neuro spora

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MUTATION INDUCTION BY DIFUNCTIONAL ALKYLATINGAGENTS IN NEUROSPORA CRASSA

TONG-MAN ONG AND F. J. DE SERRESEnvironmental Mutagenesis Branch, National Institute of Environmental Health Sciences,Research Triangle Park, North Carolina 27709

Manuscript received December 4, 1974Revised copy received March 17, 1975

ABSTRACTThe genetic characterization of ad-3 mutants of Neurospora crassa induced

by two carcinogenic difunctional alkylating agents, 1,2,4,5-diepoxypentane(DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations atthe ad-3B locus have similar complementation patterns. I n addition ta theinduction of point mutations, DEP induces a low frequency (7.5%) of multi-locus deletions, whereas DE0 induces an extremely high frequency (42.0%).The distribution of the different classes of ad-3 mutants and the frequency ofmultilocus deletion mutants among DEP-induced mutants are not significantlydifferent from those induced by the monofunctional alkylating agents EI, EMSand ICR-I77 at comparable forward-mutation frequencies. Moreover, the fre-quencies of DEP-induced ad-3B mutants showing allelic completion or havingnonpolarized complementation patterns are similar to those of ad-3B mutantsinduced by monofunctional agents. It is suggested, therefore, that the mech-anism of mutation-induction by DEP in N . crassa is similar t o that of mono-functional alkylating agents. Mutation-induction by DEO probably resultsboth from the mechanism of action of monofunctional alkylating agents andfrom inter-strand cross-linkage of the DNA molecule by the two functionalepoxy groups.

INCE the discovery of the mutagenic activity of nitrogen mustards (AUERBACHSand ROBSON1947), the mutagenicity of many alkylating agents has beeninvestigated. The mechanisms of mutation induction and other biological activ-ities of alkylating agents have been reviewed extensively (FREESE963; ORGEL1965; LAWLEY966; LOVELESS966). Based upon the number of active groups,alkylating agents are classified as mofio-, di- or polyfunctional compounds. Itseems likely that the principal difference among these groups with respect tointeraction with DNA i s that di- or polyfunctional agents per se could cause intra-or inter-strand cross-linkage of DNA (BROOKESnd LAWLEY961, 1963). Inter-strand cross-linkage of DNA could lead to an inhibition of DNA synthesis and adeletion of chromosomal segments (BROOKESnd LAWLEY964). Hence, mono-functional and di- or polyf unctional compounds might cause different spectraui genetic alterations. Several studies have shown that polyfunctional agentsproduce more chromosome breaks than monofunctional agents (FAHMYndFAHMY960; OBE 1968; NAWAR, ONZAKnd NILAN1971). Other studies, how-Genetics 80 : 475-182 July, 1975.


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DIFUNCTIONAL ALKYLATING MUAG ENS 477plementation, dikaryon and trikaryon tests. Spontaneous ad-3 mutants among DEP- and DEO-induced mutants are negligible ( < 0 . 5 % ) .Complementation test: The genotype and the complementation pattern of each mutant wasdetermined by growing each mutant with each complementation tester; the methods are sim-ilar to those described by BROCKMAN nd DE SERRES1963) . The following nine testers wereused: (1 ) a d 3 B , complon i(2-17-258) ; (2 ) a d 9 B , complon 2(2-17-1%3) (3) ad-SB, complons10-11 (2-31-8); (4 ) d 3 B , complon 15 (2-32-3); (5) &-3B, complon 16-17 (2-325); (6) a d - 3 4(1-68-13); (7) noncomplementing ad-3B, (1-112-2); (8) hist-2, nic-2, aZ-2 (74-0933-3A) ; (9)ad-2, i m s (74-OR6@44A). Testers 1 through 5 determine the complementation pa t tem of a d J Bmutants showing allelic complementation. Based on the complementation patterns, ad-3B mutantsare classified as noncomplementing, nonpolarized complementing and polarized complementing.Testers 6 through 8 determine the genotype. Tester 9 is used as a contml to test the ability ofeach mutant to form a heterokaryon. Mutants are classified as leaky if they grm by themselvesin the complementation test.Dikaryon test: Dikaryon tests were carried out by plating conidia from each mutant in Fries'minimal medium (HOROWITZnd BEADLE1943) supplemented with 100 mg adenine sulfate/land 10 mg calcium pantothenatefl. After incubation at 35" fm 2 days, the plates were exam-ined fo r the presence of co t colonies. The ad-3 mutations were induced in the heterokaryoncomponent that carries the genetic markers cot, al-2 and pan-2. The presence of co t coloniesindicates that the induced ad-3 mutants are viable in the medium supplemented with adenine.This type of mutant is classified as having a reparable point mutation ( ~ z d - 3 ~ )BROCRMAN,DE SERRESnd BARNETT969). Mutants that failed to give cot colonies in dikaryon tests weretested in the trikaryon test.Trikaryon test: Mutants that do not produce cot colonies in the dikaryon test have either apoint mutation within the ad-3A or d - 3 B locus and an independent recessive lethal mutationat another locus elsewhere in the genome ( a d 4 R+R L ) o r have a multilocus deletion coveringone or both ad-3 loci and one or more adjacent essential (irreparable) genes (ad-3IR).These twotypes of mutants can be distinguished in the trikaryon test by the method of DE SERRES1964,1968) . Trikaryons were formed by growing each mutant with each of three tester strains. Thetrikaryon tester strains are heterokaryons which carry the same genetic markers as that of theheterokaryon used fo r the forward-mutation induction experiments, except in component 11:tester 12-7-215 ( a d - 3 A m ) having a deletion covering the ad-SA locus, tester 12-5-182 (ad-3BIR)having a deletion covering the ad-3B locus and tester 12-1-8 (ad-SA ad-3B nic-2IR) having a de-letion covering the a d - 3 4 ad-3B and nic-2 loci. Mutants were classified as ad-3" if no dikaryoticcot colonies were found from the trikaryons formed with al l of the testers o r if cot colonieswere only found from the trikaryon formed with tester ad-3AIR or ad-3BIR. Mutants wereclassified as a d - 3 R f R L i f cot colonies were found from the trikaryons formed with all threetesters. If cot colonies were produced by the trikaryon formed with ad-3A'R and ad-3BrR, but notwith tester d - 3 Aa d 3B nic-ZIR, the mutant was a reparable ad-3 mutant with a recessive lethalmutation at another locus closely linked to the ad-3 region ( ~ d - 3 ~RLOI).

RESULTSThe results of genetic characterization of DEP- and DEO-induced ad-3 mutantscan be summarized as follows:Complem entation test: Among the DEP-induced ad-3 mutants tested, 35.3%

are &-SA and 60.2% are ad-3B mutants. The remaining 4.5% are ad-3 A ad-3Bdouble mutants (Table 2). Among the ad-3B point mutations, 52.1% show alleliccomplementation with 66.1% of the complementing mutants having nonpolar-ized complementation patterns (Table 3 ) .

Analysis of DEO-induced ad-3 mutants indicates 27.2% ad-3A, 50.0% ad-3Band 22.8% ad-3 A, ad-3B double mutants (Table 2). The ad-3B point mutations


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478 T.-M. O N G A N D F. J. DESERRESTABLE 2

Frequencies of genotypes of ad-3 mutants nduced by DEP andD E 0 m determined b y complementation testsad-SA ad-3B ad-JA ad-3BTotal mutants mutants double mutantsCompound ad-3 mutants (% f (% I (% I

DEP 201 35.3 (71)* 60.2 (121) 4.5 (9)DE0 162 27.2 (44) 50.0 (81) 22.8 (37)

* The number of mutants is given in parentheses.TABLE 3

Frequencies of complementation patterns of DEP- andDEO-induced ad9B point mutationsNonpolarized

Total ad-3B amongCompound mutations ( % I (% ) (% ) addB's (% )

Complementing complementingad-3B point Nonpolarized Polarized Noncomplementing complementing

DEP 119 34.5 (4.1)' 17.6 (el) 47.9 (57) 66.1 (41/62)DE0 59 37.3 (22) 18.6 (11) 44.1 (26) 66.7 (22/33)

* The number of mutants is given in parenthese's.include 55.9% which show allelic complementation, among which 66.7% havenonpolarized complementation patterns (Table 3)Dikaryon test: In the dikaryon test, 84% of the DEP-induced mutants and51% of those induced by DE0 produce cot colonies. These mutants carry pointmutations at the a d - 3 A or ad-SB locus ( a d - j R ) Tables 4 and 5 ) . Mutants whichdid not give co t colonies in dikaryon tests were tested further in the trikaryontest.Trikaryoln test: Thirteen ( 6 . 5 % ) DEP-induced mutants and seven (4.3%)DEO-induced mutants are classified as nd-SR+ RL, because they give co t coloniesfrom the trikaryons formed with all three testers. Four ( 2% ) DEP- and four(2.5% ) DEO-induced mutants, which gave cot colonies only from trikaryonsiormed with ad-3AIRand ad-3BTResters, are ad-SR4- L C 1 .

TABLE 4Results of dikaryon and trikary on tests

TotalPoint mutations multilocusTotal mutants deletionsCompound tested Tota l &-?E ad-3R + RL ad-3n -I- Lel ( a d - 3 I E )DEP 201 92.5*(186)+ 84.0 (169) 6.5 (13) 2.0 (4) 7.5 (15)DE0 162 58.0 (94) 51.2 (83) 4.3 (7) 2.5 (4) 42.0 (68)

* The frequency (% ) is based on the total mutants tested.+ The number of mutants is given in parentheses.


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DIFUNCTIONAL ALKYLATING MUAG ENSTABLE 5

Distribution of point mutations and multilocus deletions amongDEP and D E 0 induced ad-3 mutants

4 7 9

Point mutations Multilocus deletionsTotal a d 3 -~ompound mutants Total ad-3A a d J B a d - 3 A I a d - 3 B Total ad JA ad-JB ad-3A adJBDEFJ 201 186 67 119 0.56 15 4 3* 8DEQ 162 94 35 59 0.59 68 9 28* 31* One of the DEP- and 6 of the DEO-induced ad-3B multilocus-deletion mutants also canypoint mutations within the ad-SA locus and therefore a r e classified as ad JA ad-3B double mutantsin Table 2.The remaining 7.5% of the DEP- and 42%of the DEO-induced mutants do not

give cot colonies in the trikaryon test o r give cot colonies from the trikaryonformed with the ad-3ArRor ad-3BrR esters. These mutants are multilocus deletionmutants (ad-31R)(Tables4 and 5j .

DISCUSSION

Our previous studies have shown that both carcinogenic diepoxides, DEP andDEO, are mutagenic in N . crassa (ONGand DE SERRES9 7 2 ) . The results of thecomplementation tests presented in this paper show that the ratios of ad-SA toad-3B mutants among DEP- and DEO-induced ad-3 point mutations are 0 . 5 6and 0.59, respectively (Table 5 , which are not significantly different. Only4.5% of the ad-3 mutants induced by DEP, however, are ad-3A ad-3B doublemutants, whereas 22.8% of those induced by DE0 are a d 4 A ad-3B doublemutants. These results from the complementation tests are in agreement withthe results of dikaryon and trikaryon tests which show that o n l y 7.5% of DEP-induced ad-3 muiants are multilocus deletion mutants, whereas 42.0%of DEO-induced ad-3 mutants are multilocus deletion mutants. Genetic characterizationof ad-3 mutants induced by the two compounds also shows that the frequenciesof complementing ad-3B and nonpolarized complementing ad-3 B mutantsamong DEP- and DEO-inducedad-3B point mutations are similar (Table 3) andthat the frequencies of the different classes of point mutations (ad-3R,ad-3R-I-RL and a d - S R + RLcl)induced by both agents are not significantly different fromone another (Table 4). These data suggest that, except for inducing an unusuallyhigh frequency of multilocus deletions, DE0 induced a spectrum of ad-3 mutantssimilar to that induced by DEP.

In terms of the frequency of leakiness, allelic complementation, nonpolarizedcomplementation patterns and multilocus deletions, observed at comparableforward mutation frequencies, the spectrum of DEP-induced ad-3 mutants i ssimilar to those of mutants induced by monofunctional alkylating agents (EI,EMS and ICR-177) (Table 6). These results seem to suggest that the mechanismsof mutation induction by DEP in N . crassa are similar to those of monofunctionalalkylating agents. Studies by K0 L M A R K and KILBEY 1 9 6 8 ) on the induction ofadenine reversions in N . crassa have shown that EO and DEB are equally efficient


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480 T.-M. O N G A N D F. J.DE SERRESTABLE 6

Comparison between the spectra of genetic alterations in ad-3 mutants inducedby monofunctional and dif unct ionul alkylating agents

LeakymutantCompound (% )

E1* 13EMS* 11

10DEP 10DE0 7

ICR-17 7*

NonpolarizedComplementing complementing hlult ilocusa d J B ' s among a d 3 B ' s among deletionad-3B's complementingad-3B's mutants(%I (%I (%I41 67 35 3 78 151 70 1152 66 755 67 42

* D a t a from DE SERRFSt al. (1971) , MALLINGpersonal communication), ONG (1970), an dON Gan d DE SERRE~1972).in inducing reversions when the frequencies are based on survival. From theirstudies they assumed that EO and DEB induce mutations and cause lethality bysimilar mechanisms. In Drosophila melamguster, NAKAOnd AUERBACH1961)showed that the ratio of sex-linked recessive lethal mutants to translocationsinduced by EO is similar to that induced by DEB. Their results led them tosuggest that EO and DEB induce mutations and chromosome breaks via similarmechanisms.

The frequency of multilocus deletions among DEO-induced ad-3 mutants issignificantly different from that of DEP-induced mutants. It is interesting to notethat the frequency of multilocus deletions induced by DE0 is higher than thatinduced by any other physical and chemical agent tested in this system. Thesurvival of conidia after treatment with DE0 is lower than that of those treatedwith comparable concentrations of DEP. A plausible interpretation of thesedifferences is that DE0 may cause inter-strand cross-linkage of the DNAmolecule that leads to the deletion of small chromosome segments and to higherlethality. It seems, therefore, that the mutation induction by DE0 in N . crassacan be attributed in part to mechanisms similar to those of monofunctional alky-lating agents, but also to the formation of cross-linkages within the DNAmolecule.It i s generally known that the N7 of guanine is the most reactive site fo r alky-lation. Therefore, interstrand cross-linkage resulting from the reaction of DNAwith difunctional alkylating agents could be due to alkylations at the N7 of aguanine by a functional group and at the N7 of a guanine in the opposite strandby another functional group. Based on the Watson-Crick model of DNA structure,the distance from the N7 position of a guanine to the N7 position of the nearestguanine in the opposite strand is about 7.2 A". According to Drieding Stereo-models, the distances from the first carbon to the last carbon of DE0 can beextended to 8.9 A", whereas DEP can only be extended to 5.2 A". With thisdistance, it is unlikely that DEP can form cross-linkage of guanine residues inopposite strands of the DNA molecule. Such cross-linkage, however, could be


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D I F U N C TI O N A L A LK Y LA TIN G M U A G EN S 481formed by DEO. The data presented here are in agreement with these moleculardistances and suggest that the length of the carbon chain between the two func-tional groups of di- or polyfunctional alkylating agents is important in theformation of cross-linkage which in the case of DE0 lead to the production of ahigh frequency of multilocus deletions.

W e wish to thank DR. 5. D. MCKINNEY,Environmental Biology and Chemistry Branch,NIE HS , for his help in m easuring the molecular distances of D E P and DEO.LITERATURE CITED

AUE RBACH,. and J. M. ROBSON,94 7BROCKMAN,. E . and F. J. DE SERRFS, 19 63

BROCKMAN,. E., F. J. DE SERRESnd W. E. BARNETT,96 9BROOKES,. and P. D . LAWLEY,96 1

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Analysis of ad-3 mutants inducedby nitrous acid in a heterokaryon of Neurospora crassa Mutation Res. 7 : 07-314.Th e reaction of mono- and di-functional alkylating agentswith nucleic acid. Biochem. J. 80: 9 6 5 0 3 , __ 1963 Effects of alkylating agents onT, and T, bacteriophage. Biochem. J. 89: 138-144 - 1964 Alkylating agents.Brit. Med. Bull. 20: 91-95.

Genetic analysis of the s tructure of the ad-3 region of Neurospora crassaby means of irreparable recessive lethal mutations. Genetics 50: 21-30. -, 1968Genetic analysis of the extent and type of functional inactivation in irreparable recessivelethal mutations i n the ad-3 region of Neurospora crassa. Genetics 58: 69-77.Homology tests on presumed multilocus deletionsin the ad-3 region of Neurospora crassa induced by the acridine mustard ICR-170. Genetics58: 70-83.

Mutagen spe-cificity in Neurospora crassa. M utation Res. 12: 129-142.Cytogenetic analysis of the action of carcinogens andtumor inhibitors in Drosophila melanogaster. X . The na ture of the m utations induced by themesyloxy esters in relation to molecular cross-linkage. Genetics 96: 47-458.

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HOROWITZ,. H. and G. W. BEADLE,94 3 A microbiological m ethod fo r the determination ofcholine by use of a mu tant of Neurospora. J. Biol. Chem . 150: 325-333.

GLMARK ,. G. and B. J. KILBEY,1968 Kinetic studies of mu tation induction by epoxides inNeurospora crassa. Molec. Gen. Genet. 101: 89-98.LAWLEY, . D., 1966 Effects of some chemical m utag ens and carcinogens on nucleic acids, pp.89-131. In: Progress in Nucleic Acid Research and MoLecular Biology, Vol. 5. Edited byJ. H. DAVIDSON nd E. COHN.Academic Press, New York.LOVELESS, ., 1966 Genetic and Allied Effects of Alkylat ing Agents. Pennsylvania State Uni-vers ity Press, 270 pp.NAKAO,Y. and 6. UFXBACH,1961 Tes t of a possible correlation between cross-linking andchromosame break ing abilities of chemical mutagens. 2. V e r b . 92: 6 7 4 6 1 .NAWAR,M. M., C. F, KONZAK nd R. A. NILA N, 1971 Com parative studies of the biologicaleffectiveness of nitrogen mustards, ethyleneiraine and x-rays. Mutation Res. 11: 3 3 9 3 4 6 .

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482 T.-M. O N G A N D F. J . DE SERRESOBE,G., 1968 Chemische Konstitution and Mutagene Kirkung v. Verglechende Untersuckung

der Wirkung von Athyleniminen aut Menschiche Leukozytehchromosomen. Mutation Res.6: 467471.Characterization of ICR-177 induced ad-3 mutants of Neurospora crassa. Muta-

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