Mutagenesis

47 °

MUTATION I~ESEARCH

t t Y D R O X Y L A M I N E AS A MUTAGENIC AGENT FOR N E U R O S P O R A CRA SSA

H. V. M A L L I N G Biolog3' Division, Oak Ridge National Laboralor),, Oak l?idge, Tenn. ( U . S . d . ) (Received J u n e 2ist, I000)

SUMMARY

The mutagenicity of hydroxylamine (HA) has been tested in 8 different adeninerequiring mutants (ad-3B) of Neuro@ora crassa: 4 mutants ofthis 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 ICR17o 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 theothers. 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 alterations 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 characterization of HA-induced mutations in Neurospora is particularly important because it may be as specific in eukaryotes as in phagein 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, m e t h o x y 6-chloro0-(3-ethyl-2chlorethyl]-aminopropylamino)acridine dihydrochloride. NA, nitrous acid. SP, spontaneous reversion frequency in the u n t r e a t e d 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 alterations isa much simpler method to characterize the genetic effects of mutagens than any known forward-mutation technique in Neurospora 6,19. The characterization is limited b y the array of genetic alterations in each mutant comprising a tester set. However, b y selecting mutants reverting b y most common genetic alterations, it is possible to obtain a first approximation of the spectrum of geneticalterations produced b y 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 HAproduces 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...