In vitro cytotoxicity of non-cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells

A soil isolate designated 90-F-45-14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells. When activated with proteolytic processing, inclusion proteins of the isolate 90-F-45-14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC(50) value of 60.8 microg ml(-1), while showing extremely high activities on the human leukaemic T cells (MOLT-4) and the normal T cells with EC(50) values of 0.27 and 0.20 microg ml(-1), respectively. Anti-leukaemic cell activity of the 90-F-45-14 proteins was eight to nine times greater than that of the B. thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad-spectrum cytolysin. The cytopathy by the 90-F-45-14 proteins was characterized by marked cell-ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis. Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa. A 100% homology was observed in the sequence of 15 N-terminal amino acids between the proteins of 170 and 103 kDa. There was no N-terminal sequence homology between 90-F-45-14 proteins and the existing Cry/Cyt proteins of B. thuringiensis. Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.     

Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces multiple cytotoxic proteins with similar molecular masses against human cancer cells

AIMS: To prove that Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces several novel cytotoxic proteins against human leukaemic T cells. METHODS AND RESULTS: Parasporal inclusion protein was solubilized and processed by proteinase K and was separated by anion-exchange chromatography. Cytopathic effects of each fraction against MOLT-4 and Jurkat cells were monitored. CONCLUSIONS: Existence of at least two novel cytotoxic proteins was suggested and N-terminal sequences of the newly identified proteins were determined to be QSTTDVIREY and X (Y or I) (P or I) NLANELA (X indicates uncertain amino acids). Molecular masses of the two proteins were approx. 27-28 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we demonstrated that the strain 89-T-34-22 produces at least two novel cytotoxic proteins with similar molecular masses against human cancer cells. This is the first strain of B. thuringiensis which produces multiple cytotoxic proteins against human cancer cells.     

Investigation of a novel Bacillus thuringiensis gene encoding a parasporal protein, parasporin-4, that preferentially kills human leukemic T cells

A novel gene encoding a leukemic cell-killing parasporal protein, designated parasporin-4, was cloned from an isolate of Bacillus thuringiensis serovar shandongiensis. The amino acid sequence of the parasporin-4, as deduced from the gene sequence, had low-level homologies of <30% with the established B. thuringiensis Cry proteins including the three known parasporins. When the gene was expressed in a recombinant of Escherichia coli BL21(DE3), the parasporin-4 formed intracellular inclusion bodies. Alkali-solubilized and proteinase K-activated inclusion protein exhibited strong cytotoxic activity against human leukemic T cells (MOLT-4) and weak for normal T cells, but no adverse effect on human uterus cervix cancer cells (HeLa).     

Characterization of the anti-cancer-cell parasporal proteins of a Bacillus thuringiensis isolate

An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized. The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa. No homologies with the Cry and Cyt proteins of B. thuringiensis were detected based on N-terminal sequences. Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells. The protease-activated proteins showed no cytotoxicity to normal T cells.     

Identification of membrane antigen C33 recognized by monoclonal antibodies inhibitory to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1-positive T cells.

We isolated four monoclonal antibodies (MAbs), M38, M101, M104, and C33, which were capable of inhibiting syncytium formation induced in a human T-cell line, MOLT-4-#8, by coculture with human T-cell leukemia virus type 1 (HTLV-1)-positive human T-cell lines. The MAbs had, however, no inhibitory activity on syncytium formation induced in a human osteosarcoma line, HOS, by HTLV-1-positive T-cell lines. They also did not inhibit syncytium formation induced in MOLT-4-#8 by human immunodeficiency virus type 1-positive MOLT-4. All MAbs reacted with various human cell lines of lymphoid and nonlymphoid origins, including HTLV-1-positive T-cell lines. Furthermore, they all reacted with a murine A9 clone containing human chromosome 11 fragment q23-pter. Two MAbs, M104 and C33, immunoprecipitated a membrane antigen with the same molecular size. The antigen (henceforth called C33 antigen) was about 40 to 55 kDa in HTLV-1-negative Jurkat, CEM, MOLT-4, and normal peripheral blood CD4-positive human T cells and about 40 to 75 kDa in HTLV-1-positive C91/PL, TCL-Kan, MT-2, and in fresh HTLV-1-transformed CD4-positive human T-cell lines. Pulse-chase experiments revealed that C33 antigen was synthesized as a 35-kDa precursor that was then processed to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL. In the presence of tunicamycin, a 28-kDa protein was synthesized. The conversion from 35 kDa to 41 to 50 kDa in MOLT-4 and to 44 to 70 kDa in C91/PL was inhibited by monensin. Treatment with N-glycanase alone, but not with sialidase and O-glycanase in combination, completely removed the sugar moiety of C33 antigen from both HTLV-1-negative Jurkat and HTLV-1-positive C91/PL. Therefore, C33 antigen has only N-linked carbohydrates, the modification of which appears to be substantially altered in the presence of the HTLV-1 genome.     

Comparative immunogenicity of outer membrane protein K and whole-cell antigens of Vibrio parahaemolyticus for diagnosis

The immunogenicity of soluble outer membrane protein K (OmpK)- small ubiquitin-like modifier, OmpK inclusion bodies, formalin, and heat-killed Vibrio parahaemolyticus cells were prepared and studied in a mouse model. The results of whole-cell ELISA and Western blot (WB) revealed that the serum against soluble OmpK and OmpK inclusion bodies reacted only with homologous V. parahaemolyticus. Furthermore, recombinant OmpK proteins were not recognized by the serum against whole-cell V. parahaemolyticus antigens. Unexpectedly, the serum against formalin and heat-killed V. parahaemolyticus reacted broadly with homologous (an immunization strain) and heterologous (non-immunization strains) V. parahaemolyticus and Vibrio species. The WB results revealed that the serum against the two V. parahaemolyticus whole-cell antigens primarily reacted with proteins that were approximately 100, 70, 36, 28, and 22 kDa in the cell lysates from different Vibrio strains, rather than the recombinant OmpK. The 70 and 28 kDa proteins exhibited specificity to Vibrio species, while the 22 kDa protein was more specific to V. parahaemolyticus. This study showed the limitation of recombinant OmpK to prepare diagnostic antibodies and revealed several specific Omps of Vibrio sp. and V. parahaemolyticus that were promising in diagnosis and vaccine development.     

Association of two barley yellow mosaic virus (RNA 2) encoded proteins with cytoplasmic inclusion bodies revealed by immunogold localisation

Summary Antisera were raised against the RNA 2-encoded proteins of 28 kDa and 70 kDa of barley yellow mosaic virus (BaYMV) by using the corresponding cDNA sequences of a German isolate for protein overexpression inEscherichia coli BL 21 and subsequent purification. The proposed processing of a 98 kDa precursor polyprotein encoded by the long open reading frame of RNA 2 to two proteins of 28 kDa and 70 kDa could be confirmed by immunoprecipitation of the in vitro transcribed and translated cDNA-clone of RNA 2 and Western blot analysis of fragmentated protein extracts of BaYMV-infected winter barley plants. In situ localisation studies of infected leaf tissue using immunogold labeling techniques for electron microscopy revealed that both viral proteins of BaYMV (RNA 2) were associated with the crystal-like cytoplasmic inclusion bodies. No other parts of the cells and no other inclusions (pinwheelstructures or aggregated virus particles) showed any gold labeling when the 28 kDa and 70 kDa antisera were used. We suppose that both RNA 2-encoded proteins take part in the formation of the crystal-like cytoplasmic inclusion bodies which are the most dominant structures in the cytoplasm of BaYMV-infected tissue. Possible functions of the 28 kDa and 70 kDa protein of BaYMV (RNA 2) are discussed.Abbreviations PBS phosphate-buffered saline - CEA chicken egg albumin - BaYMV barley yellow mosaic virus - BaMMV barley mild mosaic virus     

Simian immunodeficiency virus from African green monkeys.

Simian immunodeficiency virus (SIV) was isolated from the total peripheral blood mononuclear cell population and the monocyte-macrophage adherent cell population of three seropositive green monkeys originating from Kenya. SIV from these African green monkeys (SIVagm) was isolated and continuously produced with the MOLT-4 clone 8 (M4C18) cell line but not with a variety of other cells including HUT-78, H9, CEM, MT-4, U937, and uncloned MOLT-4 cells. Once isolated, these SIVagm isolates were found to replicate efficiently in M4C18, SupT1, MT-4, U937, and Jurkat-T cells but much less efficiently if at all in HUT-78, H9, CEM, and MOLT-4 cells. The range of CD4+ cells fully permissive for replication of these SIVagm isolates thus differs markedly from that of previous SIV isolates from macaques (SIVmac). These SIVagm isolates had a morphogenesis and morphology like that of human immunodeficiency virus (HIV) and other SIV isolates. Antigens of SIVagm and SIVmac cross-reacted by comparative enzyme-linked immunosorbent assay only with reduced efficiency, and optimal results were obtained when homologous antibody and antigen were used. Western blotting (immunoblotting) of purified preparations of SIVagm isolate 385 (SIVagm385) revealed major viral proteins of 120, 27, and 16 kilodaltons (kDa). The presumed major core protein of 27 kDa cross-reacted antigenically with the corresponding proteins of SIVmac (28 kDa) and HIV-1 (24 kDa) by Western blotting. Hirt supernatant replicative-intermediate DNA prepared from cells freshly infected with SIVagm hybridized to SIVmac and HIV-2 DNA probes. Detection of cross-hybridizing DNA sequences, however, required very low stringency, and the restriction endonuclease fragmentation patterns of SIVagm were not similar to those of SIVmac and HIV-2. The nucleotide sequence of a portion of the pol gene of SIVagm385 revealed amino acid identities of 65% with SIVmac142, 64% with HIV-2ROD, and 56% with HIV-1BRU; SIVagm385 is thus related to but distinct from previously described primate lentiviruses SIVmac, HIV-1, and HIV-2. Precise information on the genetic makeup of these and other SIV isolates will possibly lead to better understanding of the history and evolution of these viruses and may provide insight into the origin of viruses that cause acquired immunodeficiency syndrome in humans.     

Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

Trypsin-sensitive and -resistant components in human T-cell membranes required for syncytium formation by human T-cell lymphotropic virus type 1-bearing cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) envelope proteins play an important role in viral entry into target cells. In a syncytium formation assay consisting of a coculture of HTLV-1-bearing cells and target cells, mature gp46 and gp21 proteins each inhibited syncytium formation induced by HTLV-1-bearing cells. Experiments with 125I-labeled proteins showed that 125I-gp46 bound specifically with MOLT-4 target cells even in the presence of large amounts of gp21, whereas 125I-gp21 binding to target cells was completely blocked in the presence of large amounts of gp46. These observations suggest that HTLV-1 envelope proteins in syncytium formation interact with at least two components, which are located close to each other on the cell membrane. We isolated two components from MOLT-4 cell lysate, using Sepharose 4B columns coupled with peptides corresponding to amino acids 197 to 216 and 400 to 429, respectively, of the envelope protein. One is a trypsin digestion-sensitive component of approximately 34 to 35 kDa, which interacts specifically with gp46. The other is a nonprotein component, which interacts with gp21. This component was destroyed by sodium periodate oxidation and was partitioned into the methanol-chloroform phase. These observations suggest that these two components play an important role in HTLV-1 entry into target cells via membrane fusion.     

High affinity interaction between histidine-rich glycoprotein and the cell surface type ATP synthase on T-cells

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcγ receptors do not participate in this cellular event. In the present study, we identified the α-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of α- and β-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the β-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.     

Mannose-specific lectin activity of parasporal proteins from a lepidoptera-specific Bacillus thuringiensis strain

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl- D-glucosamine, N-acetyl- D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori.     

Noninsecticidal parasporal proteins of a Bacillus thuringiensis serovar shandongiensis isolate exhibit a preferential cytotoxicity against human leukemic T cells

A Bacillus thuringiensis isolate, 89-T-34-22, belonging to the serovar shandongiensis (H22) produced noninsecticidal and nonhemolytic proteins crystallizing into irregular-shaped parasporal inclusions. The proteins showed in vitro cytotoxicity to human cells, including cancer cells, only when activated by protease treatment. The human leukemic T (MOLT-4) cells were > 100 times more susceptible than HeLa and normal T cells to the proteins of 89-T-34-22. The cytotoxicity was dose dependent and the median effective concentration for the MOLT-4 was 3.5 microg/ml. The cytopathy induced by the 89-T-34-22 proteins was characterized by remarkable condensation of the nucleus and cell-ballooning. Five major parasporal proteins of 89-T-34-22, with molecular masses in the range of 16-160 kDa, shared no similarity with the previously reported proteins in terms of the N-terminal sequence.     

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens

The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.     

Mode of action of parasporin-4, a cytocidal protein from Bacillus thuringiensis

Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cells, in particular. When cells were administrated with PS4, cell swelling and nuclear shrinkage were induced, and, the ballooned cells burst within 24 h. PSI-BLAST search showed that the protein shared homology not only with B. thuringiensis Cry toxins but also with aerolysin-type β-pore-forming toxins. Circular dichroism measurements suggested that PS4 was a β-sheet-rich protein. PS4 aggregated into oligomers on the plasma membrane of PS4-susceptible CACO-2 cells, but not on that of PS4-resistant HeLa cells. Leakage of lactate dehydrogenase and influx of extracellular FITC-dextrans were observed only in susceptible cells. The activation of effectors caspase 3 and/or 7 was not observed in PS4-treated CACO-2 cells. It was shown that cytotoxicity of the PS4 against CACO-2 cells was exhibited when treated by cyclodextrin which induces cholesterol depletion. These results suggest that PS4 is a unique β-pore-forming toxin with a cholesterol-independent activity.     

Purification,immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells

A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.DLO Centre for Plant Breeding and Reproduction Research (CPRO-DLO)     

猪繁殖与呼吸综合征病毒nsp4抗体制备与鉴定

为了获得猪繁殖与呼吸综合征病毒(PRRSV)nsp4的抗体,根据HP-PRRSV TA-12株(Gen Bank Accession No.HQ416720)的nsp4基因序列,设计并合成一对引物。用RT-PCR扩增后克隆到原核表达载体p ET-28a(+)中,构建重组质粒p ET28a-nsp4,转化至Trasseta(DE3),经IPTG诱导重组蛋白获得了高效可溶性表达,大小约为26 k Da。经镍离子亲和柱(Ni+-NTA)纯化获得了高纯度重组蛋白,将纯化的nsp4蛋白免疫新西兰大白兔制备了多克隆抗体。ELISA检测抗体效价可达106,Western blotting和IFA检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性,能够识别PRRSV感染宿主细胞中的nsp4蛋白。本研究成功制备了针对nsp4的多克隆抗体,为进一步研究nsp4的功能及PRRSV致病机制奠定了基础。     

Mannose-Specific Lectin Activity of Parasporal Proteins from a Lepidoptera-Specific <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori. Received: 21 February 2002 / Accepted: 28 March 2002     

Inducible alkyltransferase DNA repair proteins in the filamentous fungus Aspergillus nidulans.

We have investigated the response of the filamentous fungus Aspergillus nidulans to low, non-killing, doses of the alkylating agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). Such treatment causes a substantial induction of DNA alkyltransferase activity, with the specific activity in treated cells increasing up to one hundred-fold. Fluorography reveals the two main inducible species as proteins of 18.5 kDa and 21 kDa, both of which have activity primarily against O6-methylguanine (O6-MeG) lesions. In addition, two other alkyltransferase proteins can also be detected. One, of MW 16 kDa, is expressed in non-treated cells, but is not induced to the same extent as the 18.5 and 21 kDa proteins. The other, a protein of 19.5 kDa, is highly inducible and can only be detected in treated cells. Unlike the other three proteins, it acts primarily against methyl-phosphotriester (Me-PT) lesions. This is the first instance in which an MePT alkyltransferase has been detected in a eukaryotic organism and, coupled with the high level of induction of the O6-MeG alkyltransferase enzymes, this indicates that a control system similar to the bacterial adaptive response may be present in filamentous fungi.