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Inhibition of protein synthesis by activated diphtheria toxin
... While the DT-A system is efficient, there might be concerns about effects on pollinators and human health associated with the widespread usage of a diphtheria toxin gene in plants. Although eukaryotic organisms cannot actively transport DT-A inside cells in the absence of the diphtheria toxin B chain (Thorsness et al., 1991;referenced therein Collier, 1977;Pappenheimer and Gill, 1972), one DT-A molecule is enough to induce cell death (Yamaizumi et al., 1978). Another method to achieve genic male sterility is the use of cytotoxic RNase genes (Barnase and RNAse-T1 from Aspergillus oryzae) expressed in tapetum cells, the layer of nutritive cells surrounding the pollen sac, which results in the selective destruction of the tapetum during pollen formation and leads to pollenless male-sterile plants (Mariani et al., 1990). ...
Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen-specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible-to-no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand-crossed to both male-sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000-40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male-sterile tobacco, and 900-2100 seeds per male-sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI-driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.
... The failure of wild-type pollen tubes to develop on ablated stigmatic papillae cannot be ascribed to the toxic effects of stigmatic DT-A molecules on pollen grains for two reasons. First, DT-A molecules are not secreted and are not internalized by eukaryotic cells in the absence of the diphtheria toxin B subunit (Pappenheimer and Gill, 1972; Collier, 1977 ). Second , in Arabidopsis SLG 13 ::DT-A transformants, ablated papillar cells retained the capacity to support pollen tube growth (Thorsness et al., 1993). ...
Plant reproduction in crucifers is dependent on interactions that occur at the stigma surface between the male gametophyte (pollen and pollen tube) and papillar cells. To dissect these complex interactions, papillar cells were genetically ablated by targeting the expression of a toxin to appropriate cells of the flower with a flower-specific and developmentally regulated promoter. In transgenic Brassica plants that expressed the toxic gene fusion, flower morphology was normal except for aberrant papillar cell development and partial pollen sterility. Microscopic, biochemical, and functional analyses, mainly focused on papillar cell responses, revealed that papillar cells lost their ability to elongate, to synthesize cell-specific proteins, and to support pollen germination after self- or cross-pollination. This loss of stigma receptivity to pollination was mimicked by treating pistils with protein phosphatase inhibitors. Differences in the effects of genetic and chemical ablation on the pollination responses of Brassica and Arabidopsis flowers are discussed and are ascribed in part to a requirement for phosphorylation/dephosphorylation events in Brassica but not in Arabidopsis.
... Seed set was typically lower in SLG 13 ::DT-A transformants than in untransformed controls. However, because DT-A is not secreted and is not internalized by eukaryotic cells in the absence of the diphtheria toxin B chain (Pappenheimer and Gill, 1972; Collier, 1977), the reduced seed set may reflect a reduced ability of pollen to adhere to the shortened papillae, rather than to a more specific toxic effect of DT-A on pollen tube growth. DT-A expression also caused defects in anther development and pollen function. ...
A chimeric toxic gene consisting of the diphtheria toxin A chain gene fused to a promoter previously shown to direct pistil- and anther-specific expression was used to genetically target cell killing in transgenic Arabidopsis. Flowers of Arabidopsis transformants that carried the toxic gene fusion had distinct structural defects. The papillar cells at the stigma surface were stunted and were biosynthetically inactive. Anther development was also impaired by toxic gene expression, leading to abnormalities in anther dehiscence, pollen morphology, and pollen germination. The combined defects of pistil and anther rendered transformants that carried the toxic gene fusion self-sterile. However, the transformants were cross-fertile with untransformed plants: the viable pollen of ablated plants was rescued by wild-type stigmas, and, strikingly, the ablated papillar cells allowed the growth of wild-type pollen.
... The vaccine used a modified version of the diphtheria toxin, which is the key determinant of lethality. By preventing the transmission of toxigenic strains of C. diphtheriae to vaccinated individuals, widespread vaccination undoubtedly provided a selective pressure against the toxigenic strains, increasing the relative frequency of non-toxigenic strains (Schuman & Doull, 1940; Stebbins, 1940; Pappenheimer & Gill, 1972 Ewald, 1994), thereby decreasing C/I. These considerations indicate that C/I should be estimated from data obtained prior to the widespread use of vaccination. ...
Recent studies have provided evolutionary explanations for much of the variation in mortality among human infectious diseases. One gap in this knowledge concerns respiratory tract pathogens transmitted from person to person by direct contact or through environmental contamination. The sit-and-wait hypothesis predicts that virulence should be positively correlated with durability in the external environment because high durability reduces the dependence of transmission on host mobility. Reviewing the epidemiological and medical literature, we confirm this prediction for respiratory tract pathogens of humans. Our results clearly distinguish a high-virulence high-survival group of variola (smallpox) virus, Mycobacterium tuberculosis, Cornynebacterium diphtheriae, Bordetella pertussis, Streptococcus pneumoniae, and influenza virus (where all pathogens have a mean percent mortality > or = 0.01% and mean survival time >10 days) from a low-virulence low-survival group containing ten other pathogens. The correlation between virulence and durability explains three to four times of magnitude of difference in mean percent mortality and mean survival time, using both across-species and phylogenetically controlled analyses. Our findings bear on several areas of active research and public health policy: (1) many pathogens used in the biological control of insects are potential sit-and-wait pathogens as they combine three attributes that are advantageous for pest control: high virulence, long durability after application, and host specificity; (2) emerging pathogens such as the 'hospital superbug' methicillin-resistant Staphylococcus aureus (MRSA) and potential bioweapons pathogens such as smallpox virus and anthrax that are particularly dangerous can be discerned by quantifying their durability; (3) hospital settings and the AIDS pandemic may provide footholds for emerging sit-and-wait pathogens; and (4) studies on food-borne and insect pathogens point to future research considering the potential evolutionary trade-offs and genetic linkages between virulence and durability.
Self-incompatibility is a cell-cell recognition system in higher plants that is based on the ability of the pistil to discriminate “self”-pollen from “non-self”-pollen. In the simplest systems, this recognition response is controlled by a single locus — the S-locus — with multiple alleles. Pollination of a pistil with pollen bearing an S-allele recognition factor identical to that expressed in the host plant stigma or style results in rejection of the “self”-pollen. Most of the studies on the molecular genetics of self-incompatibility that are summarized in this review have had as their goal the identification and characterization of the gene product(s) associated with the self-incompatibility response. These studies have provided a great deal of new and important information about self-incompatibility — despite the fact that many critical questions remain unresolved. Taken together, the present evidence from these studies indicates that the self-incompatibility response is likely to be far more complex than suggested by historical models.
The role of the hydrophobic region of diphtheria toxin B moiety in fragment A membrane traversal has been studied using crm45. This molecule, a serologically related diphtheria toxin protein, contains a normal enzymic fragment A and the hydrophobic domain of the toxin B chain but lacks a C-terminal polypeptide needed for specific cell binding. Relatively high concentrations of crm45 are required to inhibit protein synthesis in cells however, after the loss of its hydrophobic region crm45, which still contains an active fragment A, becomes almost non-toxic. It seems thus that the non-polar peptide found in crm45 or toxin facilitates the transport of the hydrophilic fragment A across the plasma membrane.
The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli beta-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants.
Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactose-binding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens-mediated gene transfer. Yields of ricin in soluble cell extracts were 1 microg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.
DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. The introduction of this chimeric molecule into tobacco cells by electroporation lowered the level of the transient expression of the coelectroporated chloramphenicol acetyltransferase reporter gene indicating that expression of diphtheria toxin chain A in plant cells is toxic. We have developed a binary vector pGA987 which can be used for probing a variety of plant promoters.
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