Chemical Mutagen Ethyl Methanesulfonate Research Articles

Isolation and characterization of cycloheximide-resistant mosquito cell clones

Following treatment of cultured mosquito cells ( Aedes albopictus line of Singh) with the chemical mutagen ethyl methanesulfonate, we were able to isolate three cycloheximide-resistant clones. On the basis of growth kinetics, plating efficiency, and protein synthesis, these clones are 10- to 30-fold more resistant to cycloheximide than the parent cells. Cell-free lysates made from these cells retain 30–65% of their endogenous protein synthesizing ability in the presence of cycloheximide concentrations as high as 300 μg/ml. Protein synthesis in lysates from the parental cells, however, is reduced to about 10% of the control value (i.e., in the absence of drug) at 14 μg of cycloheximide/ml and was completely abolished at 75 μg/ml. These results indicate that cycloheximide resistance in these cells is likely due to an alteration in the protein synthetic machinery. This is the first description of cycloheximide-resistant insect cells, and the best example of cycloheximide resistance in cells originating from a higher eukaryotic organism.

Electron transport and chloroplast ultrastructure of a chlorophyll deficient mutant of wheat

A non-lethal chlorophyll deficient mutation was induced by use of the chemical mutagen ethyl methanesulfonate. Chloroplasts from the control and mutant plants were found to be very similar ultrastructurally. Thylakoid membrane volume was only slightly greater in plastids from the control as compared with plastids from the mutant. The chlorophyll content of the mutant was reduced by over 60%. This decrease in chlorophyll was not accompanied by a similar decrease in electron transport. Uncoupled electron transport rate based on a unit chlorophyll basis was nearly twice as great for mutant chloroplasts as for control plastids. However, electron transport rate based on a unit membrane volume was similar in mutant and control plants. At high irradiance the relative quantum requirement of the control and mutant was similar when expressed on membrane volume.

A Search for Condensed Tannins in Annual and Perennial Species of Medicago, Trigonella, and Onobrychis1

Bloat of ruminant animals feeding on fresh, green alfalfa (Medicago sativa L.) is caused by high levels of soluble plant proteins which act as foaming agents. It is postulated that bloat‐safe cultivars may be bred by introducing condensed tannins (flavolans) which would act as protein precipitants to preclude foam formation and consequent bloat. Using the vanillin‐HCl spot test, large populations of alfalfa were screened for tannins. These included several accessions, cultivars, and breeding populations. Extensive screening of 2n M. falcata L. and 4n M. sativa treated with the chemical mutagens ethyl methanesulfonate or ethyleneimine failed to reveal any mutations for tannins. Screening of 33 species (a total of 86 accessions) of annual Medicago was negative. Similarly, screening of 28 species (a total of 92 accessions) of perennial Medicago was also negative. Examination of 30 species (46 accessions) of the closely related Trigonella genus did not reveal any tannin‐containing plants. On the other hand, testing of 123 accessions of 10 species of Sainfoin (Onobrychis) showed that all plants examined within this bloat‐safe genus contained high levels of tannins.Several of the common pasture legumes, including both bloat‐causing and bloat‐safe types, were examined for tannins in leaf tissue, flower (petal) tissue and seeds. All of these legumes except alfalfa and cicer milkvetch (Astragalus cicer I.,) contained tannins in petal tissue. All of the legumes, including alfalfa, contained tannins in seedcoat tissue except that of a homozygous recessive, white‐seeded strain of alfalfa.The implications of breeding for tannins to provide a bloat‐safe alfalfa are discussed.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate: Quantitative comparison of mutation induction in Escherihia coli, V79 Chinese hamster cells and L5178Y mouse lymphoma cells, and some cytological results in vitro and in vivo

Molecular dosimetry studies were carried out to measure the extent of binding of radio-labeled ethyl groups to the DNA of Escherichia coli, V79 Chinese hamster cells and L5178Y mouse lymphoma cells treated with ethyl methanesulfonate (EMS). The results show that (1) the amount of ethylation of the DNA is similar in these cells when treatment conditions are identical, (2) the relationship between dose to DNA (ethylations per nucleotide) versus exposure (mM applied concentration) is non-linear in the sense that less alkylation of the DNA is observed at the higher exposure than would be predicted on the basis of proportionality between dose to DNA and exposure, and (3) the non-linearity of the genetic response in the bacterial cells is not reflected in a non-linearity of the alkylation of the DNA in those cells. Quantitative comparison of the frequencies of gene mutations in the various systems shows that the mutation frequency per unit DNA alkylation is heterogeneous among the mammalian cell systems and that the frequencies observed in the bacterial cells fall within the range observed with mammalian cells. Alkylation of the DNA in the bone marrow, testis and liver of Swiss randombred mice was also measured. The results support the conclusion that the distribution of the compound to the various tissues is rapid and probably uniform. Quantitative assessment of the cytological data (micronuclei, sister-chromatid exchanges, etc.) on the basis of dose was not as useful because of the low efficiency of EMS for inducing cytologically observable damage.

Isolation and characterization of six embryo-lethal mutants of Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana strain “Columbia” were soaked for 7.5 hr in an aqueous solution of the chemical mutagen ethyl methanesulfonate (0.05, 0.10, or 0.50%, v/v). Embryo-lethal mutants were identified in the resulting M-1 chimeral plants by screening the first five siliques of each plant and noting the frequency of aborted seeds. Three hundred sixty seeds were treated at each mutagen dose; the frequency of embryo-lethal mutants ranged from 1–3% of the M-1 plants grown from seeds exposed to 0.05% EMS, to 20–30% of the M-1 plants at the highest mutagen dose. Six embryo-lethal mutants identified through screening of M-1 plants were chosen for detailed studies in subsequent generations. All six mutants segregate as nonallelic, Mendelian recessive lethals, and are maintained as heterozygotes since homozygotes die as embryos. Fruits of heterozygous plants contain 25% aborted seeds and 75% phenotypically normal seeds ( 2 3 heterozygotes and 1 3 wild type). Segregation ratios are not temperature sensitive; the same frequency of aborted seeds is found in plants grown at 18, 25, and 32°C. Embryo arrest and eventual lethality in each mutant occur at a characteristic stage of early embryo development: globular-heart, globular, early globular, or preglobular. Arrested embryos from five of the six mutants resemble normal embryos at early stages of development. Developmental arrest of the embryo proper in the remaining mutant is followed by abnormal growth of the suspensor, an embryonic structure that attaches the embryo proper to the maternal tissue.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate in Escherichia coli and in V-79 chinese hamster cells

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate in Escherichia coli and in V-79 chinese hamster cells

MUTAGENESIS OF CULTURED PLANT CELLS

Experiments were designed to study the effectiveness of the chemical mutagens ethylmethane sulfonate and nitrosoguanidine on plant cells growing in liquid suspensions. Mutation frequency was defined as the number of colonies appearing on selective plates divided by the number of colonies growing on non-selective plates. The compounds tested usually increased mutation frequency by one order of magnitude over the spontaneously occurring rate, although the increase ranged from one to 140-fold. Cell killing was found to be directly correlated with mutation frequency.

Unscheduled DNA Synthesis in the Germ Cells of Male Mice Exposed In Vivo to the Chemical Mutagen Ethyl Methanesulfonate

An unscheduled DNA synthesis has been clearly demonstrated in meiotic and postmeiotic germ cell stages of the male mouse after in vivo treatment with a 250 mg/kg dose of ethyl methanesulfonate. The germ cell stages showing unscheduled DNA synthesis range from early to middle meiotic prophase stages through early to middle spermatid stages. The initiation of this synthesis, taken to be repair of chemically damaged DNA in these germ cells, is rapid, beginning within 1 hour after the injection of ethyl methanesulfonate. Unscheduled DNA synthesis has not been detected in the most mature germ cell stages, which give rise to dominant lethal mutations, nor does it occur in germ cell stages where protamine has replaced the chromosomal histones.

CYTOLOGICAL EFFECTS OF THE PESTICIDES PHOSDRIN AND BLADEX ON TRADESCANTIA AND VICIA FABA

The cytological effects of the insecticide Phosdrin (mevinphos) and the herbicide Bladex on root tips of Tradescantia and Vicia faba were observed and compared with those of the chemical mutagen ethyl methane sulfonate (EMS). In addition, plants of Vicia faba were sprayed prior to floral initiation and pollen mother cells examined for chromosomal abnormalities. Phosdrin and Bladex produced the same kinds of chromosome abnormalities as EMS, namely, fragments, bridges, multipolar anaphases and lagging chromosomes. Bladex produced a higher percentage (3.47%) of chromosome aberrations in root tips of Tradescantia than did Phosdrin (2.56%), but fewer in Vicia faba than did either EMS or Phosdrin. Bladex was more toxic to cells of Vicia faba. The greater percentage of chromosome fragments induced by both pesticides in Vicia faba may be the result of highly specific and localized action of the pesticides in heterochromatic regions. Vicia faba seedlings sprayed with Phosdrin had a greater percentage of chromosome aberrations in the pollen mother cells than were found in root tips after Phosdrin treatment. Bladex sprayed at the same concentration as Phosdrin (200, 400, 600 ppm) was lethal to the plants. The frequency of chromosomal aberrations induced by both pesticides deviated significantly from that of the control.

Ethyl methanesulfonate-induced mutations of the Sh 1 protein in maize

16 Sh 1 mutants have been obtained by treating kernels of Sh 1 Wx genotype with the chemical mutagen ethyl methanesulfonate (EMS). Immunochemical analysis of 20-day-old endosperms of newly obtained sh 1 mutants showed that 6 are CRM + (form cross reacting material with Sh 1 protein) and IO are CRM −. These mutants were further studied by starch gel electrophoresis. The occurrence of a CRM + class of sh 1 mutants, in addition to the mutants specifying electrophoretic variants of Sh 1 protein, suggests that the genetic changes induced are true gene mutations at the sh 1 locus. This study indicates that the Sh 1 protein carries out a specific function in the development of the endosperm and is not a non-specific storage-like protein since qualitative changes in the Sh 1 protein are associated with the appearance of the sh 1 phenotype.

Growth and RNA synthesis in maize seedlings treated with ethyl methanesulfonate

Corn seeds were treated with the chemical mutagen ethyl methanesulfonate and the resulting seedlings were examined for growth and RNA synthesis. Ethyl methanesulfonate treatments reduced shoot elongation, seedling growth, and chromatin-directed RNA polymerase activity of shoot tissues. The chromatin RNA polymerase activity of control seedlings peaked at 24 h. This peak was delayed up to 12 h by the chemical mutagen treatment. Saturation of embryonic axis chromatin with E. coli RNA polymerase indicated that ethyl methanesulfonate increased the amount of DNA available as template. In both the treated and control axes, the available template decreased with tissue differentiation.