The frequency of reverse mutation at the XDH loci of Aspergillus nidulans

Mutation Research

I75

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

T H E FREQUENCY OF R E V E R S E MUTATION AT T H E X D H LOCI OF

ASPERGILLUS N I D U L A N S M. J E N N I F E R H A R T L E Y *

Department of Genetics, University of Cambridge (Great Britain) ,Received May i l t h , 197 o)

SUMMARY

The chemically induced reversion frequencies of 77 mutants of Aspergillus nidulans were examined. The mutants which were at several loci concerned with nitrogen metabolism were induced by nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine and diepoxybutane and reversion was attempted by these same mutagens and diethylsulphate. The ability of the mutants to revert was affected by the inducing mutagen, the bifunctional diepoxybutane giving a much higher percentage of nonrevertible mutants than the monofunctional agents, nitrous acid and nitrosoguanidine. 4o% of the non-revertible mutants were associated with translocations and the bifunctional agent substantially increased the frequency of translocations over that given by the monofunctional agents. The frequency of reversion, among the individual revertible mutants, varied by a factor of more than IOO but even so inducing mutagen and type of mutant had a detectable influence on revertibility. Those mutants induced by diepoxybutane, that did revert, were reverted most frequently by diepoxybutane itself though this agent was less effective at reverting mutants induced by nitrous acid and nitrosoguanidine. It is suggested that diepoxybutane can induce revertible mutants by a process other than a transitional type change, possibly by a transversional type change.

INTRODUCTION

The study of reverse mutation patterns has frequently been used to elucidate the chemical changes induced by mutagens4,°,l~, 17. If reversion arises from true backmutation individual mutants might be expected to show specificity of reversion by different mutagens and likewise specificity of reversion might indicate the occurrence of true back-mutation. A mutagen is only capable of back-mutating mutants it has induced if it can cause two complementary reactions. For instance, frame-shift mutants arising from the *Present address: D e p a r t m e n t of Agricultural Botany, University of Sydney (Australia). Abbreviations: DEB, diepoxybutane; DES, diethylsulphate; NA, nitrou~ acid; NTG, N-methylN'-nitro-N-nitrosoguanidine; O.P.B. 7, orthophosphate buffer pH 7.0; XDH, xanthine dehydrogenase.

Mutation Res., io (197 o) 175-183

170

x~. ,] ENNIFEI,~ H A b V I L I " Y

a d d i t i o n or deletion of a base pair can only t)e r e t u r n e d t~) the original s t a t e b \ the r e m o v a l or a d d i t i o n of a base while b a s e - p a i r s u b s t i t u t i o n m u t a n t s require the c()m-~ p l e m e n t a r y b a s e - p a i r s u b s t i t u t i o n to give a b a c k n m t a n t . Therefore, if a m u t a g e n induces a b a s e - p a i r s u b s t i t u t i ( m it n m s t be able to react w i t h A a n d / o r T and (', a n d / o r C to give b a c k m u t a n t s from n m t a n t s it has induced. The ability, (~r otherwise, of m u t a g e n s to r e v e r t m u t a n t s t h e y h a v e p r o d u c e d m a y , therefore, give an indication of t h e t y p e a n d specificity of the changes t h e y can induce. This w o r k was carried out to see if the chemical m u t a g e n s t e s t e d react specifically with certain D N A bases or w h e t h e r their action is of a more general nature. A n u m b e r of m u t a n t s i n d u c e d b y NA, N T G a n d D E B were t e s t e d for reversion b y these s a m e m u t a g e n s a n d b y DES. The n m t a n t s selected were all concerned with nitrogen m e t a b o l i s m a n d were of t h r e e types. The cnx m u t a n t s ~8 which are u n a b l e to utilize h y p o x a n t h i n e or n i t r a t e as sole nitrogen source a n d are found at 5 loci. The hx m u t a n t s ~ which are unable to utilize h y p o x a n t h i n e as sole nitrogen source a n d are found at 2 loci a n d the u a Y m u t a n t s 6 which are unable to utilize h y p o x a n t h i n e or uric acid as sole nitrogen source a n d are found at I locus. As all these m u t a n t s lack X D H , the e n z y m e responsible for the b r e a k d o w n of h y p o x a n t h i n e , t h e y can all be referred to as X D H m u t a n t s . MATERIALS AND METHODS

All m u t a n t s to be t e s t e d for reversion were outcrossed a n d strains with a d d i t i o n a l m a r k e r s , n o t c u r r e n t l y in use in the l a b o r a t o r y , were selected for reversion t r e a t m e n t s . Conidia were grown on complete m e d i u m (3% agar), in medical flat bottles, a n d i n c u b a t e d 5-7 d a y s a t 37 °. T h e conidia were emersed in o.o2 M o r t h o p h o s p h a t e buffer p H 7.0 (O.P.B. 7) plus o.o1% Tween 8o a n d collected b y g e n t l y scraping t h e agar. T h e suspensions were v o r t e x e d t h o r o u g h l y to b r e a k eonidial chains a n d w a s h e d three times to r e m o v e a n y n u t r i e n t s t h a t m i g h t h a v e washed out of the agar. A f t e r the final washing the eonidia were r e s u s p e n d e d in the buffer a p p r o p r i a t e for the m u t a g e n t r e a t m e n t a n d a s a m p l e was r e m o v e d for control tests. N A treatment. The conidia were r e s u s p e n d e d in 9 ml 0,05 M c i t r a t e - p h o s p h a t e buffer at p H 4.6 a n d I m l o.o5 M s o d i u m n i t r i t e solution was added. N T G treatment. T h e conidia were r e s u s p e n d e d in 5 ml O.P.B. 7 a n d 5 ml N T G solution (o.oo5 M) was added. DEI3 treatment. T h e eonidia were r e s u s p e n d e d in IO ml O.P.B. 7 a n d O.Ol 5 ml D E B was a d d e d d i r e c t l y to give o.o2 M D E B , D E S treatment. The conidia were r e s u s p e n d e d in IO ml O.P.B.7 plus o.o1% Tween 80 to p r e v e n t the conidia from " c l u m p i n g " a n d o,o52 ml D E S was a d d e d d i r e c t l y w i t h vigorous mixing. F i n a l c o n c e n t r a t i o n was 0.04 M DES. All t r e a t m e n t s were carried o u t for 30 rain a t 37 °, which gave 80-90 % kill, a n d t e r m i n a t e d b y centrifuging for I o min a n d r e s u s p e n d i n g in 5 ml O.P.B. 7. Samples from t r e a t m e n t s a n d controls were t a k e n tor v i a b i l i t y e s t i m a t i o n s a n d t h e r e m a i n i n g suspensions were a d d e d to m e l t e d m i n i m a l m e d i u m , held at 45 ° a n d p o u r e d directly. This m e d i u m was supplied with h y p o x a n t h i n e as sole nitrogen source a n d s u p p l e m e n t e d w i t h a n y o t h e r r e q u i r e m e n t s of the s t r a i n u n d e r test. Viabilities were scored after 2-3 d a y s a n d m u t a t i o n plates e x a m i n e d after 5-6 d a y s when a n y colonies were c o u n t e d a n d all, or a sample, t e s t e d for genotype. M u t a t i o n R e s . , l o (197 o) I 7 5 - I S 3

REVERSE MUTATION

AT X D H

LOCI OF

A. nidulans

I77

At least lO s viable conidia were examined after each treatment except where the reversion rate was high enough for a lower number to give a reliable result. A high plating density of about lO 7 viable conidia per plate was used but loss of revertants by the GRIGGI1 effect was considered unlikely as in reconstruction experiments no inhibition of wild-type colonies was observed among mutant conidia plated at IOs viable conidia per plate. RESULTS

77 mutants induced by NA (22), NTG (31) and D E B (24) were tested for reversion by all 4 mutagens. Of these 36 were reverted by all 4 mutagens, i i were reverted by some mutagens and 30 gave no revertants from at least io 8 viable conidia (Table Ia, b and c). The proportion of mutants that could revert was significantly lower when the mutants were induced by the bifunctional agent D E B than when they were induced by the monofunctional agents NA and NTG (Table II). If it is assumed that revertible mutants arise as the result of small genetic changes, e.g. single base changes, and that non-revertible mutants arise as the result of larger chromosomal aberrations, then this result is compatible with a cross-linking action of DEB with subsequent chromosomal damage. To investigate this further, all mutants tested for reversion were also tested for the presence of translocations by the haploidisation technique of FORBES7. This showed D E B induced significantly more translocations (40%) than NTG (Io%) and NA (0%). All translocated strains were found to be nog-revertible and of all the non-revertible strains 40% were translocated I3. The spontaneous reversion frequency of the mutants was very low. An average of 3.9" lO8 viable conidia per mutant was screened for spontaneous revertants but TABLE

Ia

THE REVERSION FREQUENCIES OF REVERTIBLE 1N-A-INDUCED X D H Frequencies

MUTANTS

a r e e x p r e s s e d as t h e n u m b e r o f r e v e r t a n t s p e r i o s s u r v i v o r s .

;lI~tant No.

Reverting mulagen None NA

NTG

DEB

AC AC AC AC

3 io 14 15

1.5 0.6 o o

I 3 II 6

o 5° 3 114

9 12 37 5o7

37 39 14 4°

AC AH AU AU

17 8 ioo IOi

o o.2 o. 5 o

385 5o 16o I

55 ° 15o 3 6

87 42 20 16

26o 2o2 20 9

AU AU AU AU

lO2 lO 3 lO 5 lO6

o o o o

61 i 43 ° 278

4 6 86 705

2 39 22 io

12 9 60 200

AU AU AH AH

lO8 lO 9 15 ( u a Y ) 21 ( u a Y )

o o

97 26 58 6

ioo 54 400 2o

49 3 o I

205 14 123 48

6 mutants

o o

DES

did not revert

Mutation Res., IO (197 o) 1 7 5 - 1 8 3

178

.xj. j FNN1FF_I¢ H A R T L E V

TABLE

Ib

T H E R E V E R S I O N F R E Q U E N C I E S OF T H E R E V E R T I B L E N T ( ; - I N D U C E D

XDFt

MUTANTS

F r e q u e n c i e s e x p r e s s e d as t h e n u m b e r of r e v e r t a n t s p e r ~oS s u r v i v o r s .

Mutant N o .

Reverting mutagen None

NA

NT6

OEB

DES

o 2 o lO

2 4° 767 321

2[ 113 314 172

27 18 r26 475

2t 180 77 232

7 9 3 4

o 26 9

2 14

15 13

NC NC NC NC

6 18 21 22

NC NC NC NC

23 27 38 43

o o.4 I o

NC NC NC NH

45 5° 52 1

o 3 o o

NH NH NU NU

22 33 17 27

NU NU NU NU NU NU NH NH NH

I

II

28

o

i7

i 19 27 I

4° ioo

o ~35 5

25 52 42

2

i

o o o o

8 I 6 74

37 6 62 129

2 15 5 17

l4

334 36 42 53

o o o o

548 7 200 72

47 S ~o 54 120

56 28 17 590

99 13 t8

56 57 28 ( u a Y ) 35 ( u a Y ) 13 ( u a Y )

0. 5 o o o o

2 4 7° I o

7 17 240 9

2 o o o

4 5 31 5

2

O

I

6 mutants

TABLE

26 4

"

6

29 77

39

did not r e v e r t

Ic

T H E R E V E R S I O N F R E Q U E N C I E S OF T H E R E V E R T I B L E D E B - I N D U C E D

X D I - I MUTANTS

F r e q u e n c i e s e x p r e s s e d as t h e n u m b e r of r e v e r t a n t s p e r io* s u r v i v o r s .

Mutant N o .

R e v e r t i n g mutagen ATone NA

DC 4 DH io DU I

°.3 o o

DU 4 DU 8 DH6 (uaY)

i o o

NTG

DEB

DES

20 o

54 2

95 25

98 2

IOO

85

280

57

4° 86 o

46 265 3°

456 765 54

lO3 137 31

I8 mutants did not r e v e r t

only 12 mutants gave spontaneous revertants. With one only, NC 22 (Table Ib), was the spontaneous reversion rate at all appreciable, and this might represent a clone. The frequency of reversion among the 47 "revertible" mutants varied by more than Ioo-fold both between individual mutants and between different reverting mutagens acting on the same mutant. The frequencies of reversion were examined Mutation R e s . , i o (197 o) 1 7 5 - 1 8 3

REVERSE MUTATIONAT X D H LOCI OF A . n i d u l a n s

I79

TABLE II THE NUMBER

OF R E V E R T I B L E

AND NON-REVERTIBLE

XDH

MUTANTS INDUCED

BY

NA, N T G

AND

DEB

Inducing mutage~,

Number of mutants examined

Number of mutants reverting

N*~mber of mutants not reverting

Revertible mutants ( % )

NA NTG DEB

22 31 24

16 25 6

6 6 i8

73 81 25

Total

77

47

3°

179

H c t c r o g c n e i t y Z28 = 16.676 ( P < o . o o I ) .

on the basis of type of mutant, inducing mutagen and reverting mutagen (Table III). The overall frequency of reversion of the cnx, h x and u a Y mutants was 88, 29 and 91 revertants per lO 8 viable conidia respectively while the overall frequency of reversion of mutants induced b y NA, NTG and D E B was 94, 68 and 118 revertants per lO 8 viable conidia respectively. An analysis of variance (Table IV) of a l o g + i transformation of the frequency data showed that both the type of m u t a n t and the inducing mutagen had a significant effect on the frequency of reversion at the 5% level of significance. The effect of the reverting mutagen was not significant, though in view of the different effectiveness of these agents as forward mutagens this would have been expected if there were no differential response from the mutants. (The frequency of forward mutations induced at the X D H loci by NA, NTG and DEB was 4.4, 14.8 and 12.1 mutants per lO 4 viable conidia respectively.) There is, however, an interesting pattern in the inducingreverting relationships. Table I I I shows that DEB-induced mutants were less affected by NA and NTG while NA- and NTG-induced mutants are poorly reverted by DEB. DEB-induced mutants, however, were reverted readily by DEB and this difference is significant at the I % level of significance. DISCUSSION

The interaction between inducing and reverting mutagens possibly reflects the chemical changes brought about b y these mutagens so the possible nature of these changes must be considered. The mutagenic action of NA is believed to be caused by the deamination of those nucleic acid bases that carry amino-groups 1~. Deamination of A or C would cause AT -+ GC or GC --~ AT base-pair changes but there is evidence t h a t the former is the more frequentlS, 17. Deamination of G is believed to be lethal 2°. NTG is probably mutagenic, at least at neutral pH, via the alkaline decomposition product diazomethane 5 which is a strong methylating agent. There is, however, disagreement as to its reactivity with the nucleic acid bases. FRIEDMAN et al. 1° report methylation of guanine and thymine but no reaction with adenine and cytosine. KRIEK AND EMMELOTZ4, on the other hand, report the methylation of guanine and adenine by diazomethane while FRANKEL-CONRAT8 suggests that, though NTG affects mainly guanine, this reaction is lethal and it is the reaction with cytosine that is mutagenic. This would indicate that NTG would induce a GC ~ AT base-pair change preferentially. Mutation Res., i o (197 o) 175-183

I80

M. ,] I'-NNIFI';I,~ HAR'II.I:",

'fABLIL 11 l THE FREQUENCY OF REVERSION IN I)UCI£D BY NA, N'I'(;, l)b,'l~ : x D liES ov ~1UTANT,'q A'I"Till.. ~Hi, hX AND zga}" LOCI INDUCED BY N \ , NT(; ANI) I)I£B The figures s h o w n a r e t h e m e a n s of tile frequencies exhibited bv the individual "rex ertible" ii1111i/i1~ty; in each group. The total n u n i b e r of viable conidia examined X io s is shown in parentheses. Inducing, mut~gen

T y p e oj mutant

Nz~mbcr o f N u m b e r o f T h e m e a n fveque*~cy o f rel;clsio~ givcJ~ by *:ac/! mutants mutants reverting mutag, cn treated rcverh'd [-J/l NA N T(; l)I,2]~ I)/s'N

NA

cnx

8

5

hx

i

1

io8 (I2.9) II1

(17.4) uaY

NTG

13

202

02 (IO.73

(lO.l)

98 (25,5)

14I (17.9)

80 (37.8) 8 (25-3) 72

109 (lO.31 3 ((3.5) 89

77 (8.4)

73 (8.4)

(4.7) lO3

(o.6) 65

(7.5) 20

(65.o)

(i5.(,)

(i4.1)

(io.3)

(~9.o)

68

88

8I

61

4I

(32.4)

(27.2)

(31.3)

(37.2)

5

3

uaY

14

II

Total

31

25

I I

uaY

II

4

Total

24

6

77

(4 .~) 7° (I4.I) 8I (21.4)

III

1I

Total

42

(I6.6)

12

4

15 °

85

cnx

h\

5°

(6.2)

(52.2) 94 (82.5)

16

9

7s (3 .2 )

lO

22

cnx

13o (3.9) {2-4) 2o (I t.4) 56 (t7,7)

(128.1) DEB

145 (3-0 (4,7) I 38

Total

hx

8~ (2-71

47

I5

()

7

67

20

54

95

98

(S.o)

(3.1)

(0.7)

(2.1)

(2.~)

7

o

2

25

2

(12.5)

(4.1)

(3.s)

(i.35

(3.35

158 (14.8)

57 (4-3)

1°7 (3-t)

389 (2-5)

82 (4 .6)

II8

41

80

279

7I

(35.3)

(t 1.5)

(7.9)

(5.9)

(1o.o)

83 ('245.9)

85 (09.4)

i(it (53.0)

87 (54.9)

5s ((,8.b)

T A B L E IV ANALYSIS OF VARIANCE OF T H E F R E Q U E N C Y OF R E V E R S I O N I N D U C E D B Y NA, NTG, OF M U T A N T S AT T H E c n z , h x AND u a Y LOCI I N D U C E D B Y ~ A , N T G AND D E S

Source

d. f .

T y p e of m u t a n t Inducing mutagen Reverting mutagen T y p e of m u t a n t × T y p e of m u t a n t × Inducing mutagen T y p e of m u t a n t ×

inducing m u t a g e n reverting m u t a g e n x reverting m u t a g e n inducing i n u t a g e n ;< reverting m u t a g e n

,'145

F

2 2 3

1.921 1.690 1.416

3.5 °* 3.°8 * 2.59

4 6 6 12

1.557 0.229 0.872 o.177

2.84** 0.42 1.59 0.32

Residual

152

o.5,t 7

Total

187

0.586

* Significant at the 5% level of significance. ** Significant at the i % level of significance. IVlutation R e s , , IO (197o) 175 183

D E B AND D E S

REVERSE MUTATION AT

X D H LOCI OF A. nidulans

18I

D E B might react with any of the nucleic acid bases b y amine or enol esterification with the epoxy ring(s) while DES reacts principally by ethylation of guanine 15 giving a GC -+ AT base-pair change ~. Among the revertible mutants NA-induced mutants were reverted readily, especially b y NTG (Table III) which is compatible with their arising from an A T - + G C base-pair change. These mutants could be reverted by NA if NA also reacts mutagenically with cytosine. This m a y be a less frequent reaction so that the majority of NAinduced mutants arise b y an AT -+ GC base-pair change and revert readily b y the other mutagens while a minority arise by a GC -+ AT base-pair change and revert, therefore, more readily b y NA. This, however, is only partially so; of the 4 NAinduced mutants that were reverted highly by NA only 2 did as preferentially b y NA. NTG-induced mutants were the least revertible but they were reverted most by NA. (The reversion of NTG-induced mutants was not statistically greater by NA than b y the other mutagens but in real terms this m a y well be so if the mutating effectiveness of NA is measured b y its forward mutation rate.) This would be compatible with NTG producing a GC -+ AT base-pair change which is supported by the low reversion rate given b y DES. However, approximately half the NTG-induced mutants were reverted preferentially by NTG itself, therefore, it is possible that this compound can induce either transitional type change fairly readily. If this were so an appreciable proportion of the NTG-induced mutants would be less revertible b y NA than b y the other mutagens tested. This was, in fact, so for though NA was overall the best agent for reverting NTG-induced mutants with IO of the 25 mutants tested, it was the least effective agent. If NTG induces a considerable proportion of AT --~ GC base-pair changes these should be as revertible as the NA-induced mutants and some of the NTG-induced mutants were highly revertible. The reversion pattern shown by D E B and DEB-induced mutants was distinctly different from that shown b y the other mutagens examined. This phenomenon cannot be explained on a transitional type base-pair change, so an alternative mechanism must be sought. There is the possibility that DE]3 causes small (single base) deletions but these could only be reverted b y a base insertion or possibly a double base deletion. There is no obvious mechanism by which a base pair could be inserted b y DEB and steric hindrance makes reaction with adjacent bases unlikely 1. A more feasible postulation to fit the evidence is that D E B induces preferentially a transversional type base-pair change. If DEB removed a DNA base this might be replaced, b y a repair mechanism, but assuming random replacement this would give a 2 : I ratio of transversional : transitional type base-pair changes and the reversion data does not indicate a transitional type change. On the other hand, as BENZER3 has pointed out, adenine and guanine should be capable of forming satisfactory hydrogen bands between them but they would need greater than normal chain separation to do so. Sufficient separation might arise as a result of the esterifieation of a D E B molecule onto a nucleic acid base. The addition of this relatively large molecule might force the DNA chains apart and allow bonding to occur, during replication, between one of the purine bases, adjacent to the esterified complex, and the alternative purine rather than the complementary pyrimidine thus inducing a transversional type change. The advantage of this proposed "wedge" mechanism is that reversal could be Mutation Res., IO (197 o) i 7 5 - i 8 3

182

.xl. J ENNIlrE]~ HARII.I~.Y

induced by the same process. This could account for the (~ revertible DEB-induced mutants being reverted preferentially by DEB and, to some extent, for the fact that DEB was the least effective nmtagen on 25 of the other mutants, but it d.es not explain the IO occasions when D E B gave the highest reversion rate for NA- and NTG-induced mutants. It is obvious that reversion, in this system, does not occur by a unique process. Of the I I "revertible" mutants that did not revert by all mutagens, only 2 showed a high frequency of reversion by any mutagen. These 2, induced by NA and NTG, were both highly revertible by NTG and not at all by DEB. With the other 9, reversion was low throughout, so the fact they have not reverted by one mutagen Inav mean that reversion was difficult and that insufficient numbers were screened. The majority of revertible mutants were reverted by all 4 mutagens. It is possible, therefore, that there was an appreciable level of second-site suppression. Secondsite suppression is rare in Neurospora 1" and it does occur, rarely, in Aspergillus ~2. It is possible that there was a back-ground level of second-site suppression which might be less mutagen-specific than back-mutation and the mutant sites might vary in their susceptibility to suppression either by the number of suppressor sites or the -~-ulnerability of the site(s), which would affect the level of back-ground reversion. This could account for the appearance of revertants where back-mutation would not be expected, on chemical considerations, and would tend to level out differences in reversion rate. However, the fact that these differences were detectable, indicates that many of the revertants may have been true back mutants. Though there is evidence in the literature of the mutagens having a specificity for particular DNA bases, the evidence presented here is also indicative of the mutagens examined being able to react with at least one member of each DNA base pair, though probably at a differential rate. There is no obvious explanation of the differential revertibility of mutants of the different types as the revertibility of individual mutants should be determined by the chemical change involved and not by the location of the mutant. However, the effectiveness of suppressor mutations might vary between genes as this might be dependent on the nature of the enzyme produced. If this is so then second-site suppression must also be appreciable. These results are interesting in that they show the great variability that can occur between phenotypically similar mutants which have been induced by the same mutagen. This variability should be considered when assessing the effectiveness of mutagens by the use of reversion systems. Differences shown by such systems may reflect individual mutant differences and not the general mutagenic capacity of the mutagen. ACKNOWLEDGEMENTS The author would like to thank the Agricultural Research Council for financial support and Prof. J. M. THODAY for laboratory facilities.

Mulation Res., io (197o) 175-183

REVERSE MUTATION AT X D H LOCI OF A . n i d u l a n s

183

REFERENCES i ALEXANDER, P., S. F. COUSENS AND K. A. STACEY, The reaction of the mutagenic alkylating agents with proteins and nucleic acids, Ciba Found. Syrup., Drug Resistance Micro-organisms, 1958, pp. 294-318. 2 ]3AUTZ, E., AND E. FREESE, On the mutagenic effect of alkylating agents, Proc. Natl. Acad. Sci. (U.S.), 46 (196o) 1585-1594. 3 ]3ENZER, S., On the topography of the genetic fine structure, Proc. Natl. Acad. Sci. (U.S.) 47 (1961) 4o3-415 . 4 CALVORI, C., AND G. MORPURGO, Analysis of induced mutations in Aspergillus nidulans, I. UV- and HNOa-induced mutations, Mutation Res., 3 (1966) 145-151. 5 CERDA-OLMEDO, E., AND P. C. HANAWALT, Diazomethane as the effective agent in nitrosoguanidine mutagenesis and lethality, Z~Iol. Sen. Genet., t o i (I968) 191-2o2. 6 DARLINGTON, A. J., c. SCAZZOCCHIOAND J. A. PATEMAN, ]3iochemical and genetical studies of purine breakdown in Aspergillus, Nature, 2o6 (1965) 599-600. 7 FORBES, E., Use of mitotic segregation for assigning genes to linkage groups in Aspergillus nidulans, Heredity, 13 (1959) 67-80. 8 FRANKEL-CONRAT, H., Structural and mutagenic studies with viral RNA, in HSIEN-WEN LI (Ed.), Recent Developments in Biochemistry. 9 FREESE, E., The difference between spontaneous and base-analogue induced mutations of phage T 4, Proc. Natl. Acad. Sci. (U.S.), 45 (1959) 622-633. io FRIEDMAN, O. M., G. N. MANAPATRA, B. DASH AND R. STEVENSON, Studies on the action of diazomethane on deoxyribonucleic acid, Biochim. Biophys. Acta, IO3 (1965) 286-297. II GRIGG, G. W., Back mutation assay method in microorganisms, Nature, I69 (1952) 98-ioo. i2 HARTLEY, x'V[.J., Reversion of non-nitrate utilizing (nia D) mutants of Aspergillus nidulans, Mutation Res., 7 (1969) 163-17 o. 13 HARTLEY, M. J., AND ]3. M. REVER, in preparation. 14 KRIEK, E., AND P, EMMELOT, Methylation of deoxyribonucleic acids by diazomethane, Biochim. Biophys. Acta, 91 (1964) 59-66. 15 LAWLEY, P. D., The hydrolysis of methylated deoxyguanylic acid at pH 7 to yield 7-methylguanine, Proc. Chem. Soc., (1957) 29o-291. i6 MALLING, H. V., AND F. J. DE SERRES, Correlation between base-pair transition and complementation pattern in nitrous acid-induced ad-3B mutants of Neurospora crassa, Mutation Res., 5 (I968) 359-371. 17 MALLING, H. V., AND F. J. DE SERRES, Identification of genetic alteration induced by ethyl inethanesulfonate in Neurospora crassa, Mutation Res., 6 (1968) I7I-I93. 18 PATEMAN, J. A., D. J. CovE, ]3. M. REVER AND D. B. ROBERTS, A common co-factor for nitrate reductase and xanthine dehydrogenase which also regulates the synthesis of nitrate reductase, Nature, 2Ol (1964) 58-60. 19 SCHUSTER, H., The reaction of nitrous acid with deoxyribonucleic acid, Biochem. Biophys. Res. Communs., 2 (196o) 320-323. 20 V1ELMETTER, W., AND H. SCHUSTER, The base specificity of mutations induced by nitrous acid in phage T2, Biochem. Biophys. Res. Communs., 2 (196o) 324-328.

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