Structure and expression pattern of a human MTG8/ETO family gene, MTGR1.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

Base substitution spectra of nalidixylate resistant mutations induced by monochromatic soft X and 60Co gamma-rays in Bacillus subtilis spores.

Bacillus subtilis spores were exposed to three types of photons, monochromatic soft X-rays with the energy corresponding to the absorption peak of phosphorus K-shell electron (2,153 eV) and with the slightly lower energy (2,147 eV), and 60Co gamma-rays. From the irradiated spores, 233 mutants exhibiting nalidixic acid resistance were isolated, and together with 94 spontaneous mutants, the sequence changes in the 5'-terminal region of the gyrA gene coding for DNA gyrase subunit A were determined. Among eighteen alleles of the gyrA mutations, eight were single-base substitutions, nine were tandem double-base substitutions, and one was a double substitution skipping a middle base pair. About 6% of the radiation-induced mutations were tandem double-base substitutions, whereas none was observed among the spontaneous ones. Among spontaneous mutations, A:T and G:C pairs were equally subjected to mutations, whereas the substitutions from G:C pairs and those to A:T pairs predominated among those induced with soft X-rays. The peak-energy X-rays were more effective in killing and causing mutations than the low-energy X-rays, however, there seemed no base-change events uniquely attributable to phosphorus K-shell absorption.

Genomic structure of the human RBP56/hTAFII68 and FUS/TLS genes.

We previously isolated RBP56 cDNA by PCR using mixed primers designed from the conserved sequences of the RNA binding domain of FUS/TLS and EWS proteins. RBP56 protein turned out to be hTAFII68 which was isolated as a TATA-binding protein associated factor (TAF) from a sub-population of TFIID complexes (Bertolotti A., Lutz, Y., Heard, D.J., Chambon, P., Tora, L., 1996. hTAFII68, a novel RNA/ssDNA-binding protein with homology to the proto-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 15, 5022-5031). The RBP56/hTAFII68, FUS/TLS and EWS proteins comprise a sub-family of RNA binding proteins, which consist of an N-terminal Ser, Gly, Gln and Tyr-rich region, an RNA binding domain, a Cys2/Cys2 zinc finger motif and a C-terminal RGG-containing region. Rearrangement of the FUS/TLS gene and the EWS gene has been found in several types of malignant tumors, and the resultant fusion proteins play an important role in the pathogenesis of these tumors. In the present study, we determined the genomic structure of the RBP56/hTAFII68 gene. The RBP56/hTAFII68 gene spans about 37kb and consists of 16 exons from 33bp to 562bp. The longest exon, exon 15, encodes the C-terminal region containing 19 repeats of a degenerate DR(S)GG(G)YGG sequence. While the structure of the FUS/TLS gene has been reported previously, we determined the total DNA sequence of the FUS/TLS gene, consisting of 12kb. The RBP56/hTAFII68, FUS/TLS and EWS genes consist of similar numbers of exons. Comparison of the structures of these three genes showed that the organization of exons in the central part encoding a homologous RNA binding domain and a cysteine finger motif is highly conserved, and other exon boundaries are also located at similar sites, indicating that these three genes most likely originate from the same ancestor gene.

The AML1-MTG8 leukemic fusion protein forms a complex with a novel member of the MTG8(ETO/CDR) family, MTGR1.

The AML1-CBFbeta transcription factor complex is essential for the definitive hematopoiesis of all lineages and is the most frequent target of chromosomal rearrangements in human leukemia. In the t(8;21) translocation associated with acute myeloid leukemia (AML), the AML1(CBFA2/PEBP2alphaB) gene is juxtaposed to the MTG8(ETO/CDR) gene. We show here that the resultant AML1-MTG8 gene product specifically and strongly interacts with an 85-kDa phosphoprotein. Molecular cloning of cDNA indicated that the AML1-MTG8-binding protein (MTGR1) is highly related to MTG8 and similar to Drosophila Nervy. Comparison of amino acid sequences among MTGR1, MTG8, and Nervy revealed four evolutionarily conserved regions (NHR1 to NHR4). Ectopic expression of AML1-MTG8 in L-G murine myeloid progenitor cells inhibits differentiation to mature neutrophils and induces cell proliferation in response to granulocyte colony-stimulating factor (G-CSF). Analysis with C-terminal deletion mutants of AML1-MTG8 indicated that the region of 51 residues (488 to 538), which contains NHR2, is essential for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis indicates that this region is required for AML1-MTG8 to form a stable complex with MTGR1. Overexpression of MTGR1 stimulates AML1-MTG8 to induce G-CSF-dependent proliferation of L-G cells and to interfere with AML1-dependent transcription. These results suggest that AML1-MTG8 could function as a complex with MTGR1 and that the complex might be important in promoting leukemogenesis.

Induction of unique tandem-base change mutations in bacterial spores exposed to extreme dryness.

Despite the remarkable resistance to desiccation, Bacillus subtilis spores manifest indications of DNA damage when being kept in an extremely dry environment made by high vacuum. Spores of strain TKJ3422 (uvrA10 spl-1 recA4) with triple repair defects lost colony-forming capacity dependent on the duration and strength of the exposure. Mutations to rifampicin resistance were induced in the spores of the strain HA101 with wild-type repair capability and the strain TKJ6312 (uvrA10 spl-1) with double repair defects. The majority of nalidixic acid-resistant mutations induced by the exposure to high vacuum belonged to one particular allele gyrA12 carrying a tandem-base change, 5'-CA to 5'-TT, at codon 84 of the gyrA gene coding for DNA gyrase subunit A. This allele has never been found among more than 500 mutants obtained by various treatments other than vacuum exposure. These results indicate forced dehydration of DNA in the microenvironment of the spore core causes unique damage leading to lethal and mutagenic consequences.

Cloning and mapping of a human RBP56 gene encoding a putative RNA binding protein similar to FUS/TLS and EWS proteins.

The EWS gene was found at the chromosome breakpoints in Ewing sarcoma, and the FUS/TLS gene was found at the breakpoints of myxoid liposarcoma and acute myeloid leukemia. These genes encode proteins that carry a highly homologous RNA binding domain. Fusion proteins made of the N-terminal half of EWS or FUS/TLS and transcriptional regulatory proteins, also derived from genes located at breakpoints, have been suggested to be involved in the pathogenesis of tumors. By PCR amplification of human Namalwa cell cDNA using degenerate primers made from the conserved amino acid sequences in the RNA binding domain of EWS and FUS/TLS, we obtained a cDNA fragment (RBP56 cDNA), the predicted amino acid sequences of which were similar but not identical to those of EWS and FUS/TLS. Using this fragment as a probe, we obtained two isoforms of cDNAs consisting of 2144 and 2153 bp, respectively, which encode proteins consisting of 589 and 592 amino acid residues, respectively. The predicted amino acid sequences of RBP56 protein have a serine-, tyrosine-, glutamine-, and glycine-rich region in the N-terminal region, an RNA binding domain and a C2C2 finger motif in the central region, and degenerate repeats of DR(S)GG(G)-YGG sequences in the C-terminal region. The expression of RBP56 mRNA was observed in all of the human fetal and adult tissues examined, as was the expression of EWS and FUS/TLS mRNAs. The RBP56 gene was mapped to chromosome 17q11.2 to q12.

Experimental correspondence between spore dosimetry and spectral photometry of solar ultraviolet radiation.

The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelenght interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (+/- SD) between them was 1.24 (+/- 0.16) for 14 data points showing high inactivation rates (> 0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis.

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 bp DNA segment from a 5' portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.

Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.

In Bacillus subtilis, the adaptive response to DNA alkylation depends on the ada operon, which consists of the adaA and adaB genes, which encode methylphosphotriester DNA methyltransferase (AdaA protein) and O6-methylguanine DNA methyltransferase (AdaB protein), respectively. A structural gene (alkA) that encodes 3-methyladenine DNA glycosylase was found upstream of the ada operon, but in the opposite orientation. This cluster of genes was mapped at about 235 kb from the SfiI recognition site near the origin of replication in the physical map of the B. subtilis chromosome. Disruption of the alkA gene sensitized cells to N-propyl-N'-nitro-N-nitrosoguanidine, while its overproduction rendered cells highly resistant to N-propyl-N'-nitro-N-nitrosoguanidine, indicating that lethal DNA damage produced by bulky alkylating agents was effectively counteracted by AlkA glycosylase. Transcription of the alkA gene was induced by treating adaA+ cells with methylating agents concurrent with transcription of the ada operon. This was accomplished by using methylated AdaA protein bound to a 30-bp segment in the middle of the 100-bp sequence between the transcriptional start sites of the alkA gene and ada operon. Thus, in this organism, the adaptive response to DNA alkylation is achieved by autologous activation of a divergent regulon composed of the genes for a DNA glycosylase and two species of DNA alkyltransferase.

Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents.

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.

Bacillus subtilis ada operon encodes two DNA alkyltransferases.

By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively. These two genes overlap by 11 bp and comprise a small operon. The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells. From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues. Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E. coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.

Isolation of a Bacillus subtilis mutant defective in constitutive O6-alkylguanine-DNA alkyltransferase.

A mutant of Bacillus subtilis defective in the constitutive activity of O6-alkylguanine-DNA alkyltransferase was isolated from a strain (ada-1) deficient in the adaptive response to DNA alkylation. Cells carrying the mutation dat-1 which was responsible for the defect in constitutive activity exhibited hypersensitivity for lethality and mutagenesis when challenged with methyl-nitroso compounds. The constitutive activity is independent of the adaptive response, and seems to function as a basal defense against environmental alkylating agents.

Bacillus subtilis gene coding for constitutive O6-methylguanine-DNA alkyltransferase.

We have cloned a Bacillus subtilis DNA fragment that could correct the defect in a constitutive O6-methylguanine-DNA alkyltransferase (Dat1). This fragment also corrected the hypersensitivity of the strain TKJ6951(ada-1 dat-1) to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In the fragment, the gene activity resides in a region of about 850 bp which contains an open reading frame capable of coding for a protein of 165 amino acid residues. The amino acid sequence of this protein exhibits striking similarity to those of E. coli O6-methylguanine-DNA alkyltransferases (Ogt and Ada proteins). We conclude that this is a structural gene for the Dat1 protein, which is distinct from inducible DNA alkyltransferases involved in the adaptive response. The dat-1 mutation was shown to be caused by a structural rearrangement affecting the coding region, and the 0.8 kb transcripts of this gene were detected in dat+ cells but not in dat mutant cells.

Multiple species of Bacillus subtilis DNA alkyltransferase involved in the adaptive response to simple alkylating agents.

Three molecular species of methyl-accepting proteins exist in Bacillus subtilis cells, which collect methyl groups from methylated DNA. A 20-kilodalton (kDa) protein was constitutively present in the cells of the ada+ (proficient in adaptive response) strain as well as in those of six ada (deficient in adaptive response) mutant strains and was assigned to the O6-methylguanine:DNA methyltransferase. Another species of O6-methylguanine:DNA methyltransferase, which had a molecular size of 22 kDa, emerged after adaptive treatment of the ada+ but not any of the ada mutant cells. A 27-kDa methyl-accepting protein, which preferred methylated poly(dT) to methylated calf thymus DNA as a substrate, was assigned to the methylphosphotriester:DNA methyltransferase. It was produced, after adaptive treatment, in the cells of ada+, ada-3, ada-4, and ada-6 strains but not in the cells of ada-1, ada-2, or ada-5 strains. These results support and extend our proposition that ada mutants can be classified into two groups; one (the ada-4 group) is defective only in the inducible synthesis of O6-methylguanine:DNA methyltransferase (22-kDa protein), and the other (the ada-1 group) is deficient in the adaptive response in toto. The finding that inducible and constitutive methyltransferases reside in different molecular species of methyl-accepting proteins is intriguing compared with the regulatory mechanisms of the adaptive response to simple alkylating agents in other organisms.

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents.

Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada+) cells contained similar constitutive levels of O6-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada+ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada+, augmented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N'-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.

Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents.

Three mutant strains exhibiting hyper-sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, but not to methyl methanesulfonate, were selected by a replica method from mutagenized spores of Bacillus subtilis. All three were totally deficient in the adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine with regard to both lethality and mutagenesis. The activity to destroy O6-methylguanine residues in the methylated DNA was not elevated in the mutant cells by the pretreatment with sublethal concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. This deficiency corresponded to the persistence of O6-methylguanine residues in the DNA of both control and pretreated mutant cells challenged with the drug. The lethal and mutagenic sensitivity of the mutant strains were observed only for methyl- or ethyl-nitroso compounds that are thought to be active as inducers and are also active in O-alkylation. Except for the insensitivity to methyl methanesulfonate, the phenotypes of these mutants look very similar to those of ada mutants isolated previously in Escherichia coli.