plasmid with the vir-region allows introduction of the manipulated T-DNA into plant cells. In this way, sophisticated binary vector systems for plant genetic engineering can be developed.
Smooth microsomal membranes were isolated from immature cotyledons of Vicia faba and Phaseolus vulgaris as well as from immature
endosperms of Bomi barley and its mutantlys 3a. Isolated Bomi barley endosperm polysomes direct the synthesis of hordein precursor polypeptides. Functional reconstitution
of Bomi barley rough microsomes employing such polysomes and smooth microsomal membranes resulted in the ability of the membranes
to perform signal peptide cleavage and co-translational transport of hordein precursor polypeptides into the lumen of the
microsomes. Only a small portion of the hordein precursor polypeptides was processed and transported when the reconstitution
was performed with smooth microsomal membranes isolated from cotyledons of Vicia faba and Phaseolus vulgaris or from the endosperms
oflys 3a. A precursor polypeptide to the 50 kD vicilin was translated in vitro from mRNA of immature Vicia faba cotyledons. In
the presence of Vicia faba smooth microsomal membranes the precursor polypeptide was co-translationally processed to the size
of the polypeptide synthesized by rough microsomal membranes. Smooth microsomal membranes from cotyledons of Phaseolus vulgaris
and from the endosperms of Bomi orlys 3a barley failed to process detectable amounts of the vicilin precursor polypeptide. Dog pancreatic microsomal membranes
cleaved significant amounts of the hordein precursor polypeptides whereas only a small portion of the vicilin precursor polypeptides
was processed.
The biogenesis of protein bodies is examined in cotyledons of soybean (Glycine max, Merr) at the time when reserve protein is beginning to accumulate in the cotyledons. Reserve protein is deposited in the central vacuoles of parenchyma cells and new protein bodies arise from the central vacuole by pinching-off small masses of reserve protein surrounded by a portion of the tonoplast.
Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.
Thesis (Ph. D.)--University of Colorado, 1985. Includes bibliographical references (leaves [180]-192).
Translation of polyadenylated mRNA of Chlamydomonas reinhardtii in a cell-free wheat germ system resulted in the synthesis of numerous discrete polypeptides. Among them was a species with molecular weight 20,000 that was immunoprecipitated specifically by antibodies raised against the authentic small subunit (16,500 daltons) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase(dimerizing), EC 4.1.1.39]. Since the immunoprecipitated polypeptide has a larger molecular weight by approximately 3500 than the small subunit (S) it was identified as a putative biosynthetic precursor (pS). Post-translational conversion of pS by a specific endoprotease yielded two detectable products: one apparently identical in size to S and the other, a small peptide, presumably representing the remainder of pS. The endoprotease requires sulfhydryl groups for its activity and is present in a C. reinhardtii postribosomal supernatant as well as in a free polysome fraction. The latter could account for the observation that completion of nascent chains in free polysomes yielded S but not pS. We propose that pS is an extrachloroplastic form of S and that the small peptide portion plays a role in the transfer of S into the chloroplast.
The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.
Many newly synthesized proteins must be translocated across a membrane to reach their final destinations. Translocation requires a signal on the protein itself, a loose conformation of the protein, energy, and receptor-like components in the cytosol and on the target membrane.
We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.
A precursor for sporamin A, the storage protein of the tuberous roots of sweet potato deposited in the vacuole, is synthesized on membrane-bound polysomes and has an extra peptide of 37 amino acids at the N-terminus of the mature form, which can be divided into an N-terminal putative signal peptide sequence (residues −37 to −17) and a segment enriched with charged amino acids (residues −16 to −1) [Hattori, T., et al. (1985) Plant Mol. Biol. 5, 313–320]. We examined the in vitro processing of the sporamin A precursor using a messenger RNA derived from a full-length cDNA by the SP6 transcription system. When the in vitro translation in a wheat germ cell-free system was carried out in the presence of dog pancreas microsomal membranes, the precursor polypeptide (Mr= 24000) was processed into an intermediate form still larger than the mature polypeptide (Mr= 20 000). The processed intermediate form was also produced by addition of microsomal membranes from sweet potato and potato in the translation reaction, although less efficiently compared to dog membranes. Moreover, Escherichia coli cells expressing sporamin precursor accumulated a polypeptide with the same electrophoretic mobility as the intermediate form produced in vitro.
A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae. This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S. cerevisiae. The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene. A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site. A CYC1::alpha-gliadin transcript of approx. 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region. The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence. The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx. 0.1% of the total cellular protein. The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin. This is consistent with recognition and cleavage of the signal peptide by yeast. Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Patatin is a family of glycoproteins that accounts for up to 40% of the total soluble protein in potato tubers. We isolated and characterized 25 patatin genomic clones. All of these exhibit different restriction patterns, but can be divided into two classes based on the presence (class II) or absence (class I) of a 22-bp sequence within the 5'-untranslated region. We determined the complete nucleotide sequence of the class-I clone PS20 and the 5'-flanking sequence of seven additional clones. The transcribed region of PS20 spans 3197 bp and is divided by six introns. The seven exons encode a transcript which is identical to the cDNA, cloned in pGM203 [Mignery et al., Nucl. Acids Res. 12 (1984) 7987-8000]. The 5'-flanking sequences of both class-I and class-II patatin genes are highly homologous up to bp position -87 and then diverge. This conserved region contains the CAAT and TATA homologies as well as a homology to the core enhancer sequence. Within a class there are additional large regions of homology, but differences exist that allow the patatin genes to be further divided into several discrete subclasses. S1 nuclease protection experiments with both class-I and class-II probes showed that class-I transcripts are the predominant species present in tubers. Class-II transcripts are present in tubers, but are 50-100-fold less abundant. In potato roots, class-II transcripts are the predominant species and few, if any, class-I transcripts are present.
Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.
We have isolated recombinant lambda clones containing intact major tuber protein (patatin) genes and flanking sequences from
the commercial tetraploid variety Marls Piper. The gene is composed of seven exons and six introns, spread over 4 kb of DNA.
Nuclease mapping defined the 5' end of the mRNA approximately 45 bp upstream of the initiation codon. The 5' end of the gene
is preceeded by a canonical TATA box sequence. The three known patatin genes encode proteins of nearly identical Mr but very different isoelectric points. The sequence of the gene does not indicate a role for patatin as one of the globulin
class of plant storage proteins.
The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.
Variations in length and composition of the charged N-terminal, central hydrophobic and polar C-terminal regions in a large sample of signal sequences have been mapped, both as a function of the overall length of the sequence, and in an absolute sense, i.e. various "extremes" have been sought. The results show subtle differences between eukaryotic and prokaryotic sequences, but the general impression of signal sequences as being highly variable is reinforced. Criteria for a "minimal" signal sequence are suggested and discussed.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.
[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p-benzoquinone-derived precursor for I. Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides. The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions. The participation of the carboxylate of Glu-461 in catalysis is discussed.
Xenopus oocytes injected with poly(A)-containing RNA from developing cotyledons of field beans (Vicia faba L. var. minor) synthesize precursor polypeptides to the major storage globulins legumin and vicilin. These polypeptides are secreted into the medium without proteolytic cleavage of the legumin propolypeptides into the mature disulfide-linked alpha and beta chains. Similarly, storage globulin polypeptides from pea (Pisum sativum L.) and french bean (Phaseolus vulgaris L.) were secreted from oocytes. Inhibition of glycosylation by tunicamycin does not prevent secretion. This first report on the secretion of plant polypeptides by Xenopus oocytes shows that (a) intracellular deposition of storage proteins in membrane-bounded organelles (protein bodies) of plants and extracellular secretion have step(s) in common, and (b) the cell, in addition to the mRNA, determines the final destination of these polypeptides.
Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription. Expression of the lac genes in the absence of the inducer arabinose indicates that transcription originates within the inserted fragment; non-expression of lac with arabinose present indicates that transcription is terminated by the fragment. Using different cloning vectors, DNA fragments generated by a wide variety of restriction endonucleases can be inserted between ara and lac. This procedure has been used to identify and isolate endonuclease-generated DNA fragments from theEscherichia coli chromosome, various R plasmids, bacteriophage T5 and Drosophila melanogaster that contain nucleotide sequences capable of functioning as promoters in E.coli. A characteristic level of lac expression is determined by the amount of transcription that proceeds to the lac genes from a promoter located within each fragment. The effects of genetic regulatory mechanisms acting on a promoter can be assayed by alterations in the level of lac expression.These cloning vectors were also used to bring structural genes located within an inserted DNA fragment under the control of the ara promoter. Insertion of HindIII endonuclease-generated fragments carrying the tetracycline-resistance determinant of pSC101 or the sulfonamide-resistance determinant of the R6-5 plasmid into such vectors resulted in arabinose-induced resistance to tetracycline or sulfonamide.