Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene.

We have previously generated microphthalmic mice lacking lens fiber cells by targeting the expression of the diphtheria toxin A (DT-A) gene in transgenic mice with regulatory sequences associated with the mouse gamma 2-crystallin gene. Because of the extreme toxicity of DT to animal cells and the potential leakiness of many tissue-specific regulatory regions, we investigated whether there might be an experimental advantage in using a mutant, attenuated form of the DT-A gene (tox-176) fused to gamma 2-crystallin regulatory sequences to ablate fiber cells in the ocular lens. In contrast to the microphthalmia observed in transgenic animals carrying the native DT-A gene, independent lines of mice transgenic for the gamma 2tox176 construct displayed predominantly cataracts or clinical anophthalmia. These contrasting phenotypes were transmitted within each pedigree, although for some lines some phenotypic heterogeneity among offspring was noted. The difference in phenotype between cataractous and clinically anophthalmic transgenic lines could not be ascribed to differences in the transgene copy number. Instead, the results suggest that transgene expression and hence the extent of genetic ablation are modulated by the site of chromosomal integration and, to a lesser extent, by epigenetic events. They also suggest that the attenuated gamma 2tox176 construct can integrate into chromosomal regions that are particularly favorable for expression without compromising embryological development and therefore that the tox-176 gene may be more versatile and effective than the wild-type DT-A gene for achieving genetic ablation with a broad range of cell- or tissue-specific regulatory sequences.     

The complete nucleotide sequence of the gene coding for diphtheria toxin in the corynephage omega (tox+) genome.

A segment of corynephage omega (tox+) DNA, containing the gene for diphtheria toxin (tox) was fragmented with restriction enzymes and the fragments cloned into M13 vectors for nucleotide sequence determination. A long open reading frame was shown to encode the tox gene by comparing the predicted amino acid sequence with that of peptides derived from the mature toxin molecule. Analysis of the nucleotide sequence shows RNA polymerase and ribosome binding signals preceding a GTG codon in the open reading frame: if this is the correct starting signal for translation, then a 25 amino acid signal peptide can be predicted for the toxin molecule.     

Isolation and characterization of tox mutants of corynebacteriophage beta.

Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct form the structural gene for toxin, and that the CRM- strains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed.     

The Synthesis of Insect specific Neurotoxin Gene tox 34 and Acquirement of Transgenic tobacco

A 762 bp mite (Pyemotes tritici) insect-specific neurotoxin tox34 gene fragment encoding mature protein was fully modified based on plant gene codon usage, tox34 was inserted into bacterial expression vector pBV221. Electrophoresis of protein mixture extracted from the bacteria treated with heat-induction showed that specific protein bands migrated with apparent molecular weight of 32 ~ 33 kD in denaturing gels. Tox34 was inserted into plant high-expression vector and then transferred into tobacco (Nicotiana tabacum L. cv. SRI ) using the Agrobacterium tumefaciens (Smith et Tomnsend) Conn binary vector system, followed by the obtainment of the transgenic tobacco plants. The PCR Southern blot assay indicated that the intact toxin gene was integrated into the tobacco genome. GUS assay of transgenic seedling roots showed positive results. The feeding trail of transgenic plants using bollworms (Heliothis armigeva Hubner) manifested strong toxity to lowstar bollworm larvae.     

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.     

A superactive hormonotoxin prepared with truncated diphtheria toxin

A chemically truncated form of diphtheria toxin, DT51, which lacks the cell-binding site but retains the membrane-translocating function, was covalently linked to luteinizing hormone (LH) and compared to similar conjugates containing diphtheria toxin (DT) or diphtheria toxin A-chain (DTA). The DT51 hormonotoxin killed cells possessing an LH receptor at concentrations similar to that of DT hormonotoxin and orders of magnitude lower than DTA hormonotoxin. The DTA hormonotoxin exhibited an LD-50 similar to that of previously reported hormonotoxins which employed DTA, ricin A-chain, or gelonin as toxic moieties.     

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.     

Effect of diphtheria toxin on the viability of phagocytes and B-lymphocytes in animals sensitive and insensitive to it

It is well known, that mechanism of diphtheria toxin (DT) action triggers only if toxin penetrates into acid endosome after binding with specific receptor--heparin-binding epidermal grows factor like grows factor (HB-EGF) on the cell surface. We have suggested that DT is capable to penetrate either into B-lymphocytes, which have specific immunoglobulin receptors for DT or into phagocytes, which are able to phagocytosis of DT, because in both of these cases toxin get in endosome with conditions suitable for its activation. To check this hypothesis the comparative studies with insensitive to DT mice lacking specific receptor for DT, and with sensitive to DT guinea pigs were performed. Influence of DT on vitality of phagocytes and B-cells with different specificity from mice and guinea pigs was studied. B-cells were obtained from animals immunized by control antigen--ovalbumine and recombinant diphtheria toxoid--DT without N-terminal 28 aminoacid residues responsible for toxic effect. The results obtained have showed that DT can penetrate into phagocytes and B-cells specific to DT and kill these cells even if they lack classic receptor for DT. This fact evidences that DT is potentially able to inhibit self-directed antibody response and keep from participation of phagocytes in the protection of organism from infection.     

Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa.

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.     

System for the Investigation of the Bacteriophage-directed Synthesis of Diphtherial Toxin

Lytic corynebacteriophage betahv64(tox+) has been characterized, and methods for studying the expression of its tox(+) gene in nontoxinogenic Corynebacterium diphtheriae strain C7(s)(-)(tox-) described. During one cycle of viral growth there was a 1 million-fold increase in extracellular toxin. Both the conditions of the experiment and the use of purified phage, free from toxin, support the conclusion that all of the toxin was newly formed. This toxin was immunochemically indistinguishable from standard toxin produced by the PW8(r)(Pdi)(tox+) strain. Chloramphenicol was found to be an effective agent for synchronizing the initiation of viral growth. Once chloramphenicol was removed, intracellular toxin appeared and continued to increase throughout the latent period. Proflavine, added early in the latent period, blocked phage maturation without similarly affecting yields of toxin. Iron exerted a limited inhibitory effect on final toxin levels attained.     

Orientation of the tox gene in the prophage of corynebacteriophage beta.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRM- has also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.     

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.     

Fusion Protein of Interleukin 4 and Diphtherial Toxin with High Cytotoxicity to Cancer Cells

One means to improve the selectivity of cancer therapyis by directing foreign protein with activity againsttherapeutic targets that displays different expression levelon malignant cells from normal cells. There have hadsubstantial efforts to rationally de…     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells

Receptor of human interleukin 4 (hlL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R,a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.     

白喉毒素-白细胞介素2重组嵌合毒素的克隆表达及特异性细胞毒作用的研究

应用PCR技术分别扩增出编码白喉毒素氨基端 389个氨基酸 (DT3 89)的基因片段及人IL 2全基因 ,将两基因串连插入 pET3a载体 ,构建成含有DT3 89 IL 2融合基因的表达载体 ,转化大肠杆菌BL2 1,经表达、纯化后 ,用3 H Leucine掺入法测定其对HUT 10 2细胞的蛋白合成抑制作用。SDS PAGE电泳分析表明 ,表达产物分子质量 (Mr)约为 5 8kD ;重组嵌合毒素能够特异性地抑制高表达IL 2受体的HUT 10 2细胞的蛋白生物合成 ,且有一定的剂量反应关系 ,其细胞半数抑制浓度 (IC50 )约为 3 3× 10 -11mol/L。为进一步研制特异性的抗IL 2受体高表达肿瘤和相关疾病的药物打下了基础。     

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.     

Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae.

We have isolated and partially characterized a beta-phage mutant lysogen of Corynebacterium diphtheriae, C7(betatoxct1+), which is partially insensitive to iron inhibition of diphtheria toxin production. tox expression by C7(betatoxct1+) was found to be partially constitutive. In the presence of concentrations of iron that almost completely inhibit the expression of diphtheria toxin by the wild type, C7(beta), the level of toxin production by C7(betatoxct1+) was found to be at least 25 times that of the parent. The purified tox gene product of C7(betatoxct1+) was immunologically and electrophoretically identical to, and equally as toxic as, diphtheria toxin purified from C7(beta). In addition, the partial N-terminal amino acid sequence was found to be identical to diphtheria toxin. This data strongly suggests that the mutation allowing for the constitutive expression of tox in C7(betatoxct1+) is outside of the structural gene. Furthermore, the constitutive expression of diphtheria toxin was found to be cis dominant in the double lysogen C7(betacrm45+/betatoxct1+). The data presented is consistent with the existence of a tox operator locus.