- Email: [email protected]
Allelic complementation among nitrous acid-induced ad-3B mutants of Neurospora crassa
Mutation Research
415
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
ALLELIC COMPLEMENTATION AMONG NITROUS ACID-INDUCED ad-3B MUTANTS OF NEUROSPORA CRASSA F. J. BE SERRES, H. E. BROCKMAN,* W. E. BARNETT AND H. G. KDLMARK**
Biology Division, Oah Ridge National Laboratory, Oah Ridge, Tenn. (U.S.A.) (Received December I9th, 1966)
SUMMARY
Complementation tests on a random sample of NA-induced ad-3B mutants of Neurospora crassa have shown that 72.1% show allelic complementation and have either nonpolarized or polarized complementation patterns, the majority (6o.4%) being in the nonpolarized group. The ratio of nonpolarized to polarized complementation patterns is thus 5 to I. In addition, a total of 24.3% are leaky, have nonpolarized patterns, and account for 4o.3~o of the mutants in this class. The facts that only 3o of the 65 mutants with nonpolarized patterns and only 5 of the 13 mutants with polarized patterns are different have been interpreted as indicating a nonrandom distribution of altered sites in the ad-3B locus. The discovery of many nonleaky NA-induced mutants with nonpolarized patterns has led to a revision of an earlier complementation map of the ad-3B cistron published previously 8. A tester set of predominantly nonleaky ad-3B mutants with nonpolarized complementation patterns which define a linear complementation map of 17 complons has been selected for future work. The complementation data from the present analysis in combination with the reversion data of MALLING1B indicate a direct correlation between the percentage of mutations resulting from base-pair substitution and the percentage showing allelic complementation.
INTRODUCTION
The mutagenic activity of nitrous acid '(NA) on viral systems has been studied extensivelyn,13,18 and it has been shown to result from deamination of certain nucleic acid bases and to produce mutations predominantly by base-pair substitutions (BPS) of the type AT -~ GC and GC -+ AT. TESSMAN19 has shown that NA can also produce * Present address: Department of Biological Sciences, Illinois State University, Normal, Ill., U.S.A ** Present address: Institute of Physiological Botany, The University of Uppsala, Uppsala, Sweden. Abbreviations: BPS, base-pair substitutions; NA, nitrous acid.
Mutation Res., 4 (1967) 415-424
416
F.J. DE SERRES et al.
mutations that map as extensive deletions and that these account for about 8% of the NA-induced rII mutants of T 4 bacteriophage. Studies on the mutagenic effect of NA on Neurospora suggest that the above general spectrum of genetic alterations is also found in eukaryotes (as prokaryotes). MALLING1~has shown that at least 84% of the NA-induced ad-3 mutants of Neurospora crassa are BPS and that at least 6%, out of the remaining 16% with unidentified genetic alterations, may be due to intralocal deletions. The RNA code-word assignments designated by NIRENBERG and by KHORANA and their coworkersl, 2° show that most base-pair transitions give a new triplet which either codes for the same amino acid or another amino acid and that there are only 2 codons (UAA and UAG) which code for no amino acid. From these data it would appear that most base-pair transitions would code for (I) the same amino acid or a new amino acid which would not affect the function of the protein (sense mutation) or (2) a new amino acid which would affect the function of the protein (missense mutation) 22. However, only a minority of the possible base-pair transitions would completely inactivate the protein as a result of premature chain termination because they code for no amino acid (nonsense mutation). We do not know what ratio of sense to missense to nonsense mutations to expect after NA-treatment. However, it should be possible to estimate the ratio of missense to nonsense mutations by an analysis of the gene products specified by NA-induced mutants. Missense and nonsense mutations should have different effects o11 the type of gene product specified in terms of (I) the ability to show allelic complementation and the type of complementation pattern and (2) the level of residual enzyme activity 7. This paper is the first in our series of attempts to characterize the genetic effects of NA in Neurospora crassa at the molecular level. NA-induced ad-3B mutants have been analyzed for leakiness and allelic complementation by heterokaryon tests. These tests indicate that 72% of NA-induced ad-3B mutants show allelic complementation, that the ratio of nonpolarized to polarized complementation patterns is about 5 to I, and that 24% of the ad-3B mutants are leaky. METHODS
Strains. New ad-3 mutants were induced in wild-type strain OR 74 A (Oak Ridge vegetative isolate of ST74A ) (ref. 3). The leaky and nonleaky ad-3B mutants used as testers in heterokaryon tests for allelic complementation were all induced in wild-type strain 74 A or closely related derivatives s. The origin of the ad-3A mutants used as controls in the heterokaryon tests was described previously 4& Isolation of mutants. Cultures of wild type were grown on Fries' minimal medium (ref. 12) for 7 days at 25 °. Conidial suspensions were made in ice-cold water, filtered 2 times through wetted absorbent cotton pads (o.5-1.o cm thick) in a Buchner funnel, washed 2 times and adjusted to I. lO 7conidiaper ml in o.i M acetate buffer pH 4.6. To start the treatment 4 mg NaNO2 were added to 11.6 ml samples of conidial suspension, giving a final concentration of o.oo5 M N a N Q . Treatment was at 25 ° in a constant temperature water bath. Conidial suspensions in acetate buffer alone were used as controls. Treatment was terminated by taking samples out of the treatment vessels and pipetting them into Ioo ml cold phosphate buffer at pH 7 to stop the reaction. Mutation Res., 4 (1967) 415-424
ad-3B MUTANTS
COMPLEMENTATION O F N A - I N D U C E D
417
Control and treated conidia were analyzed for the presence of purple adenine- 3
(ad-3) mutants by the direct method 9 modified by the addition of 12.5 mg adenine sulfate/l, 250 mg L-arginine. HCI/I, 1.5°/0 L-sorbose, o.15% agar and o.I~o sucrose to basal medium~L 12-1 flasks containing IO l of this medium were autoclaved for 50 min (18 pounds, 252o F), the time required for maximum viability under these conditions TM. Inocula were planned to give a maximum of 1.2. lO 6 colonies per IO l of medium. Ad-3 mutant colonies were isolated following 7 days of incubation with aeration in the dark at 3 o°. A homokaryotic culture of each mutant was obtained by plating conidia in Fries' basal medium which contained l.-o °//o sorbose, o.1°/0 sucrose, and 2 mg adenine sulfate/1. In this medium adenine-requiring homokaryotic colonies are morphologically distinguishable from wild-type and heterokaryotic colonies. Heterokaryon tests for allelic complementation. The genotypes of the NA-induced ad-3 mutants were determined with an ad-3A mutant and a noncomplementing ad-3B mutant. The ad-3B mutants were then screened for allelic complementation with a series of 30 complementing ad-3B mutants (two nonleaky and 28 leaky) as testers. The complementation pattern of each tester is shown in Fig. I and these mutants ed-J3 ClSTRON MUTANT NUMBERS
11121314151et'~1010110
3-10-153
m
3-10-114 3 - 1 0 - 500 3-10-112
I [
2-11-5 1-230-83 2-11-131,2-11-163, 2 - t l q O 2-16-22 2-15-72 3-10-109 3-10 -132 3 -10-16 3-10-70 2-11-6 1-234-108 2-11-223 2-11-140 3-10-384 3-I0-446 3-10-506 3-10-67 1- 2 3 4 - 1 4 8 3-10-470 1 -155-11, 1-1t2-8 3-27-16, 1-230-69,2-16-18
~--
11l ~211311411511611711BI191201
I I
I I
I
I
I I I I I I
1 I'
'
I
[
I I----'1 I I I I
] I
1
k______2 f"
I
I [
Fig. i. C o m p l e m e n t a t i o n p a t t e r n s of ad-3B m u t a n t s used as testers in h e t e r o k a r y o n tests for allelic c o m p l e m e n t a t i o n of NA-induced ad-3B m u t a n t s . I , Nonleaky m u t a n t ; C3, leaky m u t a n t .
were selected from a larger sample described previously 8. To retard growth by leaky mutants alone, complementation tests were made in liquid minimal medium in IO mm × 75 mm test tubesS, s. All tests were incubated at 24-26 ° and responses were scored periodically for a period of 21 days. Tests for leaky NA-induced ad-3 mutants. Tests to determine the percentage of M~lation Res., 4 (1967) 415-424
F.J. DE SERRES et al.
418
leaky ad-3B mutants were made as indicated above but with a total of 2. 5 • lO 6conidia per tube. RESULTS AND DISCUSSION
Selection of ad-3 mutants for genetic analysis The ad-3 mutants selected for analysis were obtained from treatment of conidia with 0.005 M NAN02. The treatment times, levels of survival, and forward-mutation frequencies are given in Table I. The frequency of spontaneous mutants in this experiTABLE I ad- 3 MUTANTS NaNO~, p H 4.6, 25 °) All m u t a n t n u m b e r s begin w i t h prefix 2 - i 7. T H E ORIGIN OF N A - I N D U C E D
I N D U C E D IN W I L D - T Y P E STRAIN O R
Range of mutant numbers
Treatment time (rain)
Per cent survival
Mutations per ~o ~ survivors
2 - 1 o ; 788-854 11-43 44-125; 855-1o27 126-17o
iio iio 16o 16o
41.6 34.3 23.2 14.6
29.4 37.4 127. 5 128.1
74 A (0.0o5
M
ment was not significantly higher than the expected frequency 2 of spontaneous forward mutation of o.38" io 6. Thus, a IOO- to 4oo-fold enrichment was obtained with treatment, and the incidence of mutants of spontaneous origin in the sample of 199 NA-induced mutants selected for genetic analysis is negligible.
Screening tests for allelic complementation among NA-induced ad-3 mutants The complementation tests on 199 NA-induced ad-3 mutants selected at random for genetic analysis showed (I) that 52/199 mutants were ad-3A mutants and (2) that 147/199 were ad-3B mutants. No other genotypes were found among the mutants recovered as purple-pigmented colonies. When the ad-3B mutants were tested for allelie complementation with the tester set illustrated in Fig. I, lO6/147 mutants, or 72.I%, showed allelic complementation. Even more interesting to us were tlae facts (I) that the complon 6 coverage of the majority of the complementing mutants appeared to be quite limited, and (2) that the majority of the complementing mutants were nonleaky.
The development of a nonleaky tester set to study allelic complementation among NAinduced ad-3B mutants The discovery of a series of highly complementing mutants induced in the same isolate of wild-type strain OR 74 A (all of them most probably resulting from BPS) led us to t r y to replace the leaky mutant tester set shown in Fig. I with a series of nonleaky or only slightly leaky NA-induced mutants for further experimentation. This decision was based on 2 very different but equally important considerations: (i) The fact that m a n y of the 28 leaky mutants in the tester set grow extensively on minimal medium introduces uncertainty on the scoring of these mutants in combination. When scoring complementation tests involving l e a k y + l e a k y ' combinations, the amount of growth shown in each pairwise combination must be compared with that Mutation Res., 4 (1967) 415-424
COMPLEMENTATION OF NA-INI)UCED ad-3B MUTANTS
419
shown by each m u t a n t individually to determine whether the combination gives a positive or negative complementation response. Since the growth shown by leaky mutants alone appears at different points in time, the total time over which the complementation tests can be scored varies considerably for different pairwise combinations. Thus, the scoring of complementation tests made with the leaky mutant tester set is technically difficult and time consuming and thus not as well suited for rapid screening tests as a nonleaky tester set. (2) D a t a from a completely different experiment on (a) the leaky ad-3A and ad-3B m u t a n t samples from which the present tester set was derived s and (b) the NA-induced ad-3A and ad-3B mutants showed quite clearly (a) that m a n y of the mutants in the selected sample of leaky mutants used as testers carry modifiers that affect the leakiness of these mutants on minimal medium (DE SERRES, unpublished observations) and (b) that these modifiers are not present in any of the 147 NAinduced ad-3B mutants from the 2-17 experiment (DE SERRES, unpublished observations).
Allelic complementation among NA-induced ad-3B mutants Heterokaryon tests for allelic complementation were made on a representative sample of complementing NA-induced ad-3B mutants, in addition to several from the noncomplementing group in all possible pairwise combinations, to determine whether the interaction matrix obtained was that expected on the basis of a linear complementation map. In addition, 4 mutants from the original tester set (2 nonleaky mutants (3-1o-153 and 1-23o-83) and two leaky mutants (3-1o-384 and 2 - I 6 - i 8 ) ) which do not have modifiers (as discussed above) affecting their growth on minimal medium were also included to provide points of reference between the original and revised complementation maps. All combinations were tested in the same experiment and the results are shown in the form of a complementation map in Fig. 2. Basically, the results of this experiment are in good agreement with the earlier experiments on leaky ad-3B mutants 8. All mutants can be represented as a continuous straight line, and 3 general classes of complementation patterns were found: (I) polarized-patterns extending from complon I and ending at various points on the right side of the complementation map, (2) nonpolarized-patterns of all other complementing mutants, and (3) noncomplementing-patterns shown by mutants that complement none of the ad-3B testers. One striking feature of this complementation map is the high percentage of nonleaky mutants among those mutants with nonpolarized complementation patterns. Another is the fact that 3 of the mutants are located to the left of m u t a n t 3-1o-153 in a region not represented by the original tester set (Fig. I). This is a point of particular interest that will be discussed in a later section.
The distribution of NA-induced ad-3B mutants on the complementation map There is a noticeable difference on the complementation map of the ad-3B cistron between the distribution of the NA-induced mutants and that of the earlier sample of leaky mutants. Many NA-induced mutants map in the region covered by complons 2-4 on the original m a p and to the left of m u t a n t 1-23o-83 (covering complons 5-8 on the original map). By contrast, relatively few NA-induced mutants are found in the region to the right of m u t a n t 1-23o-83. The basis for this difference is
Mutation Res., 4 (1967) 415-424
420
F.j. ad-3B CISTRON
-~
f
MUTANT NUMBERS OLD TESTERS ] NA-INDUCE9 MUTANTS
= ~I4I~I~I~IsI~I,oI,,~,~L,~
,.,
,81,-,
,8
"r-'-i
2-17-799,158 x 2-17-118 x 2 - 1 7 - 9 1 t 33,793
3-10-153
,
REV,SEO
DE SERRES el al.
mmm
EEC]
2q7-t28, x 73,91 80,85 2-17-61 ~, 89,906,912 2 t7- 165 2-17 72x t00,155 2-17-898 x 2 17-39,x 95 2-17-71 2-17-122 2-17-t17 x 2-17-63,81 2-17-51 x 2-17-129 x 2-17-37 x 2-17-114 × 2-17-130 x
t m m m m m
]
[
"m
NON .~ POLARIZED
m m m
2-17- 79x17 2-17-68 x 2-17-34 x
m m
m m
1-230-83 x
m (
2-17-30 '( 2-17-94 x
[ZX]
3-10-384 x
m
2-17-47 x 2-17-82 X, t12
r---q EX~
2 -16-1B 2-17-98 2 17-40,78 2-17-59,t63,792
POLARIZED
2-17-147, ~2, f 6 , 4 8 2-17-8 2-17-13,156,162,55, 6t0
NONCOMPLEf" MEN rING I
l q g . 2. The correlation b e t w e e n the c o m p l e m e n t a t i o n p a t t e r n s of selected n o n l e a k y or slightly l e a k y ad-3 B testers and selected N A - i n d u c e d ad-3B m u t a n t s on the original and revised c o m p l e m e n t a t i o n m a p of the ad-3B cistron. All m u t a n t s were tested in all possible pairwise c o m b i n a t i o n s in c o m p l e m e n t a t i o n tests. × , m u t a n t s selected for n e w tester set.
not known but could be due to nonrandomness either in the sample of leaky mutants from which the leaky tester set was derived or in the present sample of NA-induccd mutants. The additional discovery of mutants located to the left of complon I on the original map led us to revise our points of reference on the complementation map of the ad-3B cistron, as shown in the revised map (Fig. 2) for all subsequent work. We do not know which map is "correct" in the sense of one or the other best representing the structure of the ad-3B cistron or the polypeptide chain specified by the ad-3B cistron. But at that point in the analysis, we felt that any bias would become apparent in the continued analysis of allelic complementation at this locus.
Selection of a tester set and analysis of a random sample of NA-induced ad-3B mutants Representative nonleaky or slightly leaky ad-3B mutants were selected from the revised complementation map (Fig. 2) to provide a tester set for the analysis of a random sample of NA-induced mutants. Two nonallellic mutants at the ad-3 A locus (2-17-15) and hist-3 locus (1-193-14) were also included as controls. ¢Ylutalion Res., 4 (~967) 4 1 5 - 4 2 4
COMPLEMENTATION OF N A - I N D U C E D
ad-3B
421
MUTANTS
A series of experiments to test the effect of variation in pH, temperature, and in various supplements to the basal medium 17 (DE SERRES, unpublished results; 1V[oRPURGO AND DE SERRES,unpublished results) showed that the maximum number of positive responses was obtained in heterokaryon tests for allelic complementation when the concentration of conidia/ml was increased to I.O. lO 5, the basal medium was supplemented with o.i fig adenine sulfate/ml and 200 fig casamino acids/ml and the heterokaryon tests were incubated at 3 o°. (Under these conditions, however, both testers for eomplon 20 grew too rapidly alone to provide reliable data and were therefore omittcd ) The analysis of a random sample of i i i NA-induced ad-3B mutants under these optimal conditions i~ shown in Fig. 3. od-3B CtSTRON ~,.T*,,
..~.s
[q-lT[
3 I 4 I ~ I ~ I 7 I ~ I 9 I,o I1t [,z 1,31,4
,511~ I iT I
°"~".
TOTAL NUM8ER
2-17 t58 118
t
m
1
33 73,76,80,85,91.128 61 165 31,72,100,155 39,95 2, 5, 71,74 9,21 ~22 53, t17,150 56,63,81,86,1!6 !48 51 129 37 1!4 133 24 17,22,79,156 54,106 68 34 25,29,30.35 75 120, 143 146,164 94 3,11,53.103 99 47 82,112 107,169
t
m m
6 1
t 4 2 4 2
m m m m
I I
1
3 6 1 NON POLARIZED
1
m
604°~
1 1
m
1 I
I
1
I
4 2
m I
m
1
m
1
i
9 1
r---n I
4 1
I
m
1 2 r---1
67 [
40,59,70 163 12 6 4 6
47
]
POLARIZED 11,7%
1
i
i
3
i
1
5
8 87,153 6,7,13 14,18.23 26,10, 36,45,48,55,57 85, , ~ , 9 7 , t01,105, tO9 126,127 i32,t 36,t37 138, 142,149,159,162,166
t3
NONCOMPL[*.fMENTING ~.
31
27 9 %
Fig. 3: C o m p l e m e n t a t i o n p a t t e r n s of a r a n d o m s a m p l e of N A - i n d u c e d v i s e d m a p of t h e ad-3B c i s t r o n .
ad-3B m u t a n t s o n t h e re-
Characteristics of allelic complementation of NA-induced ad-3B mutants The complementation tests with the revised tester set on a random sample of NA-induced ad-3B mutants showed that 72.1% (8O/Ill) exhibit allelic complemenM~lation Res., 4 (I967) 4 1 5 - 4 z 4
422
F.J. DE SERRES et al.
tation and have either polarized or nonpolarized complementation patterns, the majority (6o.4%) of the ad-3B mutants in the nonpolarized group. By chance, the percentage of mutants showing allelic complementation (72.1% 8o/111) with the revised tester set (Fig. 2) is identical with the percentage (72.1%-1o6/147) obtained with the original tester set (Fig. I) on a larger sample of mutants. Another feature of the results of these tests is the large number of mutants with identical complementation patterns. The fact that only 3 ° of the 67 mutants with nonpolarized patterns are different can be interpreted as indicating a nonrandom distribution of altered sites in the ad-3Blocus. A similar result, in which only 5 of the 13 mutants have different patterns, was obtained with mutants with polarized patterns. A nonrandom distribution of NA-induced mutants on the recombination m a p has been reported for the r I I mutants of T 4 bacteriophage 13 and the ads mutants of Schizosaccharomyces pombe 14, and hence a nonrandom distribution of NA-induced ad-3B mutants on the complementation map is not completely unexpected.
Correlation between leakiness and complementation pattern The same type of correlation between leakiness and complementation pattern was found in the present tests (Fig. 3) as in earlier studies with ad-3B mutants 6& All of the 27 leaky mutants have nonpolarized complementation patterns. Leaky mutants account for 40.3% (27/67) of the mutants in this class or for 24.3% (27/111) of the total sample of NA-induced ad-3B mutants. One particularly interesting feature of the NA-induced leaky ad-3B mutants is that they do not grow as extensively on minimal medium as the leaky mutants in the original tester set (Fig. i). Furthermore, they can be more readily characterized under the "optimal conditions" discussed above, under which the majority of the mutants in the original tester set are useless because they grow extensively alone.
The correlation between complementation pattern and genetic alteration at the molecular level Earlier studies on the specific revertibility of NA-induced ad-3B mutants 15 showed that 84% result from BPS, and the data from the present analysis show that at least 72°/o of the NA-induced ad-3B mutants show allelic complementation. The point of interest here is whether there is any correlation between the type of compleinentation pattern and the type of genetic alteration at the molecular level. Do all mutants with nonpolarized patterns result from BPS ? What is the array of genetic alterations among mutants with nonpolarized patterns ? Among noncomplementing mutants ? Previous data 7 showed a correlation between mutagenic origin and percentage of allelic complementation as well as type of complementation pattern. The specificity found in that analysis only implies specificity at the molecular level ; therefore, in our more recent work, we are attempting to obtain direct evidence by means of studies on the specific revertibility of NA-induced ad-3B mutants after treatment with various chemical mutagens. These studies will be presented in another paper; but, in brief, they show a striking correlation between complementation pattern and genetic alteration 16 and show, in addition, that mutants with an AT base-pair at the mutant site occur more often among the noncomplementing mutants and mutants with polarized complementation patterns than among mutants with nonpolarized complementation patterns (MALLING AND DE SERRES, in preparation). Mutation Res., 4 (1967) 415-424
COMPLEMENTATION OF N A - I N D U C E D a d - 3 B MUTANTS
423
CONCLUSIONS
The complementation data from the present experiments show that there is a direct correlation between the percentage of mutants showing allelic complementation and the percentage of mutants resulting from BPS. The fact that the majority of the complementing mutants have nonpolarized patterns is also in good agreement with the hypothesis', 8 that such mutants are missense mutations resulting from basepair transitions. The fact that 2413% of the NA-induced mutants are leaky shows that there is a direct correlation between this percentage and the percentage of mutants resulting from BPS. Both of these tests provide a simple assay for this class of genetic alteration. In summary, it seems reasonable to conclude that the present experiments with NA-induced ad-3B mutants show that tests for allelic complementation and leakiness provide a highly significant assay for the presence of mutations resulting from BPS in unknown samples of mutants. ACKNOWLEDGEMENTS
Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation, and supported in part by a NAS-NRC Postdoctoral Research Fellowship to H. E. BROCKMAN. REFERENCES I BRIMACOMBE, R., J. TRUPIN, M. NIRENBERG, P. LEDER, M. BERNFIt~LD AND T. JAOUNI, R N A codewords a n d p r o t e i n s y n t h e s i s , V I I I . Nucleotide s e q u e n c e s of s y n o n y m codons for arginine, valine, cysteine a n d alanine, Proc. Natl. Acad. Sci. (U.S.), 54 (1965) 954-960. 2 BROCKMAN, H. E., AND F. J. DE SERRES, I n d u c t i o n of ad-3 m u t a n t s of Neurospora crassa b y 2 - a m i n o p u r i n e , Genetics, 48 (1963) 597-604. 3 CASE, M. E., H. E. BROCKMAN AND F. J. DE SERRES, F u r t h e r i n f o r m a t i o n on t h e origin of t h e Yale a n d O a k Ridge w i l d - t y p e s t r a i n s of Neurospora crassa, Neurospora Newsletter, 8 (1965) 25-26. 4 DE SERRES, F. J., Studies w i t h purple a d e n i n e mt~tants of Neurospora crassa, IV. L a c k of c o m p l e m e n t a t i o n b e t w e e n different ad-3 A m u t a n t s in h e t e r o k a r y o n s a n d pseudowild types, Genetics, 45 (196o) 555-566. 5 DE SERI~ES, F. J., A p r o c e d u r e for m a k i n g h e t e r o k a r y o n t e s t s in liquid m i n i m a l m e d i u m , Neurospora Newsletter, I (1962) 9-1o. 6 DE SERRES, F. J., S t u d i e s w i t h p u r p l e a d e n i n e m u t a n t s in Neurospora crassa, V. E v i d e n c e for allelic c o m p l e m e n t a t i o n a m o n g ad- 3 m u t a n t s , Genetics, 48 (1963) 351-36o. 7 DE SERRES, F. J., M u t a g e n e s i s a n d c h r o m o s o m e s t r u c t u r e , J. Cellular and Comp. Physiol., 64, Suppl. I (1964) 33-42. 8 DE SERRES, F. J., T h e utilization of l e a k y ad-3 m u t a n t s of Neurospora crassa in h e t e r o k a r y o n te~ts for allelic c o m p l e m e n t a t i o n , Mutation Res., 3 (1966) 3-12. 9 DE SERRES, F. J., AND H. G. KOLMARK, A direct m e t h o d for d e t e r m i n a t i o n of f o r w a r d - m u t a tion r a t e s in Neurospora crassa, Nature, 182 (1958) 1249-125o. io DE SERRES, F. J., H. G. KOLMARK AND H. E. BROCKMAN, F a c t o r s influencing t h e s u r v i v a l of Neurospora crassa conidia in sorbose-sucrose media, Nature, 193 (1962) 556-557 . I I FREESE, E., Molecular m e c h a n i s m of m u t a t i o n , in J. H. TAYLOR, Molecular Genetics, P a r t I, A c a d e m i c Press, N e w York, 1963, pp. 2o7-27o. 12 HOROWlTZ, N. H., AND G. W. BEADLE, A microbiological m e t h o d for t h e d e t e r m i n a t i o n of choline b y use of a m u t a n t of N e u r o s p o r a , J. Biol. Chem., 15 ° (1964) 325-333 . 13 KRIEG, D. R., Specificity of chemical m u t a g e n e s i s , Progr. Nucleic Acid Res., 2 (1963) 125-168. 14 LEUPOLD, U., AND H. GUTZ, Genetic fine s t r u c t u r e in Schizosaccharomyces, Proc. ~zth, Intern. Congr. Genet., 2 (1965) 31-35 . 15 MALLING, H. V., I d e n t i f i c a t i o n of t h e genetic a l t e r a t i o n s in n i t r o u s a c i d - i n d u c e d ad-3 m u t a n t s of Neurospora crassa, Mutation Res., 2 (1965) 32o-327.
Mutation Res., 4 (1967) 415-424
424
F.j.
DE SERRES el al.
16 5IALLING, H. V., AND F. J. DE SERRES, Relation between c o m p l e m e n t a t i o n p a t t e r n s and genetic alterations in nitrous acid-induced ad 313 m u t a n t s of Neurospora crass& Alutation lees., 4 (1967) 425 44 °. 17 MORI'URGO, (;., AND Ig. J. DF SERRES, Factors influencing allelie c o m p l e m e n t a t i o n a m o n g ad-3B n l u t a n t s of Neurospora, Genetics, 47 (I962) 972 973. 18 ORGEL, L, E,~ The chemical basis of mutation, Advan. in En=ymology, 27 {s965) 289-346. 19 TESSMAN, J., The induction of large deletions by nitrous acid, J. Mol. Biol., 5 (1962) 442 44520 SOLL, D., E. OHTSUKA, D. S. JONES, R, LOHRMANN, H. HAYATSU, S. NISHIMURA AND H. (;. [{HORANA, Studies on polynucleotides, X L I X . Stimulation of the binding of aminoacyls R N A ' s of ribosomes by ribotrinucleotides and a s u r v e y of codon assignments for 20 aminoacids, Proc. Natl. Acad. ,qci. (U.S.), 54 {1965) I378 I385. 21 \VESTERGAARD, ~I., AND H. ][~. MITCHELL, Ncttyospoya, V. A synthetic medinnl favoring sexual reproduction, AnT. y. Botany, 34 (1947) 573 578. 22 YANOFSKY, C., Amino acid replacements associated with m u t a t i o n and recombination in the A gent and their relationship to in vitro coding data, Cold SpJing ~Iarbor Syrup. Quant. Biol., 28 (1963) 581-588.
Mutation Res., 4 (1967) 415-424
415
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
ALLELIC COMPLEMENTATION AMONG NITROUS ACID-INDUCED ad-3B MUTANTS OF NEUROSPORA CRASSA F. J. BE SERRES, H. E. BROCKMAN,* W. E. BARNETT AND H. G. KDLMARK**
Biology Division, Oah Ridge National Laboratory, Oah Ridge, Tenn. (U.S.A.) (Received December I9th, 1966)
SUMMARY
Complementation tests on a random sample of NA-induced ad-3B mutants of Neurospora crassa have shown that 72.1% show allelic complementation and have either nonpolarized or polarized complementation patterns, the majority (6o.4%) being in the nonpolarized group. The ratio of nonpolarized to polarized complementation patterns is thus 5 to I. In addition, a total of 24.3% are leaky, have nonpolarized patterns, and account for 4o.3~o of the mutants in this class. The facts that only 3o of the 65 mutants with nonpolarized patterns and only 5 of the 13 mutants with polarized patterns are different have been interpreted as indicating a nonrandom distribution of altered sites in the ad-3B locus. The discovery of many nonleaky NA-induced mutants with nonpolarized patterns has led to a revision of an earlier complementation map of the ad-3B cistron published previously 8. A tester set of predominantly nonleaky ad-3B mutants with nonpolarized complementation patterns which define a linear complementation map of 17 complons has been selected for future work. The complementation data from the present analysis in combination with the reversion data of MALLING1B indicate a direct correlation between the percentage of mutations resulting from base-pair substitution and the percentage showing allelic complementation.
INTRODUCTION
The mutagenic activity of nitrous acid '(NA) on viral systems has been studied extensivelyn,13,18 and it has been shown to result from deamination of certain nucleic acid bases and to produce mutations predominantly by base-pair substitutions (BPS) of the type AT -~ GC and GC -+ AT. TESSMAN19 has shown that NA can also produce * Present address: Department of Biological Sciences, Illinois State University, Normal, Ill., U.S.A ** Present address: Institute of Physiological Botany, The University of Uppsala, Uppsala, Sweden. Abbreviations: BPS, base-pair substitutions; NA, nitrous acid.
Mutation Res., 4 (1967) 415-424
416
F.J. DE SERRES et al.
mutations that map as extensive deletions and that these account for about 8% of the NA-induced rII mutants of T 4 bacteriophage. Studies on the mutagenic effect of NA on Neurospora suggest that the above general spectrum of genetic alterations is also found in eukaryotes (as prokaryotes). MALLING1~has shown that at least 84% of the NA-induced ad-3 mutants of Neurospora crassa are BPS and that at least 6%, out of the remaining 16% with unidentified genetic alterations, may be due to intralocal deletions. The RNA code-word assignments designated by NIRENBERG and by KHORANA and their coworkersl, 2° show that most base-pair transitions give a new triplet which either codes for the same amino acid or another amino acid and that there are only 2 codons (UAA and UAG) which code for no amino acid. From these data it would appear that most base-pair transitions would code for (I) the same amino acid or a new amino acid which would not affect the function of the protein (sense mutation) or (2) a new amino acid which would affect the function of the protein (missense mutation) 22. However, only a minority of the possible base-pair transitions would completely inactivate the protein as a result of premature chain termination because they code for no amino acid (nonsense mutation). We do not know what ratio of sense to missense to nonsense mutations to expect after NA-treatment. However, it should be possible to estimate the ratio of missense to nonsense mutations by an analysis of the gene products specified by NA-induced mutants. Missense and nonsense mutations should have different effects o11 the type of gene product specified in terms of (I) the ability to show allelic complementation and the type of complementation pattern and (2) the level of residual enzyme activity 7. This paper is the first in our series of attempts to characterize the genetic effects of NA in Neurospora crassa at the molecular level. NA-induced ad-3B mutants have been analyzed for leakiness and allelic complementation by heterokaryon tests. These tests indicate that 72% of NA-induced ad-3B mutants show allelic complementation, that the ratio of nonpolarized to polarized complementation patterns is about 5 to I, and that 24% of the ad-3B mutants are leaky. METHODS
Strains. New ad-3 mutants were induced in wild-type strain OR 74 A (Oak Ridge vegetative isolate of ST74A ) (ref. 3). The leaky and nonleaky ad-3B mutants used as testers in heterokaryon tests for allelic complementation were all induced in wild-type strain 74 A or closely related derivatives s. The origin of the ad-3A mutants used as controls in the heterokaryon tests was described previously 4& Isolation of mutants. Cultures of wild type were grown on Fries' minimal medium (ref. 12) for 7 days at 25 °. Conidial suspensions were made in ice-cold water, filtered 2 times through wetted absorbent cotton pads (o.5-1.o cm thick) in a Buchner funnel, washed 2 times and adjusted to I. lO 7conidiaper ml in o.i M acetate buffer pH 4.6. To start the treatment 4 mg NaNO2 were added to 11.6 ml samples of conidial suspension, giving a final concentration of o.oo5 M N a N Q . Treatment was at 25 ° in a constant temperature water bath. Conidial suspensions in acetate buffer alone were used as controls. Treatment was terminated by taking samples out of the treatment vessels and pipetting them into Ioo ml cold phosphate buffer at pH 7 to stop the reaction. Mutation Res., 4 (1967) 415-424
ad-3B MUTANTS
COMPLEMENTATION O F N A - I N D U C E D
417
Control and treated conidia were analyzed for the presence of purple adenine- 3
(ad-3) mutants by the direct method 9 modified by the addition of 12.5 mg adenine sulfate/l, 250 mg L-arginine. HCI/I, 1.5°/0 L-sorbose, o.15% agar and o.I~o sucrose to basal medium~L 12-1 flasks containing IO l of this medium were autoclaved for 50 min (18 pounds, 252o F), the time required for maximum viability under these conditions TM. Inocula were planned to give a maximum of 1.2. lO 6 colonies per IO l of medium. Ad-3 mutant colonies were isolated following 7 days of incubation with aeration in the dark at 3 o°. A homokaryotic culture of each mutant was obtained by plating conidia in Fries' basal medium which contained l.-o °//o sorbose, o.1°/0 sucrose, and 2 mg adenine sulfate/1. In this medium adenine-requiring homokaryotic colonies are morphologically distinguishable from wild-type and heterokaryotic colonies. Heterokaryon tests for allelic complementation. The genotypes of the NA-induced ad-3 mutants were determined with an ad-3A mutant and a noncomplementing ad-3B mutant. The ad-3B mutants were then screened for allelic complementation with a series of 30 complementing ad-3B mutants (two nonleaky and 28 leaky) as testers. The complementation pattern of each tester is shown in Fig. I and these mutants ed-J3 ClSTRON MUTANT NUMBERS
11121314151et'~1010110
3-10-153
m
3-10-114 3 - 1 0 - 500 3-10-112
I [
2-11-5 1-230-83 2-11-131,2-11-163, 2 - t l q O 2-16-22 2-15-72 3-10-109 3-10 -132 3 -10-16 3-10-70 2-11-6 1-234-108 2-11-223 2-11-140 3-10-384 3-I0-446 3-10-506 3-10-67 1- 2 3 4 - 1 4 8 3-10-470 1 -155-11, 1-1t2-8 3-27-16, 1-230-69,2-16-18
~--
11l ~211311411511611711BI191201
I I
I I
I
I
I I I I I I
1 I'
'
I
[
I I----'1 I I I I
] I
1
k______2 f"
I
I [
Fig. i. C o m p l e m e n t a t i o n p a t t e r n s of ad-3B m u t a n t s used as testers in h e t e r o k a r y o n tests for allelic c o m p l e m e n t a t i o n of NA-induced ad-3B m u t a n t s . I , Nonleaky m u t a n t ; C3, leaky m u t a n t .
were selected from a larger sample described previously 8. To retard growth by leaky mutants alone, complementation tests were made in liquid minimal medium in IO mm × 75 mm test tubesS, s. All tests were incubated at 24-26 ° and responses were scored periodically for a period of 21 days. Tests for leaky NA-induced ad-3 mutants. Tests to determine the percentage of M~lation Res., 4 (1967) 415-424
F.J. DE SERRES et al.
418
leaky ad-3B mutants were made as indicated above but with a total of 2. 5 • lO 6conidia per tube. RESULTS AND DISCUSSION
Selection of ad-3 mutants for genetic analysis The ad-3 mutants selected for analysis were obtained from treatment of conidia with 0.005 M NAN02. The treatment times, levels of survival, and forward-mutation frequencies are given in Table I. The frequency of spontaneous mutants in this experiTABLE I ad- 3 MUTANTS NaNO~, p H 4.6, 25 °) All m u t a n t n u m b e r s begin w i t h prefix 2 - i 7. T H E ORIGIN OF N A - I N D U C E D
I N D U C E D IN W I L D - T Y P E STRAIN O R
Range of mutant numbers
Treatment time (rain)
Per cent survival
Mutations per ~o ~ survivors
2 - 1 o ; 788-854 11-43 44-125; 855-1o27 126-17o
iio iio 16o 16o
41.6 34.3 23.2 14.6
29.4 37.4 127. 5 128.1
74 A (0.0o5
M
ment was not significantly higher than the expected frequency 2 of spontaneous forward mutation of o.38" io 6. Thus, a IOO- to 4oo-fold enrichment was obtained with treatment, and the incidence of mutants of spontaneous origin in the sample of 199 NA-induced mutants selected for genetic analysis is negligible.
Screening tests for allelic complementation among NA-induced ad-3 mutants The complementation tests on 199 NA-induced ad-3 mutants selected at random for genetic analysis showed (I) that 52/199 mutants were ad-3A mutants and (2) that 147/199 were ad-3B mutants. No other genotypes were found among the mutants recovered as purple-pigmented colonies. When the ad-3B mutants were tested for allelie complementation with the tester set illustrated in Fig. I, lO6/147 mutants, or 72.I%, showed allelic complementation. Even more interesting to us were tlae facts (I) that the complon 6 coverage of the majority of the complementing mutants appeared to be quite limited, and (2) that the majority of the complementing mutants were nonleaky.
The development of a nonleaky tester set to study allelic complementation among NAinduced ad-3B mutants The discovery of a series of highly complementing mutants induced in the same isolate of wild-type strain OR 74 A (all of them most probably resulting from BPS) led us to t r y to replace the leaky mutant tester set shown in Fig. I with a series of nonleaky or only slightly leaky NA-induced mutants for further experimentation. This decision was based on 2 very different but equally important considerations: (i) The fact that m a n y of the 28 leaky mutants in the tester set grow extensively on minimal medium introduces uncertainty on the scoring of these mutants in combination. When scoring complementation tests involving l e a k y + l e a k y ' combinations, the amount of growth shown in each pairwise combination must be compared with that Mutation Res., 4 (1967) 415-424
COMPLEMENTATION OF NA-INI)UCED ad-3B MUTANTS
419
shown by each m u t a n t individually to determine whether the combination gives a positive or negative complementation response. Since the growth shown by leaky mutants alone appears at different points in time, the total time over which the complementation tests can be scored varies considerably for different pairwise combinations. Thus, the scoring of complementation tests made with the leaky mutant tester set is technically difficult and time consuming and thus not as well suited for rapid screening tests as a nonleaky tester set. (2) D a t a from a completely different experiment on (a) the leaky ad-3A and ad-3B m u t a n t samples from which the present tester set was derived s and (b) the NA-induced ad-3A and ad-3B mutants showed quite clearly (a) that m a n y of the mutants in the selected sample of leaky mutants used as testers carry modifiers that affect the leakiness of these mutants on minimal medium (DE SERRES, unpublished observations) and (b) that these modifiers are not present in any of the 147 NAinduced ad-3B mutants from the 2-17 experiment (DE SERRES, unpublished observations).
Allelic complementation among NA-induced ad-3B mutants Heterokaryon tests for allelic complementation were made on a representative sample of complementing NA-induced ad-3B mutants, in addition to several from the noncomplementing group in all possible pairwise combinations, to determine whether the interaction matrix obtained was that expected on the basis of a linear complementation map. In addition, 4 mutants from the original tester set (2 nonleaky mutants (3-1o-153 and 1-23o-83) and two leaky mutants (3-1o-384 and 2 - I 6 - i 8 ) ) which do not have modifiers (as discussed above) affecting their growth on minimal medium were also included to provide points of reference between the original and revised complementation maps. All combinations were tested in the same experiment and the results are shown in the form of a complementation map in Fig. 2. Basically, the results of this experiment are in good agreement with the earlier experiments on leaky ad-3B mutants 8. All mutants can be represented as a continuous straight line, and 3 general classes of complementation patterns were found: (I) polarized-patterns extending from complon I and ending at various points on the right side of the complementation map, (2) nonpolarized-patterns of all other complementing mutants, and (3) noncomplementing-patterns shown by mutants that complement none of the ad-3B testers. One striking feature of this complementation map is the high percentage of nonleaky mutants among those mutants with nonpolarized complementation patterns. Another is the fact that 3 of the mutants are located to the left of m u t a n t 3-1o-153 in a region not represented by the original tester set (Fig. I). This is a point of particular interest that will be discussed in a later section.
The distribution of NA-induced ad-3B mutants on the complementation map There is a noticeable difference on the complementation map of the ad-3B cistron between the distribution of the NA-induced mutants and that of the earlier sample of leaky mutants. Many NA-induced mutants map in the region covered by complons 2-4 on the original m a p and to the left of m u t a n t 1-23o-83 (covering complons 5-8 on the original map). By contrast, relatively few NA-induced mutants are found in the region to the right of m u t a n t 1-23o-83. The basis for this difference is
Mutation Res., 4 (1967) 415-424
420
F.j. ad-3B CISTRON
-~
f
MUTANT NUMBERS OLD TESTERS ] NA-INDUCE9 MUTANTS
= ~I4I~I~I~IsI~I,oI,,~,~L,~
,.,
,81,-,
,8
"r-'-i
2-17-799,158 x 2-17-118 x 2 - 1 7 - 9 1 t 33,793
3-10-153
,
REV,SEO
DE SERRES el al.
mmm
EEC]
2q7-t28, x 73,91 80,85 2-17-61 ~, 89,906,912 2 t7- 165 2-17 72x t00,155 2-17-898 x 2 17-39,x 95 2-17-71 2-17-122 2-17-t17 x 2-17-63,81 2-17-51 x 2-17-129 x 2-17-37 x 2-17-114 × 2-17-130 x
t m m m m m
]
[
"m
NON .~ POLARIZED
m m m
2-17- 79x17 2-17-68 x 2-17-34 x
m m
m m
1-230-83 x
m (
2-17-30 '( 2-17-94 x
[ZX]
3-10-384 x
m
2-17-47 x 2-17-82 X, t12
r---q EX~
2 -16-1B 2-17-98 2 17-40,78 2-17-59,t63,792
POLARIZED
2-17-147, ~2, f 6 , 4 8 2-17-8 2-17-13,156,162,55, 6t0
NONCOMPLEf" MEN rING I
l q g . 2. The correlation b e t w e e n the c o m p l e m e n t a t i o n p a t t e r n s of selected n o n l e a k y or slightly l e a k y ad-3 B testers and selected N A - i n d u c e d ad-3B m u t a n t s on the original and revised c o m p l e m e n t a t i o n m a p of the ad-3B cistron. All m u t a n t s were tested in all possible pairwise c o m b i n a t i o n s in c o m p l e m e n t a t i o n tests. × , m u t a n t s selected for n e w tester set.
not known but could be due to nonrandomness either in the sample of leaky mutants from which the leaky tester set was derived or in the present sample of NA-induccd mutants. The additional discovery of mutants located to the left of complon I on the original map led us to revise our points of reference on the complementation map of the ad-3B cistron, as shown in the revised map (Fig. 2) for all subsequent work. We do not know which map is "correct" in the sense of one or the other best representing the structure of the ad-3B cistron or the polypeptide chain specified by the ad-3B cistron. But at that point in the analysis, we felt that any bias would become apparent in the continued analysis of allelic complementation at this locus.
Selection of a tester set and analysis of a random sample of NA-induced ad-3B mutants Representative nonleaky or slightly leaky ad-3B mutants were selected from the revised complementation map (Fig. 2) to provide a tester set for the analysis of a random sample of NA-induced mutants. Two nonallellic mutants at the ad-3 A locus (2-17-15) and hist-3 locus (1-193-14) were also included as controls. ¢Ylutalion Res., 4 (~967) 4 1 5 - 4 2 4
COMPLEMENTATION OF N A - I N D U C E D
ad-3B
421
MUTANTS
A series of experiments to test the effect of variation in pH, temperature, and in various supplements to the basal medium 17 (DE SERRES, unpublished results; 1V[oRPURGO AND DE SERRES,unpublished results) showed that the maximum number of positive responses was obtained in heterokaryon tests for allelic complementation when the concentration of conidia/ml was increased to I.O. lO 5, the basal medium was supplemented with o.i fig adenine sulfate/ml and 200 fig casamino acids/ml and the heterokaryon tests were incubated at 3 o°. (Under these conditions, however, both testers for eomplon 20 grew too rapidly alone to provide reliable data and were therefore omittcd ) The analysis of a random sample of i i i NA-induced ad-3B mutants under these optimal conditions i~ shown in Fig. 3. od-3B CtSTRON ~,.T*,,
..~.s
[q-lT[
3 I 4 I ~ I ~ I 7 I ~ I 9 I,o I1t [,z 1,31,4
,511~ I iT I
°"~".
TOTAL NUM8ER
2-17 t58 118
t
m
1
33 73,76,80,85,91.128 61 165 31,72,100,155 39,95 2, 5, 71,74 9,21 ~22 53, t17,150 56,63,81,86,1!6 !48 51 129 37 1!4 133 24 17,22,79,156 54,106 68 34 25,29,30.35 75 120, 143 146,164 94 3,11,53.103 99 47 82,112 107,169
t
m m
6 1
t 4 2 4 2
m m m m
I I
1
3 6 1 NON POLARIZED
1
m
604°~
1 1
m
1 I
I
1
I
4 2
m I
m
1
m
1
i
9 1
r---n I
4 1
I
m
1 2 r---1
67 [
40,59,70 163 12 6 4 6
47
]
POLARIZED 11,7%
1
i
i
3
i
1
5
8 87,153 6,7,13 14,18.23 26,10, 36,45,48,55,57 85, , ~ , 9 7 , t01,105, tO9 126,127 i32,t 36,t37 138, 142,149,159,162,166
t3
NONCOMPL[*.fMENTING ~.
31
27 9 %
Fig. 3: C o m p l e m e n t a t i o n p a t t e r n s of a r a n d o m s a m p l e of N A - i n d u c e d v i s e d m a p of t h e ad-3B c i s t r o n .
ad-3B m u t a n t s o n t h e re-
Characteristics of allelic complementation of NA-induced ad-3B mutants The complementation tests with the revised tester set on a random sample of NA-induced ad-3B mutants showed that 72.1% (8O/Ill) exhibit allelic complemenM~lation Res., 4 (I967) 4 1 5 - 4 z 4
422
F.J. DE SERRES et al.
tation and have either polarized or nonpolarized complementation patterns, the majority (6o.4%) of the ad-3B mutants in the nonpolarized group. By chance, the percentage of mutants showing allelic complementation (72.1% 8o/111) with the revised tester set (Fig. 2) is identical with the percentage (72.1%-1o6/147) obtained with the original tester set (Fig. I) on a larger sample of mutants. Another feature of the results of these tests is the large number of mutants with identical complementation patterns. The fact that only 3 ° of the 67 mutants with nonpolarized patterns are different can be interpreted as indicating a nonrandom distribution of altered sites in the ad-3Blocus. A similar result, in which only 5 of the 13 mutants have different patterns, was obtained with mutants with polarized patterns. A nonrandom distribution of NA-induced mutants on the recombination m a p has been reported for the r I I mutants of T 4 bacteriophage 13 and the ads mutants of Schizosaccharomyces pombe 14, and hence a nonrandom distribution of NA-induced ad-3B mutants on the complementation map is not completely unexpected.
Correlation between leakiness and complementation pattern The same type of correlation between leakiness and complementation pattern was found in the present tests (Fig. 3) as in earlier studies with ad-3B mutants 6& All of the 27 leaky mutants have nonpolarized complementation patterns. Leaky mutants account for 40.3% (27/67) of the mutants in this class or for 24.3% (27/111) of the total sample of NA-induced ad-3B mutants. One particularly interesting feature of the NA-induced leaky ad-3B mutants is that they do not grow as extensively on minimal medium as the leaky mutants in the original tester set (Fig. i). Furthermore, they can be more readily characterized under the "optimal conditions" discussed above, under which the majority of the mutants in the original tester set are useless because they grow extensively alone.
The correlation between complementation pattern and genetic alteration at the molecular level Earlier studies on the specific revertibility of NA-induced ad-3B mutants 15 showed that 84% result from BPS, and the data from the present analysis show that at least 72°/o of the NA-induced ad-3B mutants show allelic complementation. The point of interest here is whether there is any correlation between the type of compleinentation pattern and the type of genetic alteration at the molecular level. Do all mutants with nonpolarized patterns result from BPS ? What is the array of genetic alterations among mutants with nonpolarized patterns ? Among noncomplementing mutants ? Previous data 7 showed a correlation between mutagenic origin and percentage of allelic complementation as well as type of complementation pattern. The specificity found in that analysis only implies specificity at the molecular level ; therefore, in our more recent work, we are attempting to obtain direct evidence by means of studies on the specific revertibility of NA-induced ad-3B mutants after treatment with various chemical mutagens. These studies will be presented in another paper; but, in brief, they show a striking correlation between complementation pattern and genetic alteration 16 and show, in addition, that mutants with an AT base-pair at the mutant site occur more often among the noncomplementing mutants and mutants with polarized complementation patterns than among mutants with nonpolarized complementation patterns (MALLING AND DE SERRES, in preparation). Mutation Res., 4 (1967) 415-424
COMPLEMENTATION OF N A - I N D U C E D a d - 3 B MUTANTS
423
CONCLUSIONS
The complementation data from the present experiments show that there is a direct correlation between the percentage of mutants showing allelic complementation and the percentage of mutants resulting from BPS. The fact that the majority of the complementing mutants have nonpolarized patterns is also in good agreement with the hypothesis', 8 that such mutants are missense mutations resulting from basepair transitions. The fact that 2413% of the NA-induced mutants are leaky shows that there is a direct correlation between this percentage and the percentage of mutants resulting from BPS. Both of these tests provide a simple assay for this class of genetic alteration. In summary, it seems reasonable to conclude that the present experiments with NA-induced ad-3B mutants show that tests for allelic complementation and leakiness provide a highly significant assay for the presence of mutations resulting from BPS in unknown samples of mutants. ACKNOWLEDGEMENTS
Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation, and supported in part by a NAS-NRC Postdoctoral Research Fellowship to H. E. BROCKMAN. REFERENCES I BRIMACOMBE, R., J. TRUPIN, M. NIRENBERG, P. LEDER, M. BERNFIt~LD AND T. JAOUNI, R N A codewords a n d p r o t e i n s y n t h e s i s , V I I I . Nucleotide s e q u e n c e s of s y n o n y m codons for arginine, valine, cysteine a n d alanine, Proc. Natl. Acad. Sci. (U.S.), 54 (1965) 954-960. 2 BROCKMAN, H. E., AND F. J. DE SERRES, I n d u c t i o n of ad-3 m u t a n t s of Neurospora crassa b y 2 - a m i n o p u r i n e , Genetics, 48 (1963) 597-604. 3 CASE, M. E., H. E. BROCKMAN AND F. J. DE SERRES, F u r t h e r i n f o r m a t i o n on t h e origin of t h e Yale a n d O a k Ridge w i l d - t y p e s t r a i n s of Neurospora crassa, Neurospora Newsletter, 8 (1965) 25-26. 4 DE SERRES, F. J., Studies w i t h purple a d e n i n e mt~tants of Neurospora crassa, IV. L a c k of c o m p l e m e n t a t i o n b e t w e e n different ad-3 A m u t a n t s in h e t e r o k a r y o n s a n d pseudowild types, Genetics, 45 (196o) 555-566. 5 DE SERI~ES, F. J., A p r o c e d u r e for m a k i n g h e t e r o k a r y o n t e s t s in liquid m i n i m a l m e d i u m , Neurospora Newsletter, I (1962) 9-1o. 6 DE SERRES, F. J., S t u d i e s w i t h p u r p l e a d e n i n e m u t a n t s in Neurospora crassa, V. E v i d e n c e for allelic c o m p l e m e n t a t i o n a m o n g ad- 3 m u t a n t s , Genetics, 48 (1963) 351-36o. 7 DE SERRES, F. J., M u t a g e n e s i s a n d c h r o m o s o m e s t r u c t u r e , J. Cellular and Comp. Physiol., 64, Suppl. I (1964) 33-42. 8 DE SERRES, F. J., T h e utilization of l e a k y ad-3 m u t a n t s of Neurospora crassa in h e t e r o k a r y o n te~ts for allelic c o m p l e m e n t a t i o n , Mutation Res., 3 (1966) 3-12. 9 DE SERRES, F. J., AND H. G. KOLMARK, A direct m e t h o d for d e t e r m i n a t i o n of f o r w a r d - m u t a tion r a t e s in Neurospora crassa, Nature, 182 (1958) 1249-125o. io DE SERRES, F. J., H. G. KOLMARK AND H. E. BROCKMAN, F a c t o r s influencing t h e s u r v i v a l of Neurospora crassa conidia in sorbose-sucrose media, Nature, 193 (1962) 556-557 . I I FREESE, E., Molecular m e c h a n i s m of m u t a t i o n , in J. H. TAYLOR, Molecular Genetics, P a r t I, A c a d e m i c Press, N e w York, 1963, pp. 2o7-27o. 12 HOROWlTZ, N. H., AND G. W. BEADLE, A microbiological m e t h o d for t h e d e t e r m i n a t i o n of choline b y use of a m u t a n t of N e u r o s p o r a , J. Biol. Chem., 15 ° (1964) 325-333 . 13 KRIEG, D. R., Specificity of chemical m u t a g e n e s i s , Progr. Nucleic Acid Res., 2 (1963) 125-168. 14 LEUPOLD, U., AND H. GUTZ, Genetic fine s t r u c t u r e in Schizosaccharomyces, Proc. ~zth, Intern. Congr. Genet., 2 (1965) 31-35 . 15 MALLING, H. V., I d e n t i f i c a t i o n of t h e genetic a l t e r a t i o n s in n i t r o u s a c i d - i n d u c e d ad-3 m u t a n t s of Neurospora crassa, Mutation Res., 2 (1965) 32o-327.
Mutation Res., 4 (1967) 415-424
424
F.j.
DE SERRES el al.
16 5IALLING, H. V., AND F. J. DE SERRES, Relation between c o m p l e m e n t a t i o n p a t t e r n s and genetic alterations in nitrous acid-induced ad 313 m u t a n t s of Neurospora crass& Alutation lees., 4 (1967) 425 44 °. 17 MORI'URGO, (;., AND Ig. J. DF SERRES, Factors influencing allelie c o m p l e m e n t a t i o n a m o n g ad-3B n l u t a n t s of Neurospora, Genetics, 47 (I962) 972 973. 18 ORGEL, L, E,~ The chemical basis of mutation, Advan. in En=ymology, 27 {s965) 289-346. 19 TESSMAN, J., The induction of large deletions by nitrous acid, J. Mol. Biol., 5 (1962) 442 44520 SOLL, D., E. OHTSUKA, D. S. JONES, R, LOHRMANN, H. HAYATSU, S. NISHIMURA AND H. (;. [{HORANA, Studies on polynucleotides, X L I X . Stimulation of the binding of aminoacyls R N A ' s of ribosomes by ribotrinucleotides and a s u r v e y of codon assignments for 20 aminoacids, Proc. Natl. Acad. ,qci. (U.S.), 54 {1965) I378 I385. 21 \VESTERGAARD, ~I., AND H. ][~. MITCHELL, Ncttyospoya, V. A synthetic medinnl favoring sexual reproduction, AnT. y. Botany, 34 (1947) 573 578. 22 YANOFSKY, C., Amino acid replacements associated with m u t a t i o n and recombination in the A gent and their relationship to in vitro coding data, Cold SpJing ~Iarbor Syrup. Quant. Biol., 28 (1963) 581-588.
Mutation Res., 4 (1967) 415-424