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  47 ° MUTATION I~ESEARCH ttYDROXYLAMINE AS A MUTAGENIC AGENT FOR NEUROSPORA CRA SSA H. V. MALLING Biolog 3 Division, Oak Ridge National Laboralor),, Oak l?idge, Tenn. (U.S.d.) Received June 2ist, I000 SUMMARY The mutagenicity of hydroxylamine (HA) has been tested in 8 different adenine- requiring mutants ad-3B) of Neuro@ora crassa: 4 mutants of this tester set revert after treatment with nitrous acid and ethyl methanesulfonate indicating that they revert by a base-pair substitution, 2 mutants revert only after treatment with ICR- 17o indicating that they revert by a base-pair insertion or deletion, and 2 mutants revert only spontaneously. HA induced reversions in 3 of the 4 mutants which revert by base-pair substitution and in none of the others. The specificity of the action of HA on the mutants in the present tester set is consistent with the hypothesis that the predominant class of genetic alterations induced by HA in Neuro@ora is base-pair transitions from guanine-cytosine to adenine thymine. Furthernlore, it was found that the reversion frequency after HA treatment increases in proportion to the square of the treatment time. INTROI) UCTION HA induces base-pair substitutions in phage and the type of the genetic altera- tions is preferentially 9 from GC to AT. The chemical reaction between HA and DNA has been analyzed by FREESE et al. 8 who found that HA reacts only with cytosine and HMC. Treatment of RNA with HA alters both uracil and cytosine; at pH 6.1 the reaction is much faster with cytosine than with uracil, whereas at higher pH the relationship is reversedlL HA has been shown to induce chromosonml damage in mammalian cells 1 , but no previous attempt has been successful in identifying the genetic alteration(s) induced by HA in eukaryotes at the molecular level. The charac- terization of HA-induced mutations in Neurospora is particularly important because it may be as specific in eukaryotes as in phage in producing mutations by base-pair substitutions resulting from GC to AT transitions. Such specificity would make HA especially well-suited for the characterization of the genetic alterations induced by Abbrevi~tions: AT, adenine thymine. I,;MS, ethvl mcthanesulfonatc. GC, guanine cytosine. HA, hydroxylamine. HMC, hy~troxymethylcytos{ne. ICR-f7o, methoxy 6-chloro 0- 3-ethyl-2- chlorethyl]-aminopropylamino)acridine dihydrochloride. NA, nitrous acid. SP, spontaneous reversion frequency in the untreated series. ~Iulalion lees., 3 I966) 47 ° 476  CHEMICAL MUTAGENICITY IN N. crassa 471 less specific mutagens at the molecular level by means of tests for specific revertibility. Tests for the induction of reversions in mutants resulting from known genetic altera- tions is a much simpler method to characterize the genetic effects of mutagens than any known forward-mutation technique in Neurospora 6,19. The characterization is limited by the array of genetic alterations in each mutant comprising a tester set. However, by selecting mutants reverting by most common genetic alterations, it is possible to obtain a first approximation of the spectrum of genetic alterations produced by a given mutagen. In this paper a tester set of ad-3B mutants consisting of 4 mutants which revert only by base-pair substitution, 2 mutants which revert only by base-pair insertion or deletion, and 2 mutants which revert only spontaneously were each treated with HA and then screened to detect reversions to wild type. The results of such tests show clearly that in Neurospora HA produces reversion only in those strains which revert by base-pair substitution. The specificity of the action of HA on the mutants in the present tester set is consistent with the hypothesis that the predominant class of genetic alteration induced by HA in Neurospora is base-pair transition from GC to AT. MATERIALS AND METHODS a) Strains The mutants with the prefix 2-17 were all induced by nitrous acid in Neurospora wild-type strain 74 A DE SERRES, BROCKMAN, BARNETT AND KOLMARK in prepara- tion). Mutant No. 5-4-1 is of spontaneous srcin (BROCKMAN, unpublished). The mu- tants have been isolated by the direct method 3. b) Preparation of the culture In all experiments the mutagenic treatment was carried out on suspensions of conidia harvested from i25-ml Erlenmeyer flasks containing 20 ml of glycerol com- plete medium 10 (IO m glycerol per liter instead of 20 ml)+adenine sulfate (25 rag/l). The flasks were inoculated and then incubated for I day at 3 °o and then for 6- 9 days at 25 °. The conidia were harvested by first shaking the cultures with glass beads (4 mm diameter) to break up the chains of conidia; they were then suspended in saline (o.9~o), filtered through a platinum strainer, washed twice by centrifugation, and then resuspended in saline. Ad-3 mutants differ from each other with respect to development of the purple pigment in the mycelium and conidia when grown on glycerol complete, but by adding 250 mg adenine sulfate per liter purple pigment accumulation is essentially eliminated. The density of the conidial suspensions were measured on a colorimeter (Spectronic 20, Bausch and Lomb, Rochester, New York) at the peak absorptions of 750 m#. c) Treatments All treatments were carried out with conidial suspensions (~ 2 ã IoT/ml) in Erlenmeyer flasks on a rotary shaker in waterbath at 25 ° to keep the conidia in sus- pension during the treatment. 5 min before quenching the conidia were centrifuged and the supernatant was decanted. At the time of quenching after treatment with either NA, EMS, or ICR-I7O, the conidia were resuspended in a salt solution of Fries' minimal 1 adjusted to pH 8 with NaOH. This procedure was repeated twice. Mutation Res. 3 1966) 470-476  47 tt. V. MAIA.ING The salt solution of FRIES minimal adjusted to pH has been found to stop the reac- tion between cells and alkylating compounds and NA immediately (MALLI~ C., un- published). d) NA trealmenls The conidia were suspended in 0.05 M sodium acetate buffer at pH 4-5. One volume of freshly prepared o.o2 M sodium nitrate solution was added to 3 volumes of conidia. The final concentration was 0.oo 5 M NaNO,,, and the treatment was quenched as described above 4o min after the start of the treatment. e) EMS treaOnent The conidia were suspended in a 0.067 M phosphate buffer at pH 7.0. The treatment was started by adding enough EMS to bring the final concentration up to o.I M; the treatment was quenched 30o rain later. f) ICR-z7o treatment ICR-I7o is the code number assigned to methoxy4)-chloro-9-(3-[ethyl-2- chlorethylil-aminopropylamino)acridine dihydrochloride by It. ,J. CREECH and co- workers at the Institute for Cancer Research, Philadelphia. The conidia were sus- pended in a o.o67 M phosphate buffer at pH 7.o. The treatment was started by adding I vol. of a freshly prepared solution of ICR-I7O (25 ° rag/1 H20) to 49 vols. of the conidia suspension which gave a final concentration of lO.58/~M. The treatment was quenched as described above 13o rain after start of the treatment. The treatment and other manipulations involving ICR-I7O and conidia were performed under red light to eliminate the photodynamic effects associated with the acridine ringVa, ~a. Plates were also incubated in the dark for at least 24 h to allow sufficient time for the conidia to give rise to small colonies. g) HA treatmenl Before the HA treatment, the conidia were suspended in 3 M NaCl and then further diluted 5 times in the HA reaction mixture of STRACK, FREESE AND FREESE 17 giving a final HA concentration of i M. 5 min before the treatment was quenched, the conidia were centrifuged and decanted, and at the quenching time i.e., 3oo rain after the start of the treatment) the conidia were resuspended in 3 M NaCl. This washing procedure was repeated twice and then the conidia were suspended in the salt solution of Fries minimal medium t adjusted to pH 8. h) Plating medium To estimate the viability of the treated and untreated conidia, they were plated in WESTERC.AARO S minimal T supplemented with sorbose (15 g/l), glucose (0. 5 g/l), fructose (0.5 g/l), casamino acid (200 rag/l), a vitamin solution as in glycerol-complete (I ml/1), and adenine sulfate (25 rag/l). To estimate the number of revertants the conidia were plated in the same sub- strate used for scoring survivors but supplemented with 0.2 rag/1 adenine sulfate in- stead of 25 rag/1 adenine sulfate. In the plates to determine survival the density of the conidia was 5 IO conidia per ml substrate in a total volume of about IOO ml. For scoring of revertants after 3/Iutation Res., 3 (I966) 47 ° 476  CHEMICAL MUTAGENICITY IN N. crassa 473 NA, EMS, or ICR-I7O treatment, the conidia were plated to a density of IO e conidia per ml and 2 ã lO 5 conidia per ml, each ill a total volume of IOO ml. For scoring of the revertants after the HA treatment, the density of the conidia was 2 ã lO 5 per ml in a total volume of 500 ml. i) Statistical test The test for significance is done according to BIRNBAUM (see ref. 2, p. 261). The number of revertants is considered as having a Poisson distribution. The probability is calculated by assuming that the following two ratios belong to the same popu- lation : Total population surviving after the treatment) (i) Total population untreated)+total population surviving after treatment) Total number of revertants in the treated population Total number of revertants in the untreated population+total number of 2) revertants in the treated population A probability lower than 5 indicates a significant difference between the number of reversions in the control and the treated series. RESULTS NDDISCUSSION  a) Suppressors Identification of the genetic alteration in individual mutants by determining the specificity in the induction of reversions after treatment with different agents will be distorted by the occurrence of suppressor mutations along with reverse muta- tions. Revertants from 20 different mutants induced in the ad-3 loci (refs. 5, II, and BARNETT, unpublished) have been analyzed for occurrence of extragenic suppressors, and none was found. We may therefore assume that suppressors occurring outside the ad-3A or ad-3B locus are rare or that they do not occur. The influence of the suppressors on the identification of the genetic alteration in individual ad-3 mutants will be discussed further in another paper MALLING AND DE SERRES n preparation, 1966 ). In addition a detailed analysis of induced reversions of ad-3B mutants is being made to provide further information on this point. How- ever, since the revertants of the mutants in the present tester set appear early and are in the main part not accumulating the purple pigments usually done by ad-3 mutants, it seems likely that the frequency of extragenic suppressors in the present analysis is low. b) Genetic alteration induced by HA The mutagenicity of HA has been studied by determining whether there is any specificity in its action activity for inducing reversions with a tester set of 8 mutants (Table I). On the basis of present data, 4 of these strains appear to revert only by base-pair substitutions (revertible by NA and EMS), 2 strains appear to revert by a base-pair insertion and/or deletion (only revertible by ICR-I7O), and 2 strains revert only spontaneously. ICR-ITO is a monofunctional acridine mustard gas; forward mutations induced by ICR-I7O in Neurospora crassa seem mainly to be base-pair insertions or deletions 4. It is therefore likely to assume that mutants which revert after treatment with ICR- Mutation Res. 3 I966) 470-476
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