methylnitronitrosoguanidine and Chromosome-Deletion

The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.

Topics: Animals; Chromosome Deletion; Chromosome Mapping; DNA, Protozoan; Drug Resistance; Genes, Protozoan; Genetic Markers; Leishmania major; Methotrexate; Methylnitronitrosoguanidine; Mutagenesis; Ploidies; Polymerase Chain Reaction; Selection, Genetic; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Virulence

By analysis of skin tumors from F1 hybrid mice we demonstrated that the genetic events that occur during tumor progression depend on the type of chemical carcinogenesis protocol used to induce tumor growth. More than 95% of tumors induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibited mutations in Ha-ras and trisomy of chromosome 7. Carcinomas induced with multiple DMBA treatments had a lower frequency of alterations on chromosome 7 (50%), but only in tumors with Ha-ras mutations, and had a much wider spectrum of alterations, including trisomy, mitotic recombination, deletion, and gene duplication. Carcinomas induced with multiple N-methyl-N'-nitro-N-nitrosoguanidine treatments only rarely exhibited alterations on chromosome 7 (8%), even if they contained mutant Ha-ras. More frequent numerical alterations of chromosome 11 were also seen in TPA-promoted tumors (23%) than in tumors induced by multiple carcinogen treatments (8%). These results show that postinitiation events are nonrandom and fit a model in which promoting agents induce numerical chromosomal alterations but in which mutagens cause more directed mutational events.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Chromosome Aberrations; Chromosome Deletion; Genes, ras; Methylnitronitrosoguanidine; Mice; Mutation; Polymorphism, Restriction Fragment Length; Skin Neoplasms; Tetradecanoylphorbol Acetate; Trisomy

Clonal lines of transformed rat liver epithelial cells, derived from a single population of cloned diploid rat liver epithelial (stem-like) cell line (WB-F344) by exposure in vitro to N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), produce hepatocellular carcinomas, hepatoblastomas and adenocarcinomas in syngeneic rats (Tsao and Grisham, Am. J. Pathol., 127, 168-181, 1987). In this study we show that these clonal lines demonstrate near-diploid (GN clones) or near-triploid (GP clones) aneuploidy and the universal occurrence of non-random chromosomal abnormalities. Marker chromosomes that involved four autosomes--a non-reciprocal translocation involving chromosomes 1 and 7 (t1q43;7q34), and addition of DNA of unknown origin to the pericentromeric regions of chromosomes 4 and 10--occurred in all of the cells of all transformed clones and in the cells of tumors that grew from them. New marker chromosomes involving the same regions of chromosomes 4 and 7 were found in several cell lines established from independent tumors. The preservation of marker chromosomes in tumor cells in the face of random loss and gain of other chromosomes suggests that these non-random aberrations were necessary for tumor formation. The presence of marker chromosomes was associated with increased expression of the c-myc gene (located at q34 on chromosome 7), the c-H-ras gene (located at q41-43 on chromosome 1) and the c-K-ras and TGF alpha genes (both located at unknown sites on chromosome 4), which we have previously shown to be highly correlated with tumorigenicity in these same transformed clonal lines (Lee et al., Cancer Res., 51, 5238-5244, 1992).

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Deletion; DNA; DNA, Neoplasm; Epithelium; Genetic Markers; Karyotyping; Liver; Liver Neoplasms, Experimental; Male; Methylnitronitrosoguanidine; Phenotype; Ploidies; Rats; Rats, Inbred F344; Tumor Cells, Cultured

Mutations in the visA gene of Escherichia coli cause the mutant bacteria to die upon illumination with visible light. We confirmed genetically that the visA gene is a structural gene for ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1). Since other mutations in the genes involved in the biosynthesis of heme can cure the photosensitivity, the light-induced cell death appears to be brought about by the accumulation of protoporphyrin IX, one of the substrates of ferrochelatase. When cells are illuminated with visible light, protoporphyrin IX seems to produce an active species of oxygen (probably 1O2) that is harmful to the cells. This defect is the same as that associated with the human disease protoporphyria.

Topics: Anaerobiosis; Bacteriophage lambda; beta-Galactosidase; Catalase; Chromosome Deletion; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Ferrochelatase; Genes, Bacterial; Genotype; Light; Methylnitronitrosoguanidine; Mutagenesis; Phenotype; Protoporphyrins; Recombinant Proteins

The adaptive response of Escherichia coli protects cells against the mutagenic and toxic effects of alkylating agents. This response is controlled by the Ada protein, which not only functions as the transcriptional activator of the ada and alkA genes but also possesses two DNA methyltransferae activities. Ada is converted into an efficient transcriptional activator by transferring a methyl group from a DNA methylphosphotriester to its own Cys-69 residue and then binds to a DNA sequence (the Ada box) present in both the ada and alkA promoters. Although the Ada protein initially appeared to regulate the ada and alkA genes in a similar fashion, our studies show that the wild-type Ada protein and its truncated derivatives can differentially regulate ada and alkA transcription. In vivo, lower levels of wild-type methylated Ada are needed to activate ada transcription than alkA transcription. In cells exposed to alkylating agents, the N-terminal half of Ada, which contains the DNA-binding domain, is sufficient for efficient activation of alkA, but not ada, transcription. Moreover, truncated derivatives containing 80-90% of Ada are extremely strong constitutive activators of ada but are only inducible activators of alkA transcription. These results suggest that the mechanism by which Ada activates ada transcription differs from that by which it activates alkA transcription.

Topics: Alkylating Agents; Bacterial Proteins; Base Sequence; beta-Galactosidase; Chromosome Deletion; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Methylnitronitrosoguanidine; Molecular Sequence Data; Mutagenesis, Site-Directed; O(6)-Methylguanine-DNA Methyltransferase; Oligodeoxyribonucleotides; Plasmids; Recombinant Fusion Proteins; Restriction Mapping; Transcription Factors; Transcription, Genetic

Escherichia coli responds to superoxide-generating agents by inducing approximately 40 proteins. We have identified a genetic locus, soxR (superoxide response), that positively regulates 9 of these proteins during superoxide stress. Induction under soxR control is at the transcriptional level, as shown with lac fusions to five paraquat-inducible promoters. Members of the soxR regulon include at least three proteins with demonstrable antioxidant roles: Mn-containing superoxide dismutase (which destroys superoxide radicals), endonuclease IV (which repairs radical-induced damages in DNA), and glucose-6-phosphate dehydrogenase (which produces NADPH). Induction of the soxR regulon also leads to diminished levels of the major outer membrane protein OmpF and alteration of the small-subunit ribosomal protein S6. These latter changes confer resistance to a variety of antibiotics. The soxR regulon may thus operate as an inducible defense against xenobiotics in general.

Topics: Anti-Bacterial Agents; Antioxidants; Chromosome Deletion; Cloning, Molecular; Deoxyribonuclease IV (Phage T4-Induced); DNA Transposable Elements; DNA-(Apurinic or Apyrimidinic Site) Lyase; Drug Resistance, Microbial; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Regulator; Glucosephosphate Dehydrogenase; Methylnitronitrosoguanidine; Operon; R Factors; Superoxide Dismutase; Superoxides; Transduction, Genetic

The proceeding of malignantly transformed rodent cells in vitro is often accompanied by regular variation of chromosome numbers and structures. In order to clarify the relationship between them, we obtained five clones C1-1-5-from BHL B4, cell line, and analysed their biological characteristics. Results showed a positive correlation between frequency of chromosome 15 monosomy, it is suggested that suppressive gene of malignant phenotype be located on chromosome 15.

Topics: Animals; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Deletion; Cricetinae; Fibroblasts; Gene Expression Regulation, Neoplastic; Lung; Mesocricetus; Methylnitronitrosoguanidine; Monosomy; Neoplasm Transplantation; Phenotype

During the course of malignant transformation of mammalian cells in vitro, a regular variation in chromosome number and structure is usually found. In order to elucidate the relationship between chromosomal changes and malignant expression, we isolated five clones from a malignant transformed Syrian hamster fibroblast cell line and analysed their biological characteristics, as well as chromosomal changes. A positive correlation between chromosome No 15 monosomy and the transformed and malignant phenotype was observed. We suggest that a suppression gene may be located on chromosome No 15, and its deletion or the loss of the chromosome may result in expression of the malignant phenotype.

Topics: Animals; Cell Line, Transformed; Chromosome Deletion; Cricetinae; Fibroblasts; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Lung; Mesocricetus; Methylnitronitrosoguanidine; Monosomy; Neoplasm Transplantation; Phenotype

The mismatch repair system of Escherichia coli K12 removes mispaired bases from DNA. Mismatch repair can occur on either strand of DNA if it lacks N6-methyladenines within 5'-GATC-3' sequences. In hemimethylated heteroduplexes, repair occurs preferentially on the unmethylated strand. If both strands are fully methylated, repair is inhibited. Mutant (dam-) strains of E. coli defective in the adenine methylase that recognizes 5'-GATC-3' sequences (Dam), and therefore defective in mismatch repair, show increased spontaneous mutation rates compared to otherwise isogenic dam+ hosts. We have isolated and characterized 91 independent mutations that arise as a consequence of the Dam- defect in a plasmid-borne phage P22 repressor gene, mnt. The majority of these mutations are A:T----G:C transitions that occur within six base pairs of the two 5'-GATC-3' sequences in the mnt gene. In contrast, the spectrum of mnt- mutations in a dam+ host is comprised of a majority of insertions of IS elements and deletions that do not cluster near Dam recognition sites. These results show that Dam-directed post-replicative mismatch repair plays a significant role in the rectification of potential transition mutations in vivo, and suggest that sequences associated with Dam recognition sites are particularly prone to replication or repair errors.

Topics: Chromosome Deletion; DNA Repair; Escherichia coli; Genes, Bacterial; Methylnitronitrosoguanidine; Methyltransferases; Mutation; Site-Specific DNA-Methyltransferase (Adenine-Specific)

Skin fibroblasts from patients with hereditary retinoblastoma (RB cells) were examined since predisposition to the tumour might be expected to involve some degree of chromosomal instability, as has been noted for several cancer-prone conditions. Spontaneous and N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced aberration frequencies were measured, the cytogenetic effects of long-term treatment with 12-O-tetradecanoyl-phorbol-13acetate (TPA) were examined and also the spontaneous and TPA-induced sister chromatid exchange (SCE) frequencies. In all the studies the RB cells behaved in a similar fashion to normal human skin fibroblasts.

Topics: Cells, Cultured; Chromosome Aberrations; Chromosome Deletion; Eye Neoplasms; Humans; Methylnitronitrosoguanidine; Ploidies; Retinoblastoma; Sister Chromatid Exchange; Skin; Tetradecanoylphorbol Acetate; Time Factors

A bacterial strain was constructed which permitted positive selection for ochre suppressor mutations as well as for the loss of suppressor function. A derivative bearing an ochre suppressor mutation was selected following mutagenesis with N-methyl-N-nitroso-N'-nitroguanidine. The suppressor-bearing strain was treated with nitrous acid to eliminate suppressor function by mutation, and a strain lacking suppressor activity was selected. The selected strain which had lost suppressor function was then subjected to mutagenesis to induce a second suppressor mutation. The alternating sequence (induction of an ochre suppressor mutation leads to induction of a mutation eliminating ochre suppressor activity) was repeated 29 and one-half times in a single strain. Some of the suppressor mutations were tentatively mapped at four locations on the chromosome. The first suppressor mutation selected maps at about minute 30 on the chromosome. The second suppressor selected maps at approximately minute 60, while the third suppressor maps nearby, possibly as far as minute 72. Among the subsequently selected suppressor mutations, all eleven which were mapped were cotransducible with the gal and nic loci near minute 36 on the chromosome and may represent more than one suppressor gene. Deletions were selected which inactivate two of the ochre suppressor alleles mapping near the gal-nic region, suggesting that one or more such genes are dispensable. Some evidence also suggests that the occurrence of either deletion mutations or transduction-mediated recombination events in the gal-nic region can cause instability of nearby suppressor alleles.

Topics: 2-Aminopurine; Chromosome Deletion; Chromosome Mapping; Chromosomes, Bacterial; Conjugation, Genetic; Methylnitronitrosoguanidine; Mutation; Salmonella typhimurium; Selection, Genetic; Suppression, Genetic; Transduction, Genetic

Topics: 2-Aminopurine; Chromosome Deletion; Genes, Viral; Hydroxylamines; Methylnitronitrosoguanidine; Mutation; T-Phages