Chemical Mutagen Ethyl Methanesulfonate Research Articles

Effect of EMS induction on some morphological traits of Okra (Abelmoschus esculentus L)

Okra (Abelmoschus esculentus L.) is the only vegetable crop of significance in the Malvaceae family and is common in tropical and subtropical regions of the world. Mutation induction has been accepted as a useful tool in plant breeding programs to provide maximum inheritable variability. During this study, the effect of chemical mutagen Ethyl Methane Sulfonate (EMS) at three levels, 0.175%, 0.350% and 0.525% and two soaking period, 18 and 24 h. studied for variation induction on agronomic traits of okra using factorial experiment in a completely randomized design. In the M2 generation, agronomic traits were recorded including plant height, pod number/plant, pod length, pod diameter, stem thickness, seed number/pod, pod weight, number of locules, 100 seed weight and seed yield/plant. Analysis of variance showed that EMS concentration was significant at the 5% level for pod length, stem thickness and seed yield/plant and was highly significant for plant height, number of locules and 100 seed weight at the 1% probability level. The interaction of dose×soaking duration were significant pod number/plant and seed yield/ plant at 5% level and the characteristic of 100 seed weight was significant at 1% probability level. Application of EMS mutation lead to increased plant height, pod number/plant, pod length, pod diameter, stem thickness, seed number/ pod, number of locules, seed yield/plant and decrease of seed weight in okra. These results demonstrating that EMS could greatly affect the most of studied characters in M2 generation which it would increase the efficiency of the breeding objectives.

Genetic characterisation and fine mapping of a chlorophyll-deficient mutant (BnaC.ygl) in Brassica napus

A chlorophyll-deficient mutant with yellow-green leaves of Brassica napus was obtained by treatment with the chemical mutagen ethyl methanesulfonate. Compared with the wild type at seedling stage, the mutant displayed decreased total chlorophyll content, less granal stacks and granal membranes. Genetic analysis confirmed that the mutant phenotype was controlled by a recessive gene, which was designated as BnaC.ygl. Mapping of the gene was subsequently conducted in two populations with yellow-green leaves (population IBC8 and IIBC4, which comprised 3,472 and 5,288 individuals, respectively). Analysis on the public simple sequence repeat markers (SSR) showed that four SSR markers linked to BnaC.YGL gene displayed polymorphism. Based on the information of these SSR markers, the BnaC.YGL gene was mapped to the linkage group N17. From a survey of amplified fragment length polymorphism (AFLP), 15 of 47 AFLP markers were successfully converted into sequence characterised amplified region (SCAR) markers. BnY5 and CB10534, the closest flanking markers, were 0.32 and 0.03 cM away from the BnaC.YGL gene, respectively. And in the two populations, 18 makers cosegregated with BnaC.YGL. BLAST analysis revealed that the sequences of the makers displayed highly conserved homology with C06 of B. oleracea. The collinearity of makers to makers on N17 and on C06 showed that there might be an inversion occurring on the N17 group. These results are expected to accelerate the process of cloning the BnaC.YGL gene and facilitate the understanding of the biological processes of chloroplast development in Brassica napus.

Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme.

In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL), 36 hours fermentation time, pH 5, 30 °C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30 °C. The Michaelis-Menton constants (K(m), Vmax, Kcat and Kcat/K(m)) were 20 mM, 45.87 U mL(-1), 1118.81 s(-1) and 55.94 s(-1) mM(-1), respectively. The enzyme was found to be thermally stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness.

Enhancement of mycolytic activity of an antagonistic Bacillus subtilis through ethyl methane sulfonate (EMS) mutagenesis

Bacillus subtilis, isolated from the chili rhizosphere, capable of producing 3 mycolytic enzymes (chitinase, \beta-1,3-glucanase, and cellulase), was mutated by the chemical mutagen ethyl methane sulfonate (EMS). Mutants were screened based on their antifungal ability on dual plate assay against Colletotrichum gloeosporioides OGC1, the causal agent of anthracnose disease in fruit crops. EMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4, and M24) showed loss of antagonism against C. gloeosporioides OGC1 and 6 (M8, M21, M22, M57, M58, and M59) exhibited increased antagonism. The remaining mutants did not show any difference in their antagonistic property. In liquid culture, the mutants exhibited varied levels of the 3 enzymes. These mutants were studied for their mycolytic enzyme activities under shake flask conditions. Of all these mutants, M57 showed a 36-fold and 4.71-fold increase in \beta -1,3-glucanase and cellulase activities, respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59, with a 5.68-fold and 1.57-fold increase in \beta -1,3-glucanase and cellulase activities, respectively, and a 2.23-fold increase in hydrolytic activity as compared to the wild type B. subtilis strain. M24 exhibited a complete loss of \beta -1,3-glucanase and a decrease in chitinase, with a concomitant decrease in levels of hydrolytic activity with C. gloeosporioides mycelia as compared to the wild type strain. The study clearly indicated the mycolytic enzyme mediated antagonism of this strain and yielded 2 hyperproducing mutants for \beta -1,3-glucanase and cellulase, 2 important enzymes for various agricultural and industrial uses.

In vitro generation of somaclonal variant plants of sugarcane for tolerance to Fusarium sacchari

KEY MESSAGE : A combination of in vitro culture and mutagenesis using ethyl methanesulfonate (EMS) followed by culture filtrate-mediated selection produced variant sugarcane plants tolerant and resistant to Fusarium sacchari. Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of the stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane. In the current study, sugarcane plants tolerant and resistant to F. sacchari PNG40 were produced by exposing embryogenic calli to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent reduced plantlet yield. Subsequent trimming of the roots of regenerated plants and their exposure to 1,500ppm CF served as a further selection treatment. Plants produced from EMS-treated calli displayed improved root re-growth in the presence of CF pressure compared with those from non-treated calli. The tolerance of CF-selected plants was confirmed in greenhouse tests by inoculation with F. sacchari PNG40, re-isolation of Fusarium spp. from undamaged tissue of asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis. The restriction of PNG40 presence to the inoculation lesion in some plants suggested their resistance to the fungus. Genotypes exhibiting symptomless endophytic colonization by PNG40 were identified and will be utilised for testing biological control strategies against E. saccharina.

Evolutionary Mechanisms of Microbial Genomes 2012

What is the driving force in the course of microbial genome evolution? What is the mechanism for distinguishing self-genome from others? These fundamental questions remain elusive although rigorous studies are underway by using comparative genomics. The special issue “Evolutionary mechanisms of microbial genomes” has been launched in 2011 and presented 11 original papers. Here, this new version in 2012 presents 10 papers (one review and nine research articles). Two papers are presented in phylogenomics. K. Oshima et al. revealed a close relationship of Aquificales to Thermotogales based on the whole-genome comparison in “Phylogenetic position of Aquificales based on the whole genome sequences of six Aquificales species.” An extensive and elaborate review of fish pathogenic bacteria has been presented by P. S. Sudheesh et al. in “Comparative pathogenomics of bacteria causing infectious diseases in fish.” Two papers are presented on subjects related to evolution of base composition in genomes. H. Nishida et al. in “Genome signature difference between Deinococcus radiodurans and Thermus thermophilus” observed distinct tetranucleotide frequencies between the genomes of D. radiodurans and T. thermophilus, potentially reflecting different evolutionary backgrounds of the two species after divergence from common ancestor. H. Nishida in “Comparative analyses of base compositions, DNA sizes, and dinucleotide frequency profiles in archaeal and bacterial chromosomes and plasmids” reported lower GC content (by up to ~10%) of plasmids compared to their host chromosomes and higher correlation of GC content and chromosome size in bacteria than in archaea. Two papers are presented about horizontal gene transfer in genome evolution. M. Jalasvuori in “Vehicles, replicators, and intercellular movement of genetic information: Evolutionary dissection of a bacterial cell” discussed a hypothesis that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve toward possessing horizontally moving replicators of various types. V. S. Pylro et al. described horizontal gene transfer events of the gene dszC involved in the cleavage of carbon-sulfur bonds in “Detection of horizontal gene transfers from phylogenetic comparisons.” An article about DNA mutation is presented by Y. Shiwa et al. in “Whole-genome profiling of a novel mutagenesis technique using proofreading-deficient DNA polymerase δ.” They compared mutations created by the chemical mutagen ethyl methanesulfonate (EMS) and the proofreading-deficient DNA polymerase δ and found that the mutations created by the proofreading-deficient DNA polymerase δ generated more diverse amino acid substitution patterns than those by EMS. Three papers are presented on subjects related to metabolic pathway. H. Nishida in “Comparative analyses of homocitrate synthase genes of ascomycetous yeasts” described gene duplications of the homocitrate synthase which have occurred multiple times during evolution of the ascomycetous yeasts. H. Nishida and M. Nishiyama in “Evolution of lysine biosynthesis in the phylum Deinococcus-Thermus” reported that bacterial lysine biosynthesis genes of the common ancestor of the Deinococcus-Thermus phylum used the α-aminoadipate pathway instead of the diaminopimelate pathway. K. Ueda et al. in “Dispensabilities of carbonic anhydrase in Proteobacteria” analyzed the distribution of carbonic anhydrase (CA) in proteobacteria, compared CA-retaining and CA-deficient genomes, and found absence of coding sequence in some strains and frame shifts in others. In closing this introduction to the special issue, we would like to express our full appreciation to all the authors and reviewers for their enormous efforts that have made the timely completion of our assignment successful. We sincerely hope that this special issue will stimulate further the investigation of evolutionary mechanisms of microbial genomes. Hiromi Nishida Shinji Kondo Hideaki Nojiri Ken-ichi Noma Kenro Oshima

Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ.

A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδ MKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδ MKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

Registration of TAM 94L-25-M24, TAM 94L-25-M25, and TAM 94L-25-M30 Mutant Upland Cotton Germplasm with Improved Fiber Length and Strength

Three putative mutant upland cotton (Gossypium hirsutum L.) germplasm lines, TAM 94L-25-M24, (Reg. No. GP-952, PI 664553), TAM 94L-25-M25 (Reg. No. GP-953, PI 664554), and TAM 94L-25-M30 (Reg. No. GP-954, PI 664555), were developed by the Cotton Improvement Laboratory, Department of Soil and Crop Sciences, Texas AgriLife Research and released in 2010. These lines were developed by treating the parent germplasm line, TAM 94L-25 (PI 631440), an upland cotton line with improved high-volume instrument (HVI) upper-half mean length (UHML), with the chemical mutagen ethyl methanesulfonate. TAM 94L-25-M24, TAM 94L-25-M25, and TAM 94L-25-M30 exhibited UHML and fiber bundle tenacity that exceeded that of the parent, TAM 94L-25. These three lines yielded as much or more than the M0 parent and exhibited other HVI fiber-quality parameters that were within acceptable ranges. TAM 94L-25-M24, TAM 94L-25-M25, and TAM 94L-25-M30 should be useful in creating cultivars with improved fiber length and strength.

Protein Gq Modulates Termination of Phototransduction and Prevents Retinal Degeneration

Appropriate termination of the phototransduction cascade is critical for photoreceptors to achieve high temporal resolution and to prevent excessive Ca(2+)-induced cell toxicity. Using a genetic screen to identify defective photoresponse mutants in Drosophila, we isolated and identified a novel Gα(q) mutant allele, which has defects in both activation and deactivation. We revealed that G(q) modulates the termination of the light response and that metarhodopsin/G(q) interaction affects subsequent arrestin-rhodopsin (Arr2-Rh1) binding, which mediates the deactivation of metarhodopsin. We further showed that the Gα(q) mutant undergoes light-dependent retinal degeneration, which is due to the slow accumulation of stable Arr2-Rh1 complexes. Our study revealed the roles of G(q) in mediating photoresponse termination and in preventing retinal degeneration. This pathway may represent a general rapid feedback regulation of G protein-coupled receptor signaling.

Identification, fine mapping and characterisation of a dwarf mutant (bnaC.dwf) in Brassica napus

In the present study, we have obtained one dwarf mutant (bnaC.dwf) from the Brassica napus inbred line T6 through chemical mutagen ethyl methanesulfonate (EMS). We have determined the phenotypic effects and genetic characteristics of dwarf mutant (bnaC.dwf). The dwarf mutant was insensitive to exogenous GA(3) for plant height, suggesting that it is significantly playing a crucial role in the gibberellins response pathway. Genetic analysis revealed that one recessive gene is responsible for controlling the phenotypic expression of dwarf mutant. Amplified Fragment Length Polymorphism (AFLP) technique was applied for selecting markers linked to the BnaC.DWF gene which assisted in screening of dwarf and normal individuals in the BC(4) population. We have screened 1,024 primer combinations and then identified nine AFLP markers linked to the BnaC.DWF gene. Identification and linkage of the markers were carried out by analysing 2,000 individuals from a larger population of the BC(4). Two markers EA10MC09 and EA12MC02 were located on the flanking region of the BnaC.DWF gene at a distance of 0.2 and 0.05 cM, respectively. Four AFLP markers EA09MG05, EA02MC07, EA01MC01 and EC04MC07 were successfully converted into Sequence Characterised Amplified Region markers namely SCA9G5, SCA2C7, SCA1C1 and SCC4C7. We further integrated BnaC.DWF linked Simple Sequence Repeat markers into two populations (Piquemal et al. Theor Appl Genet 111:1514-1523, 2005; Cheng et al. Theor Appl Genet 118:1121-1131, 2009). BnaC.DWF was mapped to the linkage region N18. The molecular markers developed from these investigations will greatly accelerate the selection process for developing dwarf varieties in B. napus by Marker Assisted Selection and genetic engineering.

Induced mutagenesis to augment the natural genetic variability of melon (<i>Cucumis melo</i> L.)

Induced mutagenesis of agricultural crops creates new variation in genes, including those affecting agriculturally important traits such as plant morphology and fruit quality. Melon (Cucumis melo L.) is a diploid species (2n = 24) with a small genome, estimated as 450 Mb, but with relatively high levels of sequence and fruit shape polymorphism. We treated seeds of ‘Noy Yizre'el’, a ‘Galia’ melon-type parental line, with the chemical mutagen EMS (ethyl methane sulfonate). The resulting M1 plants were self-pollinated to produce about 3,000 M2 families, segregating for the induced mutations that we regard as a mutation library. Phenotypic analyses revealed newly induced variation, mostly governed by single recessive mutations, affecting different plant organs, including cotyledon, leaves, flowers, and fruit, at different growth stages, from emergence to mature fruit. Several mutations show phenotypic similarities to mutations found in other plant species. Further studies are required to determine whether t...

Chemical Mutagenesis—A Promising Technique to Increase Metal Concentration and Extraction in Sunflowers

Since most of the metal-hyperaccumulating wild plants only produce very low biomass and many high-yielding crops accumulate only moderate amounts of metals, the current research is mainly focused on overcoming these limitations and the optimization of metal phytoextraction. The main goal of the present study was the improvement of metal concentration and extraction properties of Helianthus annuus L by chemical mutagenesis (the non-GMO approach). Sunflowers—hybrid cultivar Salut and inbred lines—were treated with the chemical mutagen ethyl methanesulfonate (EMS). The effect of chemical mutagenesis on metal concentration in and extraction by new sunflower M1 and M2 mutants was directly assessed on a metal-contaminated field in Rafz, Switzerland. Mutants of the M2 generation showed a 2–3 times higher metal shoot concentration than the control plants. The best M2 sunflower “giant mutant” 14/185/04 showed a significantly enhanced metal extraction ability: 7.5 times for Cd, 9.2 times for Zn, and 8.2 times for Pb in aboveground parts, as compared to the control plants. Theoretical calculations for the phytoextraction potential of new sunflower variants note that the best sunflower mutant can produce up to 26 t dry matter per hectare and remove 13.3 kg Zn per hectare and year at the sewage sludge contaminated site of Rafz; that is a gain factor of 9 compared to Zn extraction by sunflower controls. Furthermore, the use of sunflower oil and biomass for technical purposes (lubricants, biodiesel, biogas) should produce an additional value and improve the economical balance of phytoextraction.

The effects of ethyl methanesulfonate treatment on Eucalyptus pollen behaviour in vitro

Treating pollen with mutagens prior to controlled pollination may facilitate the production of mutant trees for developmental studies and eventual plantation improvement. To establish a suitable dose of the chemical mutagen ethyl methanesulfonate (EMS) for the testing of this hypothesis, pollen of Eucalyptus globulus ssp. globulus and E. grandis was studied in vitro. Pollen germination, pollen tube elongation and generative cell division were examined after 48 h of culture, following exposure to between 0 and 1,000 ppm EMS. Doses of 600 to 1,000 ppm EMS reduced pollen germination in vitro in both species. Doses of up to 1,000 ppm EMS were not observed to significantly impact on either pollen tube length, or generative cell division in vitro of either species. A dose of 600 ppm EMS in paraffin oil is predicted to induce mutation in Eucalyptus species whilst impacting minimally on seed production based on the effect on pollen germination.

Chemically selected subclones of the CEM cell line demonstrate resistance to HIV-1 infection resulting from a selective loss of NF-kappa B DNA binding proteins.

To delineate cellular genes that are required for optimal HIV-1 infection, CEM cells were subjected to treatment with the chemical mutagen ethylmethanesulfonate (EMS) and subclones were selected based on their increased resistance to HIV-1 infection and reduced syncytium formation, despite relatively normal CD4 expression (20,000 to 25,000 receptors/cell). Two subclones with this phenotype demonstrated a diminished capacity of HIV-1 long terminal repeat-chloramphenicol acetyl transferase expression either after treatment with the protein kinase C activator PMA, or through Tat-mediated transactivation. In this study, we show that the cellular levels of the NF-kappa B DNA binding proteins (but not AP1 or SP1) are markedly reduced in these cell mutants both at the mRNA and protein levels, resulting in reduced nuclear localization of p50/p65 after PMA induction or treatment with the lymphokine TNF-alpha. Transient reconstitution with a plasmid expressing p50 resulted in partial recovery of PMA-inducible LTR-chloramphenicol acetyl transferase expression. These data suggest that, at least in the CEM T cell line, a selective reduction in the NF-kappa B DNA binding proteins is sufficient to curtail HIV-1 infection.

Downstream signal transduction defects that suppress transformation in two revertant cell lines expressing activated rat neu oncogene.

The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu-induced cellular transformation, we developed revertant cells from the neu-transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethyl methanesulfonate. The morphologically normal revertant cells were first selected by their ability either to attach to culture plates or to survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2-deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu-transformed cells, revertants exhibiting defective tyrosine phosphorylation of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation.

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L.

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR(1)) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA(3), was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA(3). Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis.

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate: Quantitative comparison of the mutagenic potency in Neurospora crassa and Saccharomyces cerevisiae

Extending previous work with E. coli and mammalian cells in culture, forward-mutation frequencies induced by ethyl methanesulfonate (EMS) were quantitatively compared in Neurospora crassa and Saccharomyces cerevisiae under standardized conditions. Concomitantly, the actual dose to DNA was measured by determining the amount of radioactivity bound to DNA after treatment with tritium-labeled EMS. After exposure to EMS (2.5–50 mM), alkylation levels in N. crassa and S. cerevisiae were similar to those previously determined in E. coli and cultured mammalian cells. Consistently, there was a slightly less than proportional increase of the DNA alkylation level with the exposure concentration of the mutagen. Forward mutagenesis induced in yeast and N. crassa showed exponential kinetics with exponents of 1.5 and 2.6, respectively. These results are similar to those previously reported with E. coli, which differed from the results with cultured mammalian cells, where a linear dose-effect relationship between exposure and genetic effect was observed. These differences may reflect differences in the fate of EMS-induced adducts by cellular DNA repair systems, but are not due to initial differences in DNA alkylation levels. The fate and persistence of specific DNA adducts potentially responsible for pre-mutagenic changes are under investigation.