Diphtheria Toxin A-chain Gene Research Articles

Non-cell-autonomous regulation of petal initiation in Arabidopsis thaliana

In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.

Intravenous Delivery of piggyBac Transposons as a Useful Tool for Liver-Specific Gene-Switching.

Hydrodynamics-based gene delivery (HGD) is an efficient method for transfecting plasmid DNA into hepatocytes in vivo. However, the resulting gene expression is transient, and occurs in a non-tissue specific manner. The piggyBac (PB) transposon system allows chromosomal integration of a transgene in vitro. This study aimed to achieve long-term in vivo expression of a transgene by performing hepatocyte-specific chromosomal integration of the transgene using PB and HGD. Using this approach, we generated a novel mouse model for a hepatic disorder. A distinct signal from the reporter plasmid DNA was discernible in the murine liver approximately two months after the administration of PB transposons carrying a reporter gene. Then, to induce the hepatic disorder, we first administered mice with a PB transposon carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain gene, and poly(A) sites), and then with a plasmid expressing the Cre recombinase under the control of a liver-specific promoter. We showed that this system can be used for in situ manipulation and analysis of hepatocyte function in vivo in non-transgenic (Tg) animals.

β-Hydroxypyruvate: a new diabetogenic factor?

The incidence of type 2 diabetes mellitus (T2DM) is increasing at an alarming rate. Insulin secretory dysfunction and insulin resistance are characteristic features of T2DM. However, their relative contributions to the progression from normal glucose tolerance to impaired glucose tolerance (IGT) to T2DM remain unknown. This limits the capacity of researchers to establish therapeutic interventions for T2DM. Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are the two primary incretin hormones secreted by intestinal K and L cells, respectively. Secretion of these incretins increases postprandially and they potentiate glucose-stimulated insulin secretion (GSIS). Despite blunted incretin responses in patients with T2DM, insulinotropic responses to exogenously administered GLP-1 remain active in these patients (1). Although controversy exists, a recent study demonstrated that insulin secretory responses to GIP were retained in patients with T2DM (1,2). Additionally, incretin response to GIP is improved by reducing blood glucose levels in patients with T2DM (3). These findings support the rationale for use of incretin-based pharmacotherapies in the treatment and management of T2DM. However, identification of the mechanism by which the function of K cells contributes to the development of T2DM is still elusive and requires a good model of study. To determine the role of K cells in the regulation of metabolism, K cells were eliminated from mice by expressing the diphtheria toxin A-chain gene in GIP-producing cells (DT mice). Despite a severely impaired …

Novel expression vectors enabling induction of gene expression by small-interfering RNAs and microRNAs.

Small-interfering RNAs and microRNAs are small ∼21–22 nucleotide long RNAs capable of posttranscriptional suppression of gene expression. The synthetic siRNAs are especially designed to target pre-specified genes and are common molecular biology tools. The miRNAs are endogenous regulators of gene expression found in a wide variety of eukaryotes. miRNAs are currently utilized for diagnostics applications. Therapeutically, various miRNA-antagonizing tools are being explored and miRNAs are also utilized for cell-specific inhibition of the expression of gene therapy vectors harboring target sites for specific miRNAs. Here we show, for the first time, that siRNAs and miRNAs can be harnessed to induce gene expression. We designed special expression vectors in which target sites for artificial siRNAs or endogenous miRNAs are located between the transgene and an Upstream Inhibitory Region (UIR). We hypothesized that cleavage of the mRNA by siRNAs or miRNAs will separate the transgene from the UIR and the resulting uncapped mRNA will be capable of being translated. A UIR composed of seven open reading frames was found to be the most efficient inhibitor of the translation of the downstream transgene. We show that under such a configuration both artificial siRNAs and endogenous miRNAs were capable of inducing transgene expression. We show that using the diphtheria toxin A-chain gene, in combination with target sites for highly expressed miRNAs, specific induction of cell-death can be achieved, setting the stage for application to cancer therapy.

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Hrs, a Mammalian Master Molecule in Vesicular Transport and Protein Sorting, Suppresses the Degradation of ESCRT Proteins Signal Transducing Adaptor Molecule 1 and 2

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

Production of CETD transgenic mouse line allowing ablation of any type of specific cell population

Diphtheria toxin A-chain (DT-A) is a potent inhibitor of protein synthesis. As little as a single molecule of DT-A can result in cell death. DT-A gene driven by a tissue-specific promoter is used to achieve genetic ablation of a particular cell lineage. However, this transgenic approach often results in aberrant depletion of unrelated cells. To avoid this, we established a method for specific depletion of a cell population by controlled expression of the DT-A gene via the Cre-loxP system. We produced five transgenic mice carrying CETD construct containing loxP-flanked enhanced green fluorescent protein (EGFP) cDNA and the DT-A gene. Transfection of primary cultured cells derived from CETD transgenic fetus with Cre expression plasmid resulted in extensive cell loss, as expected. Bigenic (double transgenic) offspring obtained by crossbreeding between CETD and MNCE transgenic mice in which Cre expression is controlled by the myelin basic protein (MBP) promoter exhibited embryonic lethality, suggesting expression of Cre at embryonic stages. Intravenous injection of Cre expression vector to CETD mice led to generation of glomerular lesions, probably due to predominant depletion of glomerular epithelial cells. This Cre-loxP-based cell ablation technology is powerful and convenient method of generating mice lacking any chosen cell population.

A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid.

BackgroundVarious animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A) gene. DT-A is known to kill cells by inhibiting protein synthesis.MethodsAn expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6) and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5.ResultsH-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis.ConclusionsRepeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine.

Diphtheria toxin-mediated cell ablation reveals interregional communication during Arabidopsis seed development.

Fertilization of the female gametophyte in angiosperm plants initiates a process of coordinated development of embryo, endosperm, and seed coat that ensures the production of a viable seed. Mutant analysis has suggested that communication between the endosperm and the seed coat is an important determinant in this process. In addition, cell groups within the embryo, derived from the apical and from the basal cell, respectively, after zygote division, concertedly establish a functional root meristem, and cells in the apical region of the embryo are hypothesized to repress cell divisions in the basal cell-derived suspensor. The available evidence for these interregional communication events mostly relies on the analysis of mutant phenotypes in Arabidopsis. To provide independent and direct evidence for communication events, we used conditional domain-specific expression of the diphtheria toxin A chain (DTA) in developing Arabidopsis seeds. By using a collection of cell- or tissue-type-specific promoters, we show that the mGAL4:VP16/UAS two-component gene expression allows reliable spatiotemporal and conditional expression of the GFP:GUS reporter and the DTA gene in the developing embryo and endosperm. Expression of DTA in the protoderm of the embryo proper led to excessive proliferation of suspensor cells, sometimes resulting in the formation of secondary embryos. Endosperm-specific expression of DTA caused complete cessation of seed growth, followed by pattern defects in the embryo and embryo arrest. Taken together, the results presented here substantiate the evidence for and underline the importance of interregional communication in embryo and seed development and demonstrate the usefulness of conditional toxin expression as a method complementary to phenotypic analysis of developmental mutants.

Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L. ssp. pekinensis.

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5' noncoding (promoter) region were different, except for the sequence from -281 to -89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.

Joining chain-expressing and -nonexpressing B cell populations in the mouse.

The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

Growth inhibition of cancer cells by co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector.

We constructed a plasmid containing bovine leukemia virus (BLV)-tax gene driven by SR alpha promoter, designated as pME-BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV-LTR by pME-BLVtax was confirmed by luciferase assay. When the cells, such as COS-1, C8, and KU-1, were transfected with a plasmid pBLV-LUC1, which contained the luciferase gene under the control of BLV-LTR, and pME-BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV-LUC1 was introduced into the cells. Activation of the BLV-LTR promoter was regulated by pME-BLVtax and 0.5 microg of pME-BLVtax was sufficient for the expression of the gene under the control of BLV-LTR. Furthermore, pME-BLVtax was used to direct the cell expression of the gene for diphtheria toxin A-chain under the control of BLV-LTR (pLTR-DT) to various tumor cell lines, KU-1, C8, COS-1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co-transfection of pLTR-DT with pME-BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co-introductions of pLTR-DT with pME-BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR-DT and pME-BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.

Use of transcriptional regulatory sequences of telomerase (hTER and hTERT) for selective killing of cancer cells.

Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer.

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Genetic ablation of root cap cells in Arabidopsis.

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

Pollen Ablation of Transgenic Tobacco Plants by Expression of the Diphtheria Toxin A-Chain Gene under the Control of a Putative Pectin Esterase Promoter from Chinese Cabbage

Pollen Ablation of Transgenic Tobacco Plants by Expression of the Diphtheria Toxin A-Chain Gene under the Control of a Putative Pectin Esterase Promoter from Chinese Cabbage

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines

Objective: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. Study design: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. Results: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. Conclusions: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.

In vivo antitumor effect of cationic liposomes containing diphtheria toxin A-chain gene on cells infected with bovine leukemia virus.

A plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) in the multicloning site of pUC-18 was entrapped in cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome), and their antitumor effect on BLV-infected tumor cells was examined in vivo. The cationic TMAG-liposome containing pLTR-DT was successively injected into the tumor transplanted to nude mice. The growth of tumor was significantly inhibited by the injection of cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pUC18 plasmids showed no such effect. These results suggest that a DT-A expression plasmid under the control of BLV-LTR is highly toxic to the BLV-infected tumor cells, and that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for BLV-infected tumor cells and can be utilized for DT-A gene delivery into BLV-infected tumor cells in vivo.

Evaluation of cationic liposomes for delivery of diphtheria toxin A-chain gene to cells infected with bovine leukemia virus.

We investigated whether cationic liposomes are efficient at delivering the gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (pLTR-DT) into BLV-infected cells and are also suitable for in vivo use. The transfection activity of the cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome) and liposomes composed of phosphatidylserine (PS) (PS-liposome) was evaluated by the luciferase assay using a plasmid which contains the coding sequence of firefly luciferase under the control of the SR alpha promoter (pSR alpha/L-A delta 5). The TMAG-liposome gave highly efficient transfection in the presence of serum. On the other hand, PS-liposome showed inferior efficiency. When BLV-infected cells were co-transfected with a fixed amount of pSR alpha/L-A delta 5-entrapped TMAG-liposome and various amount of pLTR-DT-containing TMAG-liposome, the luciferase activity in the BLV-infected cells was inhibited by the addition of pLTR-DT-entrapped TMAG-liposome dose-dependently. The cationic TMAG-liposome containing pLTR-DT was successively added to BLV-infected cells in culture. The number of viable cells was markedly reduced by the cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pSR alpha/L-A delta 5 showed no such effect. pLTR-DT entrapped by the cationic TMAG-liposome was not digested by the treatment with DNase I and with serum. These results suggest that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for the BLV-infected cells and can be utilized for DT-A gene delivery into the BLV-infected cells in vivo.

A novel transient assay system demonstrates that DT-A tsm is a temperature-sensitive toxin in plant tissues

We describe a rapid transient assay system for analysing the efficacy of diphtheria toxin A chain (DT-A) derivatives as agents of genetic cell ablation in plant tissues. Because of the extreme cytotoxic effects of DT-A in eukaryotic cells we are investigating the use of temperature sensitive (DT-A tsm) and attenuated (tox 176) derivatives of DT-A to generate more intermediate phenotypes. The relative toxicities of DT-A, DT-A tsm and tox 176 were measured by determining their effects on the expression of a co-transfected luciferase reporter gene construct, using microprojectile bombardment into plant tissues. In pollen and leaves incubated at 16°C, the expression of DT-A or DT-A tsm resulted in a significant reduction in luciferase (LUC) activity. However, at 30°C the expression of DT-A tsm had no effect on the level of LUC activity, in contrast to the DT-A gene, which abolished LUC activity. Furthermore, at the lower permissive temperature DT-A tsm was between 50- and 200-fold less toxic than DT-A. Similar co-bombardment experiments showed that, in contrast to the mammalian systems, the toxicity of tox 176 was not attenuated relative to DT-A.