Interaction of the mutagenic metabolite of sodium azide, synthesized in vitro, with DNA of barley embryos.

The in vitro synthesized sodium azide mutagenic metabolite (azidoalanine) produced single-strand breaks and proteinase K-sensitive sites in isolated, germinating barley embryos. In contrast with sodium azide, the efficiency of DNA damage induction was lower, and both types of DNA lesions were totally or partially repaired in the course of subsequent 24 h incubation of the embryos. The mutagenic azide metabolite did not inhibit DNA replication, while azide did so even at doses which are not highly mutagenic. The metabolite labelled with 14C at the amino acid residue was taken up with a similar efficiency both into barley embryos germinating for 2 days and into cells of Salmonella typhimurium TA100. The majority of the radioactivity was incorporated into proteins, less into RNA and a negligible amount into DNA.

Investigation of partial sterility in advanced generation, sodium azide-induced lines of spring barley.

The partial sterility found in several advanced generation, sodium azide-induced lines of spring barley (Hordeum vulgare L.) was investigated. Plants of mutant lines were reciprocally crossed with plants of their untreated mother lines. Spike sterility was measured in the selfed offspring of the plants crossed and in F1 and F2 progeny. Pollen sterility and endosperm development were analyzed in the selfed offspring of the plants crossed. Results indicated that the sterility was inherited in the mutant lines and was not caused by translocations, inversions, endosperm lethals, embryo-endosperm lethals, or major gene mutations. Furthermore, the sterility was not cytoplasmically inherited, and was essentially eliminated in the F1 and F2 of crosses between partially sterile lines and their fertile parents. Results suggest that the sterility may be caused by an environmental interaction with deleterious, homozygous recessive, minor gene mutations that were in the heterozygous condition when the mutant lines were originally selected.

Synthesis and mutagenicity of the two stereoisomers of an azide metabolite (azidoalanine).

The L- and D-isomers of azidoalanine (azide metabolite) have been chemically synthesized with 60% yield using corresponding N-(tert-butoxycarbonyl)-serine as starting materials. The mutagenic properties of synthesized L-azidoalanine are very similar to those of azide and in vivo synthesized azidoalanine. Synthetic D-azidoalanine shows very low mutagenic activity on Salmonella typhimurium TA1530 strain compared to that of the L-isomer. Thus a stereoselective process is involved in azidoalanine mutagenicity. The data presented in this study suggest that further biochemical activation is required for L-azidoalanine to produce its mutagenic activity.

A mutagenic metabolite synthesized by Salmonella typhimurium grown in the presence of azide is azidoalanine.

A mutagenic azide metabolite was purified from the medium in which Salmonella typhimurium cells were grown in the presence of azide. This metabolite was identified to be azidoalanine based on infrared and mass spectroscopy and elemental analysis. This compound appeared to be identical to the mutagenic compound synthesized in vitro from azide and O-acetylserine by partially purified O-acetylserine sulfhydrylase. The metabolite (azidoalanine) mutagenic efficiency and spectrum in S. typhimurium was similar to that of inorganic azide. The compounds 2-azidoethylamine, 2-bromoethylamine, 3-bromopropionic acid and N-(azidomethyl) phthalimide were also mutagenic with a similar spectrum to azide and azidoalanine, but with lower efficiency. The compounds 3-azidopropylamine, 4-azidobutylamine, 3-chloroalanine and ethylamine were only weakly or nonmutagenic. Numerous other chloro, bromo and azido phthalimide derivatives tested were nonmutagenic. It is suggested that the lack of azide mutagenicity (and perhaps carcinogenicity) in mammalian cells may be due to their inability to convert azide to azidoalanine.

In vitro production of azide mutagenic metabolite in Arabidopsis, Drosophila and Neurospora.

The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530.

Lack of induction of single-strand breaks in mammalian cells by sodium azide and its proximal mutagen.

The mutagenicity of sodium azide in both higher plants and bacteria is well documented. However, in mammalian cells, research on the effects of azide on gene mutations has produced conflicting results. Furthermore, no research has been conducted on the effects of azide and its proximal mutagen (mutagenic metabolite) on DNA single-strand breaks. Experiments were designed to overcome this lack of information on azide mutagenicity and to evaluate the potential hazard of azide exposure to man. Chinese hamster V79 cells were treated with either azide or its proximal mutagen(s) for 2 or 6 h, respectively, and analyzed by alkaline elution for single-strand breaks. The data showed that neither azide nor the proximal mutagen(s) induced single-strand DNA breaks or DNA-protein cross-links. Therefore it appears that neither azide nor its proximal mutagen(s) interact directly with DNA and this suggests that azide may be an indirect-acting mutagen. Furthermore, this lack of interaction with DNA may account for azide's lack of carcinogenicity.

Mapping of the Hor-3 locus encoding D hordein in Barley.

The hordein storage proteins of barley (Hordeum vulgare L.) are of intense interest due to their genetic diversity and prominence and impact on the industrial and agricultural uses of the seed. Two major hordein loci have been previously mapped on chromosome 5 (Hor-1 and Hor-2 encoding the C and B hordeins, respectively). A third major locus, Hor-3, which codes for D hordein, has been located in the centromeric region of chromosome 5, probably on the long arm. Two allelic variants with apparent molecular weights of 83,000 and 91,000 and similar isoelectric points of 8.0 comprise the products of this locus in the barley varieties 'Advance' and 'Triple Awned Lemma'. The D hordein examined is similar in molecular weight and isoelectric point to the high molecular weight (HMW) glutenin proteins encoded by the 1B chromosome of wheat (Triticum aestivum L.).

In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts.

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction not produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by L-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55 degrees C and had a pH optimum between 7-7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.

Isolation of an azide mutagenic metabolite in Salmonella typhimurium.

A scheme that employs a cation-exchange column and high-pressure liquid chromatography (HPLC) is devised to isolate and process large quantities of azide metabolite produced by S. typhimurium TA1530 strain. The mutagenic metabolite adheres strongly to the cation-exchange column, thus providing a convenient way to separate the metabolite from unreacted azide (N3-). The metabolite is very polar and only sparingly soluble in most organic solvents. Recrystallization in a methanol-carbon tetrachloride solvent system gave rise to microcrystalline material that decomposes with charring and gas evolution at 173-176 degrees C. The infrared spectrum indicates the presence of a covalently bound azide moiety.

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells.

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted. Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10(-3) to 10(-7) M. Cells were harvested and chromosomes stained by the FPG technique. In human lymphocytes, concentrations above 10(-4) induced lethality whereas the K1 cell line was sensitive to concentrations above 10(-5) M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels. This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.

Effects of L-cysteine and O-acetyl-L-serine in the synthesis and mutagenicity of azide metabolite.

The ability of L-cysteine to inhibit azide-metabolite synthesis and mutagenicity is investigated in Salmonella typhimurium TA1530 and cys E6 strains. L-cysteine specifically inhibits the synthesis of the mutagenic azide metabolite as other compounds containing SH group did not affect the production of this metabolite. Azide mutagenicity is completely inhibited by L-cysteine at a concentration (5 mumoles/plate) where the metabolite mutagenicity was not affected. O-Acetyl-L-serine can reverse the L-cysteine mediated inhibition of the metabolite synthesis and thus mutagenicity in the same strains. These results suggest that O-acetyl-L-serine may be required to synthesize the azide metabolite or its precursor.

Pollen genetic markers for detection of mutagens in the environment.

To utilize and exploit pollen for in situ mutagen monitoring, screening and toxicology, the range of genetic traits in pollen must be identified and analyzed. Traits that can be considered include ornamentation, shape and form, male sterility viability, intraspecific incompatibility, proteins and starch deposition. To be useful for the development of mutagen detection systems proteins should be: (1) activity stainable or immunologically identifiable in the pollen, (2) the products of one to three loci, and (3) gametophytic and nuclear in origin. Several proteins including alcohol dehydrogenase in maize, which meet those criteria will be discussed. The waxy locus in barley and maize which controls starch deposition has been characterized genetically and methods have been developed for pollen screening and mutant detection. At Washington State University a waxy pollen system is being developed in barley for in situ mutagen monitoring. The basis is an improved method for staining and scoring waxy pollen mutants. Specific base substitution, frameshift, and deletion mutant lines are being developed to provide information about the nature of the mutations induced by environmental mutagens. Thirty waxy mutant lines, induced by sodium azide and gamma-rays have been selected and are being characterized for spontaneous and induced reversion frequencies, allelism, karyotype, amylose content, and UDP glucose glucosyltransferase (waxy gene product) activity. Twelve mutant alleles are being mapped by recombinant frequencies.

Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations.

Clustering of mutant pollen grains in a population of normal pollen due to premeiotic mutational events complicates translating mutation frequencies into rates. Embryo ontogeny in barley will be described and used to illustrate the formation of such mutant clusters. The nature of the statistics for mutation frequency will be described from a study of the reversion frequencies of various waxy mutants in barley. Computer analysis by a "jackknife" method of the reversion frequencies of a waxy mutant treated with the mutagen sodium azide showed a significantly higher reversion frequency than untreated material. Problems of the computer analysis suggest a better experimental design for pollen mutation experiments. Preliminary work on computer modeling for pollen development and mutation will be described.

In vivo conversion of sodium azide to a stable mutagenic metabolite in Salmonella typhimurium.

Salmonella typhimurium TA1530 and G46 strains growing in minimal medium supplemented with sodium azide produce a stable mutagenic metabolite which is not azide. The production of this metabolite is restricted to the log phase of bacteria grown in the presence of azide. The metabolite is highly mutagenic in DNA-repair defective base-substitution strains TA1530 and TA1535, but ineffective in frameshift strains TA1538 and TA1537. The metabolite induces mutations in resting cells of the TA1530 strain.

Potential of plant genetic systems for monitoring and screening mutagens.

Plants have too long been ignored as useful screening and monitoring systems of environmental mutagens. However, there are about a dozen reliable, some even unique, plant genetic systems that can increase the scope and effectiveness of chemical and physical mutagen screening and monitoring procedures. Some of these should be included in the Tier II tests. Moreover, plants are the only systems now in use as monitors of genetic effects caused by polluted atmosphere and water and by pesticides. There are several major advantages of the plant test systems which relate to their reproductive nature, easy culture and growth habits that should be considered in mutagen screening and monitoring. In addition to these advantages, the major plant test systems exhibit numerous genetic and chromosome changes for determining the effects of mutagens. Some of these have not yet been detected in other nonmammalian and mammalian test systems, but probably occur in the human organism. Plants have played major roles in various aspects of mutagenesis research, primarily in mutagen screening (detection and verification of mutagenic activity), mutagen monitoring, and determining mutagen effects and mechanisms of mutagen action. They have played lesser roles in quantification of mutagenic activity and understanding the nature of induced mutations.Mutagen monitoring with plants, especially in situ on land or in water, will help determine potential genetic hazards of air and water pollutants and protect the genetic purity of crop plants and the purity of the food supply. The Tradescantia stamen-hair system is used in a mobile laboratory for determining the genetic effects of industrial and automobile pollution in a number of sites in the U.S.A. The fern is employed for monitoring genetic effects of water pollution in the Eastern states. The maize pollen system and certain weeds have monitored genetic effects of pesticides. Several other systems that have considerable value and should be developed and more widely used in mutagen monitoring and screening, especially for in situ monitoring, are discussed. Emphasis is placed on pollen systems in which changes in pollen structure, chemistry, and chromosomes can be scored for monitoring; and screening systems which can record low levels of genetic effects as well as provide information on the nature of induced mutations. THE VALUE OF PLANT SYSTEMS FOR MONITORING AND SCREENING MUTAGENS CAN BE IMPROVED BY: greater knowledge of plant cell processes at the molecular and ultrastructural levels; relating these processes to mutagen effects and plant cell responses; improving current systems for increased sensitivity, ease of detecting genetic and chromosome changes, recording of data (including automation), and for extending the range of genetic and chromosome end points; and designing and developing new systems with the aid of previous and current botanical and genetic knowledge.

Mutagenic and chromosome-breaking effects of azide in barley and human leukocytes.

Azide (10-3 M, solution buffered at pH 3) is more effective in inducing mutations in embryonic shoots of seeds germinated between 8 and 16 h than in non-germinated seeds and in seeds germinated between 0 and 8 h and 16 to 28 h. This peak of chlorophyll-deficient seedling mutation frequency coincides with maximum frequencies of seeding lethals and DNA replication in the cells of the embryonic shoot. The mutation data suggest azide may only act on replicating DNA. Azide induced no chromosome-aberration frequencies significantly above controls in (1) embryonic shoots of barley seeds germinated for 8--12 h, (2) microspores of barley and (3) human leukocytes. It appears to be a point-mutation mutagen.

Artificial mutagenesis as an aid in overcoming genetic vulnerability of crop plants.

Artificially induced genetic variation is being used effectively to supplement or complement sources of natural origin for practical plant breeding. Thus, creating genetic variation uill become increasingly important as crop genetic resources become more difficult to obtain via plant exploration. The aritificial induction of useful genetic variation offers important elements that can be used for overcoming genetic vulnerability: (1) new, previously unknown alleles can be induced in crop plant species to broaden the base of variation; (2) useful genetic variation can be induced in modern cultivars helping to shorten breeding time or to extend production "life"; (3) characteristics of existing genetic resource stocks can be improved to make them more useful in breeding; and (4) recombination in crosses may be enhanced. The performance of induced mutant crop cultivars and the successful uses of induced genetic variation in cross breeding indicate that artificial mutagenesis will play an increasingly greater role in plant breeding.