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Correlation between base-pair transition and complementation pattern in nitrous acid-induced ad-3B mutants of neurospora crassa
Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
359
C O R R E L A T I O N B E T W E E N B A S E - P A I R T R A N S I T I O N AND COMPLEMENT A T I O N P A T T E R N IN N I T R O U S A C I D - I N D U C E D NEUROSPORA CRASSA*
ad-3B MUTANTS OF
H. V. MALLING AND F. J. DE S E R R E S Biology Division, N C I - A E C Carcinogenesis Program, Oak Ridge National Laboratory, Oah Ridge, Tenn. (U.S.A.) (Received March I2th, 1968)
SUMMARY
Nitrous acid-induced ad-3B mutants of Neurospora crassa were tested for revertibility after treatment with hydroxylamine (HA). Mutants which reverted after the treatment were classified as having guanine-cytosine (GC) at the m u t a n t site. A high frequency (86.5%) of nitrous acid-induced ad-3B mutants resulting from base-pair transitions have GC at the n m t a n t site, whereas in bacteriophage T4 most nitrous acid-induced r l I mutants have adenine thymine (AT) at the m u t a n t site. This apparent contradiction is discussed. In a random sampling of base-pair transition ad-3B mutants, we found among mutants with nonpolarized complementation pattern a lower incidence (2.3%) of AT at the m u t a n t site than among mutants with polarized complementation pattern (25.0%) and among noncomplementing mutants (55.0°/'0). Nonsense mutants, which specify the RNA triplets UAA, UAG, or UGA, and presumably the DNA anticodons ATT, ATC or ACT, should not be able to revert by a GC -+, AT transition, because by reaction with hydroxylamine the two C-containing triplets would be transformed to ATT, a nonsense triplet. The difference in the frequency of the GC pairs at n m t a n t sites among tile three different classes of complementation response indicates that nonsense mutations occur more frequently among mutants with polarized complementation patterns and among nonconiplementing mutants than among mutants with nonpolarized complementation patterns.
INTRODUCTION
Earlier studies of allelic complementation among the ad-3B mutants of Neurospora crassaS,9 showed that these m u t a n t s could be classified as (z) noncomplenlenting * Research jointly sponsored by the National Cancer Institute, and the U.S. Atomic Energy Commision under contract with the Union Carbide Corporation. Abbreviations: A, adenine; AT, adenine-thymine; C, cytosine; EMS, ethyl methanesulfonate; G, guanine; GC, guanine-cytosine; HA, hydroxylamine; HMC, hydroxymethylcytosine: I CR- t 7o, 2-methoxy-6-chloro-9- E3-ethyl- 2-chloroethyl) aminopropylanlino]acridine dihydrochloride; MNNG, N-nlethyl-N'-nitro-N-nitrosoguanidine; NA, nitrous acid; T, thymine.
Mutation Res., 5 (1968) 359-371
360
H . V . MALLING, F. J. DE SERRES
or (2) complementing with either (a) nonpolarized or (b) polarized complementation patterns. A striking correlation was found between the type of mutagens used to induce the nmtants and the spectrmn of eomplementation patternsS,~0; a similar correlation was found between the type of mutagens used to induce the mutants and the percent leakiness 9. This suggested that the type of eomplementation pattern was correlated with the type of genetic alteration, and the analysis of tim genetic alterations in NA-induced ad-3B nmtants by means of tests for specific revertibility provided conclusive proof 2a. In the latter analysis the ad-3B mutants with nonpolarized con> plementation patterns were shown to result mainly from base-pair substitutions, whereas mutants with polarized complementation patterns and noncomplementing mutants are derived from a variety of genetic alterations. In T4 bacteriophage, HA induced base-pair transitions from GC to AT ~4. In Neurospora, HA induces reversion only in some of the nmtants which revert by basepair transition, and never in those nmtants which revert by a base-pair insertion or deletion2~, °-~-. Later analysis of HA-induced ad-3B nmtants by specific revertibility test have shown that the majority of the mutants have AT at mutant site (MALLIN6, unpublished). Therefore, we have concluded that HA induces base-pair transitions preferentially from GC to AT in Neurospora and can be used in tests for specific revertibility to determine the direction of the base-pair transition. In this paper, our previous attempts both to identify the genetic alterations among NA-induced ad-3B mutants and to correlate genetic alteration with complementation pattern have been further extended by the identification of the two types of base-pair transitions within these three classes of mutants. The present data show that base-pair transitions of the type AT to GC give rise more frequently to ad-3B nmtants with nonpolarized complementation patterns, whereas GC to AT transitions give rise more frequently to mutants with polarized complementation patterns and noncomplementing mutants. The significance of these observations is discussed in relation to what is known about the genetic code for nonsense nmtations. MATERIALS AND METHODS
All ad-3B nmtants were isolated by the direct method 4 after NA treatment of the wild-type strain OR 74 Al°. The treatment conditions, survival levels, forward mutation frequencies, and complementation patterns of the mutants are described by DE SERRES et al) ° and MALLINGAND DE SERRES2a. All tieatments were carried out on freshly harvested suspensions of conidia as described by MALLING21.
HA treatment The procedure for treatment with HA has been described previously =1. In brief, the eonidia were treated for 300 rain with I M HA. HC1 (Baker Chemical Co., Phillipsburg, N.J.) dissolved in a 3 M NaC1 solution and adjusted to pH 6.2 ~.9.
The specificity of HA The revertibility of NA-induced mutants was tested after treatment with HA to attempt to identify tile direction of the base-pair transition, leading to the reverse mutation. The r I I mutants induced by HA in T~ bacteriophage are randomly distribMulalio*e Res., 5 (I968) 359-371
COMPLEMENTATION PATTERNS AND TYPE OF BASE-PAIR TRANSITION
361
uted] on the genetic map of the r I I locusX5,tL Therefore the base-pair sequence in the DNA seems to have no influence on the reaction of HA with DNA; but, on the other hand, nothing is known about how this reaction is influenced by the protein in the chromosomes of eukaryotes. Dilute solutions of HA (IO a M) in combination with oxygen give rise to oxidation products which showed a strong inactivating effect on the transforming DNA of B. subtilis ~6. At higher concentrations of HA ( > o . I M), these oxidation products were not formed. In vivo experiments 3° with the single stranded DNA phage @XI74 ) treated with lower concentrations of HA (2.5" IO-2 M) indicated that HA presumably reacted with all 4 bases since it was found that mutants resulted from base-pair transitions in both directions. Under the treatment conditions used in the present tests for specific revertibility on Neurospora, HA should react predominantly with C in the DNA ~4. However, even under these conditions, there is evidence 5 that a small amount of thymine is lost when DNA is treated with HA. Studies of HA-induced ad-3B inutants have shown that a minor fraction of the HA-induced nmtants are able to revert after treatment with O-methylhydroxylamine (MALLIN6, unpublished), which in transforming principles is assumed to be more specific than HA ~6 in the reaction with C in the DNA. This indicates that a minor nonspecificity exists for HA when used to characterize mutants with GC at the mutant site in Neurospora. The plating procedure for estimation of the viability of the treated and untreated eonidia and the revertion frequency has been described 21.
Statistical tests The tests for the significance of the difference between the spontaneous and induced reversion frequencies were performed according to BIRNBAUM1 and were briefly described elsewhere2L In previous attempts to determine the genetic alterations in ad-3B mutants by specific revertibility 23, a probability lower than 5% was chosen to indicate a significant difference between the reversion frequencies obtained in the control and in the treated series. Because there may be a certain degree of nonspecifity in the nmtagenicity of HA, the level of significance was decreased from 5°//o to I°',.o in the present analyses. R E S U LTS
The NA-indueed ad-3B mutants tested for specific revertibility in the plesent analysis were isolated by the direct method 4. They were induced by using different concentration of sodium nitrite at pH 4.6 and by using various treatment tinles. The detailed description of the method has been given previously 1°. The nmtation frequency of the ad-3B nmtants varied from approximately 3o'xo 6 to 13o-Io -6 per survivor according to the different treatments. The ad-3B mutants can either be noncomplementing, or complementing with either a nonpolarized or a polarized complenlentation pattern. A nmtant is classified as having a polarized complementation pattern when the pattern covers complon x and extends to different positions on the complementation map, usually beyond complon IO (ref. 8). The determination of the genetic alteration in the nmtants are done on the Mutation Res., 5 (1968) 3 5 9 - 3 7 E
362
H . V . MALLING, F. J. DE SERRES
b a s i s of b o t h p r e v i o u s r e v e r s i o n d a t a 2a a n d p r e s e n t d a t a b y f o l l o w i n g t h e r u l e s g i v e n i n T a b l e I. All t e s t s f o r r e v e r t i b i l i t y of t h e a d - 3 B m u t a n t s
by HA were made under the
of t h e a d - 3 B m u t a n t s
with HA
u n d e r t h e s e c o n d i t i o n s g a v e a n a v e r a g e s u r v i v a l of 4 9 . 9 % w i t h a s t a n d a r d
deviation
standard
c o n d i t i o n s d e s c r i b e d e a r l i e r 2:. T r e a t m e n t
"FABLE 1 A GUIDE FOR IDENTIFICATION OF THE GENETIC ALTERATIONSIN MUTANTS BY MEANS OF SPECIFIC REVERTIBILITY TESTS The classification of the m u t a n t s is based on calculations of the p r o b a b i l i t y t h a t the reversion frequencies obtained in the t r e a t e d series are the same as the reversion frequencies obtained in the controls. A p r o b a b i l i t y below ,9-°~.o(MALLING AND DF, SERRES=a) or : % (in the p r e s e n t paper) is considered significant.
Mutagenic treatment SPR NA EMS
ICR-±7o
Genetic alteration at mutant site; Mailing and De Serres (ref. 23)
Revertibility response to H A
Genetic alteration at mutant site; final score
*
-}-
t /o
+/o
BPS
:o
GC AT
*
(o)
o
o
BPS ?
t o
GC BPS ?
*
o
~
+ I,
BPS
+
o
GC BPS ?
t o
(;C BPS ?
5
o
** - / -
! o
(;C -/ - ?
t o
GC SI'
?
*
o
F
o
BPS ?
*
o
o
~H
-/--
*
o
o
+ 1~
+ /
+
o
o
o
SP
--
--
+ o
= ---+ / - - -BPS = SP = NON = ? = ** -* = L -14 -( ) -S P R --
?
NON
~ --
GC NON
F r e q u e n c y significantly higher t h a n t h a t occurring spontaneously. F r e q u e n c y n o t significantly higher t h a n t h a t occurring spontaneously. No r e v e r t a n t s detected. base-pair insertion or deletion. base-pair substitutions. the m u t a n t r e v e r t s only s p o n t a n e o u s l y . t h e m u t a n t does n o t revert. indicate u n c e r t a i n t y in the determination. did n o t occur. D a t a not summarized. Low reversion frequency. High reversion frequency. Reversion d a t a on the 5°o probability level. s p o n t a n e o u s reversion frequency.
of I6}/o . 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 m u t a n t s
revertible by HA obtained under
these conditions were usually less than : per :o 6 survivors (Tables II, III, IV). The mutants
which were characterized
deletion
mutants
either as nonrevertible
or base-pair insertion and
i n t h e p r e v i o u s t e s t 2a d i d n o t r e v e r t a f t e r H A t r e a t m e n t
(Tables
II, III, IV). F i v e of t h e m u t a n t s
with nonpolarized
-7:,-74,-:I8) which revert spontaneously,
Mutation Res., 5 (I968) 359-371
complementation
but not after treatment
patterns
(2-:7-2-,-5,
w i t h NA, E M S , or
COMPLEMENTATION
TABLE
PATTERNS
AND TYPE
OF BASE-PAIR
363
TRANSITION
n
REVERTIBILITY
AND DETERMINATION
WITH NONPOLARIZED
Mutant No.
OF T H E G E N E T I C A L T E R A T I O N IN N A - I N D U C E D
COMPLEMENTATION
Complon coverage
PATTERNS AFTER TREATMENT
Reversion per lO 6 survivors
ad- 31:3M U T A N T S
XVITH H A
SPR
HA
P
Genetic alteration at m u t a n t site 2VIalling and Final De Serres ( r @ 23) score
IO 9 2"1o a
BPS SP
GC GC
2--I 7- 1 5 8 118
i I
0.02 o.oi
0.59 0.07
85
2
o
o.I 5
lO -6
BPS
GC
76 8o 91 128
2 2 2 2
0.02 0.02 o.02 O.Ol
o.25 o.32 0.45 1.12
IO 8 IO 9 IO -9 lO -9
BPS BPS BPS BPS
GC GC GC GC
73 33
2 2
0.02 o.12
O. I I 0.60
2" 1 0 -4 lO -9
BPS BPS
GC GC
61
2-3
0.03
0.64
i o 10
BPS
GC
31 72 ioo 155
2-4 2 4 2 4 2- 4
o.o 5 0.03 0.02 o
0.36 0.32 0.20 o.21
io -s i o -~° l O -8 l O -6
BPS BPS BPS BPS
GC GC GC GC
39 95
2-5 2-5
o.or 0.03
0.07 0.23
3" lO-Z 6 ' lO -4
BPS ? BPS ?
GC GC
2 5 71 74
2-7 2 7 2- 7 2-7
0.03 0.02 O.OI o.oi
o. I I o.14 O.I I 0.09
9 ' lO-4 6'1o 4 2" 10 -3 IO a
SP SP SP SP
GC GC GC GC
9 21
2-II 2-11
O.ll o. I I
0.30 0.27
IO -~ IO -5
BPS BPS
GC GC
448
3-IO
0.02
o.33
7" 1 ° - a
BPS
GC
I22
3-11
0.02
o.o9
IO -6
BPS
GC
58 117 150
3-I2 3-12 3--12
0.o3 0.05 0.02
o.23 o.18 O.19
lO -8 l O -5 10 -6
BPS BPS BPS
GC GC GC
56 63 81 86 116 148
3-14 3-14 3-14 3-14 3-14 3 14
0.05 o.o 4 0.06 0.o3 0.02 0.02
O.ll o.19 0.20 o.13 0.08 0.27
8- lO a lO . 6 IO 6 2 • IO ,t 7" IO a l O -9
BPS BPS BPS BPS BPS BPS
GC GC GC GC GC GC GC
51
4-7
0.02
0.08
4" I o a
BPS ?
129
5-8
0.05
0.02
3" lO-1
BPS ?
AT
441
5-14
0.04
o.18
l O -5
BPS
GC
289 863
5 15 5-15
0.03 0.02
o.14 o.19
2"1o 3 lO -7
BPS BPS
GC GC
37 114 13o
6-7 7-9 8-14
o.oi o.14 0.08
o.91 o.61 0.45
I'IO 9 io s lO - l °
BPS ? BPS BPS
GC GC GC
17 22 79 156
9-11 9-11 9 1I 9-II
o.o2 0.02 0.05 0.04
0.29 o.21 o.15 0.30
lO - l ° 3-1o a 1"1o a IO s
BPS BPS BPS BPS
GC GC GC (;C
31ulation Res., 5 (1968) 3 5 9 37~
364
H.V.
TABLI'~
F. J. DE
SERRES
l I (continued)
J'VIulant No.
2
MALLING,
t7-24 08 lo0
Complon coverage
Reversiotzs p e r I,o ~; slcrvivors
Genetic alteration at m u t a n t site "
NPI?
HA
P
~Ialling and-De Serres ( r @ 23)
Final score
to-i[ to-I ]
0.05 o. 10
]o.-] I
6. Jo ~ 3" zo ~ lo u
BPS BPS I3PS ?
(;C AT (;C
0.0,
o. i 0 0.04 1.83
34
io
13
o.i()
o.41
4" i o - a
I:IPS
(;C
25
12
I0
O.OI
0.i2
29
12
I6
0
0.30
30
I2
tO
o.ol
0.38
35
I2
10
O.O1
0.22
75
12
z(>
o
o.3~
io lo ,o to lo
a ~ ~ s lo
I20
12
I2
O
O.10
lO
7
143
12
I()
0,0l
0.23
10 -9
BPS 13PS HPS 13PS I31)S [31'S HPS
(;( (;(" (;C (;C (;((;(" G("
164
12
l()
o.oJ
0.28
,o
BI)S
(;C
I4()
12
[0
O.O1
0,3()
9" ~o ~
131)S
(;("
94
14
17
0-03
o,i3
[ • lO a
BI)S
(;("
47
I0
l7
o.o2
o.18
5" ~o ~
131)S ?
(;C
82 99 112
I7
0.02
0.39 0. 4 ,
u 9
(;("
0.0 ~
6.1o4" i o
13I'S
17
I3I'S
(;C
[7
o.o2
0.27
2-[o
s
!'~>S
(;C
frequency
obtained
P = The probability that the reversion the SPI¢. F o r e x p l a n a t i o n , s e e T a b l e I.
,a
after
HA
c ~ c , z t m e n t , is t h e s a m e a s
ICR-I7 o-°a, reverted after HA treatment, but only at approximately one-third or less of the frequency obtained for many of the other mutants with nonpolarized complementation patterns. Since in each case the induced reversion frequency was significantly different from the control (P
The association between complementation patterns and genetic alterations of NA-induced mtt{at1[s
I d e n t i f i c a t i o n of the genetic a l t e r a t i o n s at the molecular level in N A - i n d u c e d m u t a n t s after t r e a t m e n t with NA, EMS, or ICR-x7o was r e p o r t e d previously'-% F r o m this s t u d y we concluded t h a t m u t a n t s with polarized c o m p l e m e n t a t i o n p a t t e r n s a n d n o n c o m p l e m e n t i n g m u t a n t s are d e r i v e d from a v a r i e t y of genetic alterations, whereas m u t a n t s with n o n p o l a r i z e d c o m p l e m e n t a t i o n p a t t e r n s are d e r i v e d p r e d o m i n a n t l y from 1)ase-pair s u b s t i t u t i o n s . In the present analysis we found t h a t H A i n d u c e d revert a n t s only in m u t a n t s which were c h a r a c t e r i z e d p r e v i o u s l y as b a s e - p a i r s u b s t i t u t i o n s or in n m t a n t s which r e v e r t e d only spontaneously. Of the n m t a n t s resulting from b a s e - p a i r s u b s t i t u t i o n (Table V), a significantly higher fraction of n o n p o l a r i z e d n m t a n t s r e v e r t e d after H A t r e a t m e n t t h a n did the polarized m u t a n t s (Z2 4.6, o.oe < P < o.o 5, Y a t e s correction included). An even g r e a t e r difference was found b e t w e e n the 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 in the nonc o m p l e i n e n t i n g a n d n o n p o l a r i z e d c o m p l e m e n t i n g classes (Z~ 28.2 ; P < o.ooI). W e f o u n d no significant difference b e t w e e n the fraction of H A - r e v e r t a b l e m u t a n t s a m o n g t h e polarized c o m p l e m e n t i n g a n d the n o n c o m p l e m e n t i n g m u t a n t s (Z2 -- ~.65, P o.2). Muia/io~z ICes., 5
( 1 9 ( ) 8 ) 35(-1 - ] ' ] [
COMPLEMENTATION
TABLE
PATTERNS
AND TYPE
OF BASE-PAIR
TRANSITION
365
III
R E V E R T I B I L I T Y AND DETERMINATION OF THE GENETIC ALTERATION IN N A - I N D U C E D WITH POLARIZED COMPLEMENTATION PATTERNS AETER TREATMENT WITH H A
Mutant No.
Complon coverage
2-17-98
Reversions per zo 6 survivors SPR
HA
P
MUTANTS
Genetic alteration at m u t a n t site M a l l i n g and Final De Serres (re/. 23) score
i-IO
O
0.587
4° 78 437
1-12 1-12 L-I2
o o o
o o o
I.OO i.oo i .oo
ISI
I--I2
O.I 5
0.02
I.OO
@/
59 163
1--13 I-I 3
O.OI o,oi
O o,Io
I.OO 9' 1°-5
BPS ? BPS
BPS ? G("
147 I98 498
1-14 1-14 1-14
o o o
o o o
I.OO I.OO I.OO
NON NON NON
NON NON NON
12 16 46
[-14 1-14 1-14
O.O1 0.02 o.oi
O,13 0,05 0,05
IO -5 5" I ° ~ 2 2 - IO -2
BPS BPS BPS
GC AT AT
131 213 264
1-14 1--14 1-14
O 0.03 o,oi
o O,O3 o.I I
I.OO 2 . 9 " IO -1 5" I ° - 4
BPS SP BPS
AT SP (;C
200 412 433
1-14 1--14 I-I4
0.02 O.OI 0.06
0.09 O.12 o.12
4" 1 ° - 3 i O -4 8 . IO -3
BPS BPS BPS ?
G(~', G(" GC
214
I-i 4
o.o6
o.o 4
2 . 3 ' IO -1
BPS ?
BPS ?
8 87 153 172
1-15 1-15 1-15 1--15
o.oi o.oi o.oi 0.03
o.o2 0.09 0.03 0.05
3.o" 9" 1.4" 8"
BPS ? SP -T / ? @ /- ?
BPS ? GC -- / ? ~/ ?
387
1-15
0.07
o.12
2.1 • lO `3
BPS
GC
For explanation,
TABLE
IO 5
ad-3B
lO -'1 lO-4 lO-1 10 -2
BPS
C;C
NON SP NON
N()N SP NON " /
s e e T a b l e s 1, I I .
IV
R E V E R T I B I L I T Y AND DETERMINATION OF THE GENETIC ALTERATION IN N.3t-INDUCED NONCOMP L E M E N T I N G ad-3B MUTANTS AFTER TREATMENT WITH H A
A . B a s e - p a i r substitutions Mutant No.
Reversions per zo 6 survivors
SPR
HA
P
2-17-142 149 166 230 45 96 127 228 14 18
o.12 o.16 0.09 0.o2 o.to o.o9 o 0.02 0.03 o.io
o o 0.38 o. I o 0.24 0.05 o 0.20 0.20 0.07
I.OO i.oo
For explanation,
see T a b l e s
lO -5 2.5.1o 3 lO -5 2 . 6 - IO 1 i .oo 4" IO 4 2 . lO -4 3" lO -1
Genetic alteration f or the reverse mutation mechanism Mal-ling and Final De Serres (ref. 23) score BPS BPS BPS ? BPS BPS BPS BPS BPS BPS BPS
AT AT GC GC GC AT AT GC GC AT
I, I I ,
M u t a t i o n Res., 5 ( 1 9 6 8 ) 3 5 9 - 3 7 1
366
H.V.
TABLE
MALLING,
F. J. D E S E R R E S
IV (continued)
~Iutanl
Reversions per z o ~ surviw)rs
No.
--
2 I7-26 1o5 I20 ~30 138 159 173 ~9 o 212 490
Genetic alteratio.n j o t the reverse mutalion mechanism M a i l i n g a;id -- Fina)-De Serres (r @ z3) score
. . . . . . . .
SPR
HA
P
0.08 o.t5 o.e8 u.o2 O,12 0.06 0.04 o.oz 0.07 0.23
o 0.20 0.09 o.ol 0.03 0.05 o. 1Z o.-, 7 0.20 o.1 7
I.OO 4'1o 4.(3" IO 5" IO 2 . 8 ' IO 2.2' io 8 " IO ~o 4.2' Io 2.0' to
BPS BPS BPS HPS BPS BI'S BPS BPS BPS f3PS
a '~ ~ 1 * 4 0 4 1
AT (;C :V|" AT AT AT (;C (;C (;C AT
13. Base-pa~r insertion or deletions aim ~,tnidentified alterations 2-~7-55 57 (17 too t8o :z, 240
o O o o o o o
o O o o o o o
t.oo I,OO ,.oo I .oo I.OO ~.oo 1.o()
zI8 lo 23 2[ I 217
o o o 0.00 o.oI
o.oI o o o 0.03
3.,'to I .oo 1.0o
I3 30 48 88 ~3-" 1()2
o o o.u [ o.o2 o-07 O
o.04 o
5-7"[o t.oo
o
I.oo
o o O
I.oo i.oo 1.OO
() 7 83 lol 22o
u.ol 0.04 o.o, 0.07 0.03
o.ol 0.05 0.04 (/.o3 o.o6
5"IO I.IO 2.~o 2. 4 - i o 3"[o
For explanation,
see T a b l e s
[.oo 0-to
1
1
NON N()N NON NON NON NON N()N
N()N N()N N()N N()N N()N NON NON
SP .q]) ~P SP SP
SI' SI' SI' S1 ) St'
t / t /
:/
t / T/ : /
i/
2
t/
: / t ! t 4 I
~ t i 1 2
,/
/ / / / /--
/ I/I/
1, I I.
V
TABLE
THE PERCENT OF THE DIFFERENT TYPES OF NA-INDUCED a d - 3 B MUTANTS OF BY THEIR COMPLEMENTAT[ON PATTERN AND TYPE OF GENETIC ALTERATION
Complementation pallern O,pe
Nonpolarized Polarized Noncomplementing Total
60.4 I L.7 27. 9 mo.o
Previous Note:
Percenlage
data
Total mutanls a n a lvzed 63 25 43
2. T (2) 1.4 (3) 7.2 ( I X )
I31
lo. 7
in parenthesis
58.3
(6I)
4..3 (9) 5.8 (9) 68.4
BP,g?
13ase-pair insertion or delelion
Unidentified Reverting Nonspontaneously reverting
O
O
O
(O)
is t h e a c t u a l n u m b e r
3qutation l?es., 5 ( 1 9 6 8 ) 3 5 9 371
(O)
(O)
O
(O)
L4 (3) o (o)
1.4 (3) 7.2 (11)
0.9 (2) .3.2 (5)
e.3 (5) 4-5 (7)
~.4
8.6
4.t
6.8
8o. 5 84
(I~IALLING22)
The number
Base-pair transition i~iutanl ~ wild type ~qT;Z;GC GC ~ -AT
:\Teztrospora crassa C H A R A C T E R I Z E D
of mutants
12. 7 1o
found.
6
COMPLEMENTATION PATTERNS AND TYPE OF BASE-PAIR TRANSITION
367
DISCUSSION
The general spectrum of the genetic alterations induced by NA (Table V) Determination of the general spectrum of genetic alterations induced b y a mutagen b y means of specific revertibility studies can be greatly influenced b y (I) the occurrence of suppressors, (2) hot spots, and (3) the base ratio in the gene.
The influence of suppressors Suppressors can be located either inside the gene which they suppress (intralocal suppressors) or elsewhere in the genome (extragenic suppressors). Extragenic suppressors are extremely rare among the revertants of ad-3B mutants 7,17 (BARNETT, unpublished; see MALLING2°). In E. coli some base-pair substitutions can revert by intralocal suppressors which also are base-pair substitutions 3~. A m u t a n t with an AT pair at the m u t a n t site might also revert b y a GC --> AT base-pair transition at another site (intralocal suppressor) and vice versa for a m u t a n t with a GC pair at the m u t a n t site. A mutagen which would react preferentially with AT base-pairs would, in theory, make it possible to distinguish between these different mechanisms. If we assume that almost all nonpolarized m u t a n t s are able to revert by intralocal suppressors (induced b y a base-pair transition from GC -~ AT), then all nonpolarized mutants, independent of mutagenic origin, should revert after HA treatment. However, since a much lower percentage of HA-induced ad-3B mutants with nonpolarized complementation patterns than of NA-induced mutants with the same type of pattern reverted after HA-treatment (MALLING, unpublished), it seems highly unlikely that induction of intralocal suppressors is the predominant reversion mechanism for nonpolarized ad-3B mutants.
Hot spots A previous study ~3 showed that mutants which covered complons 2- 4 have nearly the same reversion frequency after treatment with NA, EMS, or ICR-I7O. We found that all these mutants reverted with approximately the same frequency after HA-treatment. These new data support the hypothesis that these mutants might have been induced at a single site (i.e., hot spot). This was also true for the other hot spots, namely sites for mutants with complementation patterns 3-14 (6 mutants) and 12-16 (8 mutants) (Table I). All these m u t a n t s reverted b y a GC --~ AT transition. Relatively few of the polarized and noncomplementing base-pair substitution mutants have identical induced-reversion frequencies. I t seems likely then that most of the 98 NA-induced base-pair transition mutants result from alteration of different sites in the ad-3B locus.
Base ration in the DNA The high content of GC-base-pairs at the m u t a n t site among the NA-induced
ad-3B mutants in Neurospora crassa could result from a high content of AT pairs in the DNA of the ad-3B locus. However, this possibility is not likely because the ratio between the base-pairs (GC/AT) in the DNA in Neurospora is approximately I.I625,~7 ; and since the ad-3B locus specifies a structural protein 11, the simplest assumption is that the DNA in the ad-3B locus has the same base ratio as the total DNA. F~om this discussion we can conclude that NA hot spots, suppressors, and the
Mutation Res., 5 (1968) 359-37I
368
H . V . MALLING, F. J. DE SERRES
base-ratio of the D N A do not have a predominant influence on the determination of the general spectrum of genetic alterations induced b y NA. The general spectrum of genetic alterations within the NA-induced n m t a n t s are given in Table V and the results 23 have previously been discussed in detail. The numbers in the table agree with a previous analysis of NA-induced ad-3 m u t a n t s in a microconidial strain of Neurospora~°. The numbers obtained from this and previous analyses are included in Table V. The most striking and unexpected results are the high percentage of m u t a n t s with GC pairs at the m u t a n t site (Table VI [86.5°,~ (68.4/79.1) of the total n u m b e r of base-pair transition@ Our finding is not in accord with the findings (~) that the relative rate of deamination b y NA of the bases in T2 and "1"4D N A is A : C or HMC : G -I:6.3:I6~5,3x; and (2) t h a t the base ratio in phage T2 and T 4 D N A GC/AT o.515 [which deviates greatly from Neurospora (GC/AT = I . I 6 ) 2 . Nevertheless, a high percentage of the NA-induced n m t a n t s in "F4 bacteriophage has been identified as having AT at the m u t a n t site ~. (For review see KRIE6~.) TABLE
V1
CORRELATION BASE-PAIR
BETWEEN SUBSTITUTIONS
THE
TYPI';
IN
THE
OF
COMPLEMENTATION
ad-313 R E G I O N
OF
PATTERN
AND
THE
DIRECTION
OF
THE
Neurospora c r a s s a
F r e q u e n c e in p e r c e u t w i t h i n each c o m p l e m e n t a t i o n class.
Complementation pattern
Nonpolarized Polarized Nonconlplementing
Direction of base-pair transition in reverse mz~lations A 7" -~ (;C (;C --~ .4 T ~ P ) 5°~,
5'~o
3 (2) 16 (2) 5 o (lo)
O
'~ (~) 5 (~)
P)
I°b I°;i P ) I 9 (I2) -'5 (3) i (2)
o.I°~,
Total
P
tOO (63) too (r2) ioo (20)
P : is t h e p r o b a b i l i t y t h a t t h e r e v e r s i o n f r e q u e n c y o b t a i n e d a f t e r H A t r e a t m e n t is t h e s a m e as t h e r e v e r s i o n f r e q u e n c y in t h e c o n t r o l ( B i r n b a u m ' s test). N o t e : T h e n u m b e r in p a r e n t h e s i s is t h e a c t u a l n u m b e r of m u t a n t s in e a c h c a t e g o r y f o u n d .
The high frequency of GC at the n m t a n t site a m o n g the NA-induced ad-3B m u t a n t s in Neurospora m a y be explained b y the following observations: (z) After 2o h t r e a t m e n t of bacteriophage T2 with I M NaNO2 at pH 4.2, 35°J/o of the guanine, 13°,/o of the cytosine, and 3°/'o of the adenine in the D N A were deaminated ~1. W h e n pneumococcal D N A was treated i h with NA under the same conditions (sufficient to inactivate 99.9~'/o of the transforming activity), only IO I5~o of the guanine and 1-2°,"o of adenine in the D N A were deaminated. (No deamination of cytosine was detectable.) (2) The m u t a n t s in Neuro@ora were obtained after t r e a t m e n t of conidia with either o.oo 5 or o.oi M NaNO2 at p H 4.6 (concentration of HNO~:3.2- IO ~ or 6. 4. xo 4 M) ~0 for 16o min. The mutation frequency was found to be 3 o - 1 3 o ' x o -6 per survivor. Since LITMaN'S treatmenO 9 conditions are closer to the treatment conditions used in the Neurospora experiment than the t r e a t m e n t conditions used b y VIELMETTER AND SCHUSTERal in the phage experiment, we think t h a t it is reasonable to expect that the NA-induced m u t a n t s in Neurospora would have predominantly a GC base-pair at the m u t a n t site. The NA-induced r l I m u t a n t s in bacteriophage T 4 (ref. 13) were isolated io rain after t r e a t m e n t with 2 M NaNO2 at p H 5.3 (concentration of HNO2 = 2.5" Io -2 31). Mutation Res., 5 (i968) 359-371
C O M P L E M E N T A T I O N P A T T E R N S A N D T Y P E OF B A S E - P A I R T R A N S I T I O N
369
Therefore we would expect that the frequency of AT at the mutants site should be higher among the r I I mutants in bacteriophage T 4 than among the ad-3B mutants in Neurospora. On the basis of LITMAX's data, it is somewhat surprising that mutants with AT at the mutant site are found among NA-induced mutants. The NA treatments in the bacteriophage and Neurospora experiments were performed with nmeh less concentrated solution of HNO~ and/or shorter treatment time than that which gave no reaction between NA and cytosine in pneumococcal DNA in LITMAN'Sexperiments; this is especially significant since the deamination of guanine does not produce GC to AT base-pair transition aa. Mutants derived from GC to AT base-pair transitions can be nonsense mutations (see below). Mutants derived from an AT to GC base-pair transition cannot be nonsense mutations. Because nonsense mutation will result in formation of a polypeptide chain fragment, they are not likely to be leaky. However, a large fraction of mutants resulting from missense mutation are leaky. Thus, mutants with AT at the mutant site may have a higher probability of recovery than mutants with GC at the mutant site. This may explain why mutants with AT at the mutant site are recovered at unexpectedly high frequency after NA-treatment in both bacteriophage T4 and
Neurospora. Association between type of complernentation and type of base-pair at mutant site From our earlier study of the association between complementation patterns and genetic alterations of NA-indueed mutants 23, we determined that base-pair substitution mutants with polarized complementation patterns and noncomplementing base-pair substitution mutants were either nonsense mutations which give rise to polypeptide fragments or missense mutations of an amino acid essential for the tertiary structure of the protein. The RNA nonsense triplets in some strains of E. coli are UAA, UAG, and UGA2,3,32; the analogous codons and anticodons in the DNA are respectively ATT/ TAA, ATC/TAG and TCA/AGT. Since HA only reacts with C, it can induce transitions in the last two triplets; but both of these reactions yield ATT which also is a nonsense triplet. Consequently we may conclude that nonsense triplets are not able to revert by a GC to AT transition. It has been shown eonvincinglyl~, 26 that mutants carrying a nonsense mutation form only a part of the polypeptide chain specified b y the gene, and that all fragments have the same end in common. If the polypeptide fragment is large enough to show allelic complementation, nonsense mutations would be expected to lead to nmtants with a polarized complementation pattern. The fact that the base-pair AT occurs more frequently at the mutant site among mutants with polarized complementation patterns and among noncomplementing mutants than among the mutants with nonpolarized complementation patterns is interpreted as indicating that the first two classes of mutant are derived more frequently from nonsense mutations than the latter. Supersuppressors have now been found in Neurospora ~8. Since supersuppressors most likely suppress nonsense mutations, a direct, independent test of our interpretation of these data can be made by studying the ability of supersuppressors to suppress ad-3B mutants as a function of their complementation pattern. We are now attempting to study this correlation for the ad-3B mutants. .VIutation Res., 5 (~968) 3 5 9 - 3 7 1
370
H. V. MALLING, F. J. D E S E R R E S
Comparative mutagenesis studies The data from the present study on the revertibility of NA-induced base-pair substitution after treatment with HA shows that the ratio of mutants with the two types of base-pairs at the m u t a n t site (AT/GC) varied between the three different classes of complementation pattern (Table IV). This can be explained in part by the simple fact that a base-pair transition can only induce a nonsense triplet by a GC -~ AT transition, and not b y a AT -+ GC transition. The present data have especially interesting implications with regard to the diagnostic capability of the relatively simple test for allelic complementation on samples of ad-3B mutants induced by different mutagens. For example, a mutagen causing AT to GC base-pair transitions would produce a sample showing (I) a very high percentage of mutants able to show allelic complementation and (2) most or all of these mutants having nonpolarized complementation patterns. By contrast, a mutagen producing GC to AT base-pair transitions should induce mutants with (r) a much lower percentage allelic complementation and (2) a high frequency of mutants with polarized complementation patterns. Present data on allelic complemenration among MNNG provide an exact fit to first example 24, whereas the data on allelic complementation among HA-induced mutants provide an exact fit to the second (MALLINGAND DE SERRES, unpublished). ACKNOWLEDGEMENTS
The authors wish to thank Dr. M. A. KASTENBAUM for his help in preparing the statistical analysis, and Mrs. ARLEE TEASLEY for her valuable technical assistance.
RI,H"I';R1LNCES I BIRNBAUM, A., Statistical m e t h o d s for Poisson processes and exponential populations, J. Am. Statist. Assoc., 49 (1954) 254. 2 BRENNER, A., A. O. W. STRATTON AND S. KAPLAN, Genetic code: The " n o n s e n s e " triplets for chain t e r m i n a t i o n and their suppression, Nature, 206 (1965) 994 998. 3 BRENNER, S., L. BARNETT, E. R. KATZ AND I i'. H. C. CRICK, UGA: A third nonsense triplet in the genetic code, Nature, 213 (1967) 449 45 °. 4 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 by 2-aminopurine, Genetics, 48 (1963) 597-6o4. 5 BROWN, D. M., AND P. SCFIELL, The reaction of h y d r o x y l a m i n e w i t h cytosine and related conlpounds, J. :lIol. Biol., 3 (I961) 7o9. 6 CHAMPE, S. P., AND S. BENZER, Reversal of n l u t a n t p h e n o - t y p e s by 5-fluorouracil, an a p p r o a c h to nucleotide sequences in m e s s e n g e r - R N A , Proc, Natl. Acad. Sci. (U.S.), 48 (1962) 532-546. 7 DE SERRES, F. J., Studies with purple adenine m u t a n t s in Neurospora crassa, I I I . Reversions of X - r a y - i n d u c e d m u t a n t s , Genetics, 43 (1958) 187-2o6. 8 DE SERRES, F. J., Mutagenesis and c h r o m o s o m e structure, J. Cellular Comp. Physiol., 64, Suppl. I (1964) 33-42. 9 DE SERRES, F. J., The utilization of leaky ad-3 m u t a n t s of Ne~rospora crassa 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 , Mutation Res., 3 (1966) 3-12. IV DE SERRES, F. J., H. E. BROCKMAN, W. E. BARNETT AND H. G. KOLMARK, Allelic complenlentatiou anlong n i t r o u s acid-induced ad-3 B m u t a n t s of Neurospora crassa, Mutation Res., 4 (1967) 415 424 • I I FISHER, C. R., D e t e r m i n a t i o n of the enzymatic functions controlled b y ad-3A and ad-3B loci in Neurospora crassa, Genetics, 56 (1967) 560 (Abstract). I2 JL?O\VLER, n . V., AND I. ZABIN, Co-linearity of fl-galactosidase w i t h its gene b y inlmunological detection of incomplete polypeptide chains, Science, 154 (1966) IO27-IO29. 13 15"REESE, F~., On the molecular e x p l a n a t i o n s of s p o n t a n e o u s and induced m u t a t i o n s , Brookhaven 5;ymposia in Biology, No. 12 (1959) 63-73.
Mutation Res., 5(1968) 359-371
COMPLEMENTATION PATTERNS AND TYPE OF BASE-PAIR TRANSITION
371
14 FREESE, E., E. BAUTZ AND E. B. FREESE, The chemical and m u t a g e n i c specificity of h y d r o x y l amine, Proc. Natl. Acad. Sci. (U.S.), 47 (1961) 845-855. 15 FREESE, E., E. BAUTZ-FREESE AND E. BAUTZ, H y d r o x y l a m i n e as a mutagenic and i n a c t i v a t i n g agent, J. Mol. Biol., 3 (196I) 133-143. 16 FREESE, E., AND E. B. FREESE, The o x y g e n effect on deoxyribonucleic acid inactivation b y h y d r o x y l a m i n e s , Biochemistry, 4 (1965) 2419 2433. 17 I~OLMARK, G., AND •. H. GILES, C o m p a r a t i v e studies on monoepoxides as inducers of reverse m u t a t i o n s in Neurospora, Genetics, 4 ° (1955) 890 902. 18 KRIEG, D. R., Specificity of chemical mutagenesis, Progr. Nucleic Acid Res., 2 (1963) I 2 5 - I 0 8 . 19 LITMAN, R. M., Genetic a n d chemical alterations in the t r a n s f o r m i n g D N A of JPneumococcus caused b y ultraviolet light and by n i t r o u s acid, J. Chim. Phys., 58 (1961) 997 lOO4. 20 MALLING, H. V., Identification of the genetic alteration in n i t r o u s acid-induced m u t a n t s of Neurospora crassa, Mutation Res., 2 (1965) 320-327. 21 MALLING, H. V., H y d r o x y l a m i n e as a mutagenic agent for Neurospora crassa, Mutation Res., 3 (1966) 470-476. 22 MALLING, H. V., Mutagenicity of m e t h y l h y d r o x y l a m i n e s in Neurospora crassa 3/lutation Res., 4 (1967) 265-274. 23 MALLING, H. V., AND F. J. DE SERRES, Relation b e t w e e n 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 n i t r o u s acid-induced ad-3B m u t a n t s of Neurospora crassa, Mutation Res., 4 (1967) 425-44 °. 24 MALLING, H. V., AND F. J. DE SERRES, N-Methyl-N'-nitroso-N-nitroguanidine (MNNG) as a mutagenic agent for Neurospora crassa, Genetics, 56 (1967) 575 (Abstract). 25 MINAGAWA, T., B. WAGNER AND B. STR#_USS, The nucleic acid c o n t e n t of Neurospora crassa, Arch. Biochem. Biophys., 80 (1959) 442-445 • 26 SARABHAI, z~k.S., A. D. STRETTON, S. BRENNER AND A. BOLLE, Co-linearity of the gene with the polypeptide chain, Nature, 2Ol (1964) 13-17. 27 SCHILDKRAUT, C. L., J. MARMUR AND P. DOTY, D e t e r m i n a t i o n of the base cmnposition of deoxyribonucleic acid from its b u o y a n t density in CsC1, J. Mol. Biol., 4 (1962) 430-443. 28 SEALE, T. W., Evidence for s u p e r s u p p r e s s o r s in Neurospora, Genetics, 56 (1967) 586 (Abstract). 29 STRACK, H. B., E. B. FREESE AND E. FREESE, Comparison of m u t a t i o n and inactivation rates induced in bacteriophage and t r a n s f o r m i n g D N A b y various mutagens, Mutation Res., i (i964) io-2I. 3 ° TESSMAN, I., H. ISHIWA AND S. ]~UMAR, Mutagenic effect of h y d r o x y l a m i n e in vivo, Science, 148 (1965) 5o7-5o8. 31 VIELMETTER, W., AND H. SCHUSTER, Die ]3asenspezifitXt bei d e r I n d u k t i o n von M u t a t i o n e n durch salpetrige SXure im P h a g e n T2, Z. Natur[orsch., I 5 b (196o) 304 31I. 32 WEIGERT, M. G., AND A. GAREN, Base composition of nonsense condons in E. coli, Evidence f r o m amino-acid s u b s t i t u t i o n s at a t r y p t o p h a n site in alkaline p h o s p h a t a s e , Nature, 206 (1965) 992-994. 33 YANOFSKY, C., Possible R N A code w o r d s for the eight a m i n o acids t h a t can o c c u p y one position in the t r y p t o p h a n s y n t h e t a s e A protein, Biochem. Biophys. Res. Commun., 18 (1965) 898-909.
Mutation Res., 5 (1968) 359-371
359
C O R R E L A T I O N B E T W E E N B A S E - P A I R T R A N S I T I O N AND COMPLEMENT A T I O N P A T T E R N IN N I T R O U S A C I D - I N D U C E D NEUROSPORA CRASSA*
ad-3B MUTANTS OF
H. V. MALLING AND F. J. DE S E R R E S Biology Division, N C I - A E C Carcinogenesis Program, Oak Ridge National Laboratory, Oah Ridge, Tenn. (U.S.A.) (Received March I2th, 1968)
SUMMARY
Nitrous acid-induced ad-3B mutants of Neurospora crassa were tested for revertibility after treatment with hydroxylamine (HA). Mutants which reverted after the treatment were classified as having guanine-cytosine (GC) at the m u t a n t site. A high frequency (86.5%) of nitrous acid-induced ad-3B mutants resulting from base-pair transitions have GC at the n m t a n t site, whereas in bacteriophage T4 most nitrous acid-induced r l I mutants have adenine thymine (AT) at the m u t a n t site. This apparent contradiction is discussed. In a random sampling of base-pair transition ad-3B mutants, we found among mutants with nonpolarized complementation pattern a lower incidence (2.3%) of AT at the m u t a n t site than among mutants with polarized complementation pattern (25.0%) and among noncomplementing mutants (55.0°/'0). Nonsense mutants, which specify the RNA triplets UAA, UAG, or UGA, and presumably the DNA anticodons ATT, ATC or ACT, should not be able to revert by a GC -+, AT transition, because by reaction with hydroxylamine the two C-containing triplets would be transformed to ATT, a nonsense triplet. The difference in the frequency of the GC pairs at n m t a n t sites among tile three different classes of complementation response indicates that nonsense mutations occur more frequently among mutants with polarized complementation patterns and among nonconiplementing mutants than among mutants with nonpolarized complementation patterns.
INTRODUCTION
Earlier studies of allelic complementation among the ad-3B mutants of Neurospora crassaS,9 showed that these m u t a n t s could be classified as (z) noncomplenlenting * Research jointly sponsored by the National Cancer Institute, and the U.S. Atomic Energy Commision under contract with the Union Carbide Corporation. Abbreviations: A, adenine; AT, adenine-thymine; C, cytosine; EMS, ethyl methanesulfonate; G, guanine; GC, guanine-cytosine; HA, hydroxylamine; HMC, hydroxymethylcytosine: I CR- t 7o, 2-methoxy-6-chloro-9- E3-ethyl- 2-chloroethyl) aminopropylanlino]acridine dihydrochloride; MNNG, N-nlethyl-N'-nitro-N-nitrosoguanidine; NA, nitrous acid; T, thymine.
Mutation Res., 5 (1968) 359-371
360
H . V . MALLING, F. J. DE SERRES
or (2) complementing with either (a) nonpolarized or (b) polarized complementation patterns. A striking correlation was found between the type of mutagens used to induce the nmtants and the spectrmn of eomplementation patternsS,~0; a similar correlation was found between the type of mutagens used to induce the mutants and the percent leakiness 9. This suggested that the type of eomplementation pattern was correlated with the type of genetic alteration, and the analysis of tim genetic alterations in NA-induced ad-3B nmtants by means of tests for specific revertibility provided conclusive proof 2a. In the latter analysis the ad-3B mutants with nonpolarized con> plementation patterns were shown to result mainly from base-pair substitutions, whereas mutants with polarized complementation patterns and noncomplementing mutants are derived from a variety of genetic alterations. In T4 bacteriophage, HA induced base-pair transitions from GC to AT ~4. In Neurospora, HA induces reversion only in some of the nmtants which revert by basepair transition, and never in those nmtants which revert by a base-pair insertion or deletion2~, °-~-. Later analysis of HA-induced ad-3B nmtants by specific revertibility test have shown that the majority of the mutants have AT at mutant site (MALLIN6, unpublished). Therefore, we have concluded that HA induces base-pair transitions preferentially from GC to AT in Neurospora and can be used in tests for specific revertibility to determine the direction of the base-pair transition. In this paper, our previous attempts both to identify the genetic alterations among NA-induced ad-3B mutants and to correlate genetic alteration with complementation pattern have been further extended by the identification of the two types of base-pair transitions within these three classes of mutants. The present data show that base-pair transitions of the type AT to GC give rise more frequently to ad-3B nmtants with nonpolarized complementation patterns, whereas GC to AT transitions give rise more frequently to mutants with polarized complementation patterns and noncomplementing mutants. The significance of these observations is discussed in relation to what is known about the genetic code for nonsense nmtations. MATERIALS AND METHODS
All ad-3B nmtants were isolated by the direct method 4 after NA treatment of the wild-type strain OR 74 Al°. The treatment conditions, survival levels, forward mutation frequencies, and complementation patterns of the mutants are described by DE SERRES et al) ° and MALLINGAND DE SERRES2a. All tieatments were carried out on freshly harvested suspensions of conidia as described by MALLING21.
HA treatment The procedure for treatment with HA has been described previously =1. In brief, the eonidia were treated for 300 rain with I M HA. HC1 (Baker Chemical Co., Phillipsburg, N.J.) dissolved in a 3 M NaC1 solution and adjusted to pH 6.2 ~.9.
The specificity of HA The revertibility of NA-induced mutants was tested after treatment with HA to attempt to identify tile direction of the base-pair transition, leading to the reverse mutation. The r I I mutants induced by HA in T~ bacteriophage are randomly distribMulalio*e Res., 5 (I968) 359-371
COMPLEMENTATION PATTERNS AND TYPE OF BASE-PAIR TRANSITION
361
uted] on the genetic map of the r I I locusX5,tL Therefore the base-pair sequence in the DNA seems to have no influence on the reaction of HA with DNA; but, on the other hand, nothing is known about how this reaction is influenced by the protein in the chromosomes of eukaryotes. Dilute solutions of HA (IO a M) in combination with oxygen give rise to oxidation products which showed a strong inactivating effect on the transforming DNA of B. subtilis ~6. At higher concentrations of HA ( > o . I M), these oxidation products were not formed. In vivo experiments 3° with the single stranded DNA phage @XI74 ) treated with lower concentrations of HA (2.5" IO-2 M) indicated that HA presumably reacted with all 4 bases since it was found that mutants resulted from base-pair transitions in both directions. Under the treatment conditions used in the present tests for specific revertibility on Neurospora, HA should react predominantly with C in the DNA ~4. However, even under these conditions, there is evidence 5 that a small amount of thymine is lost when DNA is treated with HA. Studies of HA-induced ad-3B inutants have shown that a minor fraction of the HA-induced nmtants are able to revert after treatment with O-methylhydroxylamine (MALLIN6, unpublished), which in transforming principles is assumed to be more specific than HA ~6 in the reaction with C in the DNA. This indicates that a minor nonspecificity exists for HA when used to characterize mutants with GC at the mutant site in Neurospora. The plating procedure for estimation of the viability of the treated and untreated eonidia and the revertion frequency has been described 21.
Statistical tests The tests for the significance of the difference between the spontaneous and induced reversion frequencies were performed according to BIRNBAUM1 and were briefly described elsewhere2L In previous attempts to determine the genetic alterations in ad-3B mutants by specific revertibility 23, a probability lower than 5% was chosen to indicate a significant difference between the reversion frequencies obtained in the control and in the treated series. Because there may be a certain degree of nonspecifity in the nmtagenicity of HA, the level of significance was decreased from 5°//o to I°',.o in the present analyses. R E S U LTS
The NA-indueed ad-3B mutants tested for specific revertibility in the plesent analysis were isolated by the direct method 4. They were induced by using different concentration of sodium nitrite at pH 4.6 and by using various treatment tinles. The detailed description of the method has been given previously 1°. The nmtation frequency of the ad-3B nmtants varied from approximately 3o'xo 6 to 13o-Io -6 per survivor according to the different treatments. The ad-3B mutants can either be noncomplementing, or complementing with either a nonpolarized or a polarized complenlentation pattern. A nmtant is classified as having a polarized complementation pattern when the pattern covers complon x and extends to different positions on the complementation map, usually beyond complon IO (ref. 8). The determination of the genetic alteration in the nmtants are done on the Mutation Res., 5 (1968) 3 5 9 - 3 7 E
362
H . V . MALLING, F. J. DE SERRES
b a s i s of b o t h p r e v i o u s r e v e r s i o n d a t a 2a a n d p r e s e n t d a t a b y f o l l o w i n g t h e r u l e s g i v e n i n T a b l e I. All t e s t s f o r r e v e r t i b i l i t y of t h e a d - 3 B m u t a n t s
by HA were made under the
of t h e a d - 3 B m u t a n t s
with HA
u n d e r t h e s e c o n d i t i o n s g a v e a n a v e r a g e s u r v i v a l of 4 9 . 9 % w i t h a s t a n d a r d
deviation
standard
c o n d i t i o n s d e s c r i b e d e a r l i e r 2:. T r e a t m e n t
"FABLE 1 A GUIDE FOR IDENTIFICATION OF THE GENETIC ALTERATIONSIN MUTANTS BY MEANS OF SPECIFIC REVERTIBILITY TESTS The classification of the m u t a n t s is based on calculations of the p r o b a b i l i t y t h a t the reversion frequencies obtained in the t r e a t e d series are the same as the reversion frequencies obtained in the controls. A p r o b a b i l i t y below ,9-°~.o(MALLING AND DF, SERRES=a) or : % (in the p r e s e n t paper) is considered significant.
Mutagenic treatment SPR NA EMS
ICR-±7o
Genetic alteration at mutant site; Mailing and De Serres (ref. 23)
Revertibility response to H A
Genetic alteration at mutant site; final score
*
-}-
t /o
+/o
BPS
:o
GC AT
*
(o)
o
o
BPS ?
t o
GC BPS ?
*
o
~
+ I,
BPS
+
o
GC BPS ?
t o
(;C BPS ?
5
o
** - / -
! o
(;C -/ - ?
t o
GC SI'
?
*
o
F
o
BPS ?
*
o
o
~H
-/--
*
o
o
+ 1~
+ /
+
o
o
o
SP
--
--
+ o
= ---+ / - - -BPS = SP = NON = ? = ** -* = L -14 -( ) -S P R --
?
NON
~ --
GC NON
F r e q u e n c y significantly higher t h a n t h a t occurring spontaneously. F r e q u e n c y n o t significantly higher t h a n t h a t occurring spontaneously. No r e v e r t a n t s detected. base-pair insertion or deletion. base-pair substitutions. the m u t a n t r e v e r t s only s p o n t a n e o u s l y . t h e m u t a n t does n o t revert. indicate u n c e r t a i n t y in the determination. did n o t occur. D a t a not summarized. Low reversion frequency. High reversion frequency. Reversion d a t a on the 5°o probability level. s p o n t a n e o u s reversion frequency.
of I6}/o . 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 m u t a n t s
revertible by HA obtained under
these conditions were usually less than : per :o 6 survivors (Tables II, III, IV). The mutants
which were characterized
deletion
mutants
either as nonrevertible
or base-pair insertion and
i n t h e p r e v i o u s t e s t 2a d i d n o t r e v e r t a f t e r H A t r e a t m e n t
(Tables
II, III, IV). F i v e of t h e m u t a n t s
with nonpolarized
-7:,-74,-:I8) which revert spontaneously,
Mutation Res., 5 (I968) 359-371
complementation
but not after treatment
patterns
(2-:7-2-,-5,
w i t h NA, E M S , or
COMPLEMENTATION
TABLE
PATTERNS
AND TYPE
OF BASE-PAIR
363
TRANSITION
n
REVERTIBILITY
AND DETERMINATION
WITH NONPOLARIZED
Mutant No.
OF T H E G E N E T I C A L T E R A T I O N IN N A - I N D U C E D
COMPLEMENTATION
Complon coverage
PATTERNS AFTER TREATMENT
Reversion per lO 6 survivors
ad- 31:3M U T A N T S
XVITH H A
SPR
HA
P
Genetic alteration at m u t a n t site 2VIalling and Final De Serres ( r @ 23) score
IO 9 2"1o a
BPS SP
GC GC
2--I 7- 1 5 8 118
i I
0.02 o.oi
0.59 0.07
85
2
o
o.I 5
lO -6
BPS
GC
76 8o 91 128
2 2 2 2
0.02 0.02 o.02 O.Ol
o.25 o.32 0.45 1.12
IO 8 IO 9 IO -9 lO -9
BPS BPS BPS BPS
GC GC GC GC
73 33
2 2
0.02 o.12
O. I I 0.60
2" 1 0 -4 lO -9
BPS BPS
GC GC
61
2-3
0.03
0.64
i o 10
BPS
GC
31 72 ioo 155
2-4 2 4 2 4 2- 4
o.o 5 0.03 0.02 o
0.36 0.32 0.20 o.21
io -s i o -~° l O -8 l O -6
BPS BPS BPS BPS
GC GC GC GC
39 95
2-5 2-5
o.or 0.03
0.07 0.23
3" lO-Z 6 ' lO -4
BPS ? BPS ?
GC GC
2 5 71 74
2-7 2 7 2- 7 2-7
0.03 0.02 O.OI o.oi
o. I I o.14 O.I I 0.09
9 ' lO-4 6'1o 4 2" 10 -3 IO a
SP SP SP SP
GC GC GC GC
9 21
2-II 2-11
O.ll o. I I
0.30 0.27
IO -~ IO -5
BPS BPS
GC GC
448
3-IO
0.02
o.33
7" 1 ° - a
BPS
GC
I22
3-11
0.02
o.o9
IO -6
BPS
GC
58 117 150
3-I2 3-12 3--12
0.o3 0.05 0.02
o.23 o.18 O.19
lO -8 l O -5 10 -6
BPS BPS BPS
GC GC GC
56 63 81 86 116 148
3-14 3-14 3-14 3-14 3-14 3 14
0.05 o.o 4 0.06 0.o3 0.02 0.02
O.ll o.19 0.20 o.13 0.08 0.27
8- lO a lO . 6 IO 6 2 • IO ,t 7" IO a l O -9
BPS BPS BPS BPS BPS BPS
GC GC GC GC GC GC GC
51
4-7
0.02
0.08
4" I o a
BPS ?
129
5-8
0.05
0.02
3" lO-1
BPS ?
AT
441
5-14
0.04
o.18
l O -5
BPS
GC
289 863
5 15 5-15
0.03 0.02
o.14 o.19
2"1o 3 lO -7
BPS BPS
GC GC
37 114 13o
6-7 7-9 8-14
o.oi o.14 0.08
o.91 o.61 0.45
I'IO 9 io s lO - l °
BPS ? BPS BPS
GC GC GC
17 22 79 156
9-11 9-11 9 1I 9-II
o.o2 0.02 0.05 0.04
0.29 o.21 o.15 0.30
lO - l ° 3-1o a 1"1o a IO s
BPS BPS BPS BPS
GC GC GC (;C
31ulation Res., 5 (1968) 3 5 9 37~
364
H.V.
TABLI'~
F. J. DE
SERRES
l I (continued)
J'VIulant No.
2
MALLING,
t7-24 08 lo0
Complon coverage
Reversiotzs p e r I,o ~; slcrvivors
Genetic alteration at m u t a n t site "
NPI?
HA
P
~Ialling and-De Serres ( r @ 23)
Final score
to-i[ to-I ]
0.05 o. 10
]o.-] I
6. Jo ~ 3" zo ~ lo u
BPS BPS I3PS ?
(;C AT (;C
0.0,
o. i 0 0.04 1.83
34
io
13
o.i()
o.41
4" i o - a
I:IPS
(;C
25
12
I0
O.OI
0.i2
29
12
I6
0
0.30
30
I2
tO
o.ol
0.38
35
I2
10
O.O1
0.22
75
12
z(>
o
o.3~
io lo ,o to lo
a ~ ~ s lo
I20
12
I2
O
O.10
lO
7
143
12
I()
0,0l
0.23
10 -9
BPS 13PS HPS 13PS I31)S [31'S HPS
(;( (;(" (;C (;C (;((;(" G("
164
12
l()
o.oJ
0.28
,o
BI)S
(;C
I4()
12
[0
O.O1
0,3()
9" ~o ~
131)S
(;("
94
14
17
0-03
o,i3
[ • lO a
BI)S
(;("
47
I0
l7
o.o2
o.18
5" ~o ~
131)S ?
(;C
82 99 112
I7
0.02
0.39 0. 4 ,
u 9
(;("
0.0 ~
6.1o4" i o
13I'S
17
I3I'S
(;C
[7
o.o2
0.27
2-[o
s
!'~>S
(;C
frequency
obtained
P = The probability that the reversion the SPI¢. F o r e x p l a n a t i o n , s e e T a b l e I.
,a
after
HA
c ~ c , z t m e n t , is t h e s a m e a s
ICR-I7 o-°a, reverted after HA treatment, but only at approximately one-third or less of the frequency obtained for many of the other mutants with nonpolarized complementation patterns. Since in each case the induced reversion frequency was significantly different from the control (P
The association between complementation patterns and genetic alterations of NA-induced mtt{at1[s
I d e n t i f i c a t i o n of the genetic a l t e r a t i o n s at the molecular level in N A - i n d u c e d m u t a n t s after t r e a t m e n t with NA, EMS, or ICR-x7o was r e p o r t e d previously'-% F r o m this s t u d y we concluded t h a t m u t a n t s with polarized c o m p l e m e n t a t i o n p a t t e r n s a n d n o n c o m p l e m e n t i n g m u t a n t s are d e r i v e d from a v a r i e t y of genetic alterations, whereas m u t a n t s with n o n p o l a r i z e d c o m p l e m e n t a t i o n p a t t e r n s are d e r i v e d p r e d o m i n a n t l y from 1)ase-pair s u b s t i t u t i o n s . In the present analysis we found t h a t H A i n d u c e d revert a n t s only in m u t a n t s which were c h a r a c t e r i z e d p r e v i o u s l y as b a s e - p a i r s u b s t i t u t i o n s or in n m t a n t s which r e v e r t e d only spontaneously. Of the n m t a n t s resulting from b a s e - p a i r s u b s t i t u t i o n (Table V), a significantly higher fraction of n o n p o l a r i z e d n m t a n t s r e v e r t e d after H A t r e a t m e n t t h a n did the polarized m u t a n t s (Z2 4.6, o.oe < P < o.o 5, Y a t e s correction included). An even g r e a t e r difference was found b e t w e e n the 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 in the nonc o m p l e i n e n t i n g a n d n o n p o l a r i z e d c o m p l e m e n t i n g classes (Z~ 28.2 ; P < o.ooI). W e f o u n d no significant difference b e t w e e n the fraction of H A - r e v e r t a b l e m u t a n t s a m o n g t h e polarized c o m p l e m e n t i n g a n d the n o n c o m p l e m e n t i n g m u t a n t s (Z2 -- ~.65, P o.2). Muia/io~z ICes., 5
( 1 9 ( ) 8 ) 35(-1 - ] ' ] [
COMPLEMENTATION
TABLE
PATTERNS
AND TYPE
OF BASE-PAIR
TRANSITION
365
III
R E V E R T I B I L I T Y AND DETERMINATION OF THE GENETIC ALTERATION IN N A - I N D U C E D WITH POLARIZED COMPLEMENTATION PATTERNS AETER TREATMENT WITH H A
Mutant No.
Complon coverage
2-17-98
Reversions per zo 6 survivors SPR
HA
P
MUTANTS
Genetic alteration at m u t a n t site M a l l i n g and Final De Serres (re/. 23) score
i-IO
O
0.587
4° 78 437
1-12 1-12 L-I2
o o o
o o o
I.OO i.oo i .oo
ISI
I--I2
O.I 5
0.02
I.OO
@/
59 163
1--13 I-I 3
O.OI o,oi
O o,Io
I.OO 9' 1°-5
BPS ? BPS
BPS ? G("
147 I98 498
1-14 1-14 1-14
o o o
o o o
I.OO I.OO I.OO
NON NON NON
NON NON NON
12 16 46
[-14 1-14 1-14
O.O1 0.02 o.oi
O,13 0,05 0,05
IO -5 5" I ° ~ 2 2 - IO -2
BPS BPS BPS
GC AT AT
131 213 264
1-14 1--14 1-14
O 0.03 o,oi
o O,O3 o.I I
I.OO 2 . 9 " IO -1 5" I ° - 4
BPS SP BPS
AT SP (;C
200 412 433
1-14 1--14 I-I4
0.02 O.OI 0.06
0.09 O.12 o.12
4" 1 ° - 3 i O -4 8 . IO -3
BPS BPS BPS ?
G(~', G(" GC
214
I-i 4
o.o6
o.o 4
2 . 3 ' IO -1
BPS ?
BPS ?
8 87 153 172
1-15 1-15 1-15 1--15
o.oi o.oi o.oi 0.03
o.o2 0.09 0.03 0.05
3.o" 9" 1.4" 8"
BPS ? SP -T / ? @ /- ?
BPS ? GC -- / ? ~/ ?
387
1-15
0.07
o.12
2.1 • lO `3
BPS
GC
For explanation,
TABLE
IO 5
ad-3B
lO -'1 lO-4 lO-1 10 -2
BPS
C;C
NON SP NON
N()N SP NON " /
s e e T a b l e s 1, I I .
IV
R E V E R T I B I L I T Y AND DETERMINATION OF THE GENETIC ALTERATION IN N.3t-INDUCED NONCOMP L E M E N T I N G ad-3B MUTANTS AFTER TREATMENT WITH H A
A . B a s e - p a i r substitutions Mutant No.
Reversions per zo 6 survivors
SPR
HA
P
2-17-142 149 166 230 45 96 127 228 14 18
o.12 o.16 0.09 0.o2 o.to o.o9 o 0.02 0.03 o.io
o o 0.38 o. I o 0.24 0.05 o 0.20 0.20 0.07
I.OO i.oo
For explanation,
see T a b l e s
lO -5 2.5.1o 3 lO -5 2 . 6 - IO 1 i .oo 4" IO 4 2 . lO -4 3" lO -1
Genetic alteration f or the reverse mutation mechanism Mal-ling and Final De Serres (ref. 23) score BPS BPS BPS ? BPS BPS BPS BPS BPS BPS BPS
AT AT GC GC GC AT AT GC GC AT
I, I I ,
M u t a t i o n Res., 5 ( 1 9 6 8 ) 3 5 9 - 3 7 1
366
H.V.
TABLE
MALLING,
F. J. D E S E R R E S
IV (continued)
~Iutanl
Reversions per z o ~ surviw)rs
No.
--
2 I7-26 1o5 I20 ~30 138 159 173 ~9 o 212 490
Genetic alteratio.n j o t the reverse mutalion mechanism M a i l i n g a;id -- Fina)-De Serres (r @ z3) score
. . . . . . . .
SPR
HA
P
0.08 o.t5 o.e8 u.o2 O,12 0.06 0.04 o.oz 0.07 0.23
o 0.20 0.09 o.ol 0.03 0.05 o. 1Z o.-, 7 0.20 o.1 7
I.OO 4'1o 4.(3" IO 5" IO 2 . 8 ' IO 2.2' io 8 " IO ~o 4.2' Io 2.0' to
BPS BPS BPS HPS BPS BI'S BPS BPS BPS f3PS
a '~ ~ 1 * 4 0 4 1
AT (;C :V|" AT AT AT (;C (;C (;C AT
13. Base-pa~r insertion or deletions aim ~,tnidentified alterations 2-~7-55 57 (17 too t8o :z, 240
o O o o o o o
o O o o o o o
t.oo I,OO ,.oo I .oo I.OO ~.oo 1.o()
zI8 lo 23 2[ I 217
o o o 0.00 o.oI
o.oI o o o 0.03
3.,'to I .oo 1.0o
I3 30 48 88 ~3-" 1()2
o o o.u [ o.o2 o-07 O
o.04 o
5-7"[o t.oo
o
I.oo
o o O
I.oo i.oo 1.OO
() 7 83 lol 22o
u.ol 0.04 o.o, 0.07 0.03
o.ol 0.05 0.04 (/.o3 o.o6
5"IO I.IO 2.~o 2. 4 - i o 3"[o
For explanation,
see T a b l e s
[.oo 0-to
1
1
NON N()N NON NON NON NON N()N
N()N N()N N()N N()N N()N NON NON
SP .q]) ~P SP SP
SI' SI' SI' S1 ) St'
t / t /
:/
t / T/ : /
i/
2
t/
: / t ! t 4 I
~ t i 1 2
,/
/ / / / /--
/ I/I/
1, I I.
V
TABLE
THE PERCENT OF THE DIFFERENT TYPES OF NA-INDUCED a d - 3 B MUTANTS OF BY THEIR COMPLEMENTAT[ON PATTERN AND TYPE OF GENETIC ALTERATION
Complementation pallern O,pe
Nonpolarized Polarized Noncomplementing Total
60.4 I L.7 27. 9 mo.o
Previous Note:
Percenlage
data
Total mutanls a n a lvzed 63 25 43
2. T (2) 1.4 (3) 7.2 ( I X )
I31
lo. 7
in parenthesis
58.3
(6I)
4..3 (9) 5.8 (9) 68.4
BP,g?
13ase-pair insertion or delelion
Unidentified Reverting Nonspontaneously reverting
O
O
O
(O)
is t h e a c t u a l n u m b e r
3qutation l?es., 5 ( 1 9 6 8 ) 3 5 9 371
(O)
(O)
O
(O)
L4 (3) o (o)
1.4 (3) 7.2 (11)
0.9 (2) .3.2 (5)
e.3 (5) 4-5 (7)
~.4
8.6
4.t
6.8
8o. 5 84
(I~IALLING22)
The number
Base-pair transition i~iutanl ~ wild type ~qT;Z;GC GC ~ -AT
:\Teztrospora crassa C H A R A C T E R I Z E D
of mutants
12. 7 1o
found.
6
COMPLEMENTATION PATTERNS AND TYPE OF BASE-PAIR TRANSITION
367
DISCUSSION
The general spectrum of the genetic alterations induced by NA (Table V) Determination of the general spectrum of genetic alterations induced b y a mutagen b y means of specific revertibility studies can be greatly influenced b y (I) the occurrence of suppressors, (2) hot spots, and (3) the base ratio in the gene.
The influence of suppressors Suppressors can be located either inside the gene which they suppress (intralocal suppressors) or elsewhere in the genome (extragenic suppressors). Extragenic suppressors are extremely rare among the revertants of ad-3B mutants 7,17 (BARNETT, unpublished; see MALLING2°). In E. coli some base-pair substitutions can revert by intralocal suppressors which also are base-pair substitutions 3~. A m u t a n t with an AT pair at the m u t a n t site might also revert b y a GC --> AT base-pair transition at another site (intralocal suppressor) and vice versa for a m u t a n t with a GC pair at the m u t a n t site. A mutagen which would react preferentially with AT base-pairs would, in theory, make it possible to distinguish between these different mechanisms. If we assume that almost all nonpolarized m u t a n t s are able to revert by intralocal suppressors (induced b y a base-pair transition from GC -~ AT), then all nonpolarized mutants, independent of mutagenic origin, should revert after HA treatment. However, since a much lower percentage of HA-induced ad-3B mutants with nonpolarized complementation patterns than of NA-induced mutants with the same type of pattern reverted after HA-treatment (MALLING, unpublished), it seems highly unlikely that induction of intralocal suppressors is the predominant reversion mechanism for nonpolarized ad-3B mutants.
Hot spots A previous study ~3 showed that mutants which covered complons 2- 4 have nearly the same reversion frequency after treatment with NA, EMS, or ICR-I7O. We found that all these mutants reverted with approximately the same frequency after HA-treatment. These new data support the hypothesis that these mutants might have been induced at a single site (i.e., hot spot). This was also true for the other hot spots, namely sites for mutants with complementation patterns 3-14 (6 mutants) and 12-16 (8 mutants) (Table I). All these m u t a n t s reverted b y a GC --~ AT transition. Relatively few of the polarized and noncomplementing base-pair substitution mutants have identical induced-reversion frequencies. I t seems likely then that most of the 98 NA-induced base-pair transition mutants result from alteration of different sites in the ad-3B locus.
Base ration in the DNA The high content of GC-base-pairs at the m u t a n t site among the NA-induced
ad-3B mutants in Neurospora crassa could result from a high content of AT pairs in the DNA of the ad-3B locus. However, this possibility is not likely because the ratio between the base-pairs (GC/AT) in the DNA in Neurospora is approximately I.I625,~7 ; and since the ad-3B locus specifies a structural protein 11, the simplest assumption is that the DNA in the ad-3B locus has the same base ratio as the total DNA. F~om this discussion we can conclude that NA hot spots, suppressors, and the
Mutation Res., 5 (1968) 359-37I
368
H . V . MALLING, F. J. DE SERRES
base-ratio of the D N A do not have a predominant influence on the determination of the general spectrum of genetic alterations induced b y NA. The general spectrum of genetic alterations within the NA-induced n m t a n t s are given in Table V and the results 23 have previously been discussed in detail. The numbers in the table agree with a previous analysis of NA-induced ad-3 m u t a n t s in a microconidial strain of Neurospora~°. The numbers obtained from this and previous analyses are included in Table V. The most striking and unexpected results are the high percentage of m u t a n t s with GC pairs at the m u t a n t site (Table VI [86.5°,~ (68.4/79.1) of the total n u m b e r of base-pair transition@ Our finding is not in accord with the findings (~) that the relative rate of deamination b y NA of the bases in T2 and "1"4D N A is A : C or HMC : G -I:6.3:I6~5,3x; and (2) t h a t the base ratio in phage T2 and T 4 D N A GC/AT o.515 [which deviates greatly from Neurospora (GC/AT = I . I 6 ) 2 . Nevertheless, a high percentage of the NA-induced n m t a n t s in "F4 bacteriophage has been identified as having AT at the m u t a n t site ~. (For review see KRIE6~.) TABLE
V1
CORRELATION BASE-PAIR
BETWEEN SUBSTITUTIONS
THE
TYPI';
IN
THE
OF
COMPLEMENTATION
ad-313 R E G I O N
OF
PATTERN
AND
THE
DIRECTION
OF
THE
Neurospora c r a s s a
F r e q u e n c e in p e r c e u t w i t h i n each c o m p l e m e n t a t i o n class.
Complementation pattern
Nonpolarized Polarized Nonconlplementing
Direction of base-pair transition in reverse mz~lations A 7" -~ (;C (;C --~ .4 T ~ P ) 5°~,
5'~o
3 (2) 16 (2) 5 o (lo)
O
'~ (~) 5 (~)
P)
I°b I°;i P ) I 9 (I2) -'5 (3) i (2)
o.I°~,
Total
P
tOO (63) too (r2) ioo (20)
P : is t h e p r o b a b i l i t y t h a t t h e r e v e r s i o n f r e q u e n c y o b t a i n e d a f t e r H A t r e a t m e n t is t h e s a m e as t h e r e v e r s i o n f r e q u e n c y in t h e c o n t r o l ( B i r n b a u m ' s test). N o t e : T h e n u m b e r in p a r e n t h e s i s is t h e a c t u a l n u m b e r of m u t a n t s in e a c h c a t e g o r y f o u n d .
The high frequency of GC at the n m t a n t site a m o n g the NA-induced ad-3B m u t a n t s in Neurospora m a y be explained b y the following observations: (z) After 2o h t r e a t m e n t of bacteriophage T2 with I M NaNO2 at pH 4.2, 35°J/o of the guanine, 13°,/o of the cytosine, and 3°/'o of the adenine in the D N A were deaminated ~1. W h e n pneumococcal D N A was treated i h with NA under the same conditions (sufficient to inactivate 99.9~'/o of the transforming activity), only IO I5~o of the guanine and 1-2°,"o of adenine in the D N A were deaminated. (No deamination of cytosine was detectable.) (2) The m u t a n t s in Neuro@ora were obtained after t r e a t m e n t of conidia with either o.oo 5 or o.oi M NaNO2 at p H 4.6 (concentration of HNO~:3.2- IO ~ or 6. 4. xo 4 M) ~0 for 16o min. The mutation frequency was found to be 3 o - 1 3 o ' x o -6 per survivor. Since LITMaN'S treatmenO 9 conditions are closer to the treatment conditions used in the Neurospora experiment than the t r e a t m e n t conditions used b y VIELMETTER AND SCHUSTERal in the phage experiment, we think t h a t it is reasonable to expect that the NA-induced m u t a n t s in Neurospora would have predominantly a GC base-pair at the m u t a n t site. The NA-induced r l I m u t a n t s in bacteriophage T 4 (ref. 13) were isolated io rain after t r e a t m e n t with 2 M NaNO2 at p H 5.3 (concentration of HNO2 = 2.5" Io -2 31). Mutation Res., 5 (i968) 359-371
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Therefore we would expect that the frequency of AT at the mutants site should be higher among the r I I mutants in bacteriophage T 4 than among the ad-3B mutants in Neurospora. On the basis of LITMAX's data, it is somewhat surprising that mutants with AT at the mutant site are found among NA-induced mutants. The NA treatments in the bacteriophage and Neurospora experiments were performed with nmeh less concentrated solution of HNO~ and/or shorter treatment time than that which gave no reaction between NA and cytosine in pneumococcal DNA in LITMAN'Sexperiments; this is especially significant since the deamination of guanine does not produce GC to AT base-pair transition aa. Mutants derived from GC to AT base-pair transitions can be nonsense mutations (see below). Mutants derived from an AT to GC base-pair transition cannot be nonsense mutations. Because nonsense mutation will result in formation of a polypeptide chain fragment, they are not likely to be leaky. However, a large fraction of mutants resulting from missense mutation are leaky. Thus, mutants with AT at the mutant site may have a higher probability of recovery than mutants with GC at the mutant site. This may explain why mutants with AT at the mutant site are recovered at unexpectedly high frequency after NA-treatment in both bacteriophage T4 and
Neurospora. Association between type of complernentation and type of base-pair at mutant site From our earlier study of the association between complementation patterns and genetic alterations of NA-indueed mutants 23, we determined that base-pair substitution mutants with polarized complementation patterns and noncomplementing base-pair substitution mutants were either nonsense mutations which give rise to polypeptide fragments or missense mutations of an amino acid essential for the tertiary structure of the protein. The RNA nonsense triplets in some strains of E. coli are UAA, UAG, and UGA2,3,32; the analogous codons and anticodons in the DNA are respectively ATT/ TAA, ATC/TAG and TCA/AGT. Since HA only reacts with C, it can induce transitions in the last two triplets; but both of these reactions yield ATT which also is a nonsense triplet. Consequently we may conclude that nonsense triplets are not able to revert by a GC to AT transition. It has been shown eonvincinglyl~, 26 that mutants carrying a nonsense mutation form only a part of the polypeptide chain specified b y the gene, and that all fragments have the same end in common. If the polypeptide fragment is large enough to show allelic complementation, nonsense mutations would be expected to lead to nmtants with a polarized complementation pattern. The fact that the base-pair AT occurs more frequently at the mutant site among mutants with polarized complementation patterns and among noncomplementing mutants than among the mutants with nonpolarized complementation patterns is interpreted as indicating that the first two classes of mutant are derived more frequently from nonsense mutations than the latter. Supersuppressors have now been found in Neurospora ~8. Since supersuppressors most likely suppress nonsense mutations, a direct, independent test of our interpretation of these data can be made by studying the ability of supersuppressors to suppress ad-3B mutants as a function of their complementation pattern. We are now attempting to study this correlation for the ad-3B mutants. .VIutation Res., 5 (~968) 3 5 9 - 3 7 1
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H. V. MALLING, F. J. D E S E R R E S
Comparative mutagenesis studies The data from the present study on the revertibility of NA-induced base-pair substitution after treatment with HA shows that the ratio of mutants with the two types of base-pairs at the m u t a n t site (AT/GC) varied between the three different classes of complementation pattern (Table IV). This can be explained in part by the simple fact that a base-pair transition can only induce a nonsense triplet by a GC -~ AT transition, and not b y a AT -+ GC transition. The present data have especially interesting implications with regard to the diagnostic capability of the relatively simple test for allelic complementation on samples of ad-3B mutants induced by different mutagens. For example, a mutagen causing AT to GC base-pair transitions would produce a sample showing (I) a very high percentage of mutants able to show allelic complementation and (2) most or all of these mutants having nonpolarized complementation patterns. By contrast, a mutagen producing GC to AT base-pair transitions should induce mutants with (r) a much lower percentage allelic complementation and (2) a high frequency of mutants with polarized complementation patterns. Present data on allelic complemenration among MNNG provide an exact fit to first example 24, whereas the data on allelic complementation among HA-induced mutants provide an exact fit to the second (MALLINGAND DE SERRES, unpublished). ACKNOWLEDGEMENTS
The authors wish to thank Dr. M. A. KASTENBAUM for his help in preparing the statistical analysis, and Mrs. ARLEE TEASLEY for her valuable technical assistance.
RI,H"I';R1LNCES I BIRNBAUM, A., Statistical m e t h o d s for Poisson processes and exponential populations, J. Am. Statist. Assoc., 49 (1954) 254. 2 BRENNER, A., A. O. W. STRATTON AND S. KAPLAN, Genetic code: The " n o n s e n s e " triplets for chain t e r m i n a t i o n and their suppression, Nature, 206 (1965) 994 998. 3 BRENNER, S., L. BARNETT, E. R. KATZ AND I i'. H. C. CRICK, UGA: A third nonsense triplet in the genetic code, Nature, 213 (1967) 449 45 °. 4 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 by 2-aminopurine, Genetics, 48 (1963) 597-6o4. 5 BROWN, D. M., AND P. SCFIELL, The reaction of h y d r o x y l a m i n e w i t h cytosine and related conlpounds, J. :lIol. Biol., 3 (I961) 7o9. 6 CHAMPE, S. P., AND S. BENZER, Reversal of n l u t a n t p h e n o - t y p e s by 5-fluorouracil, an a p p r o a c h to nucleotide sequences in m e s s e n g e r - R N A , Proc, Natl. Acad. Sci. (U.S.), 48 (1962) 532-546. 7 DE SERRES, F. J., Studies with purple adenine m u t a n t s in Neurospora crassa, I I I . Reversions of X - r a y - i n d u c e d m u t a n t s , Genetics, 43 (1958) 187-2o6. 8 DE SERRES, F. J., Mutagenesis and c h r o m o s o m e structure, J. Cellular Comp. Physiol., 64, Suppl. I (1964) 33-42. 9 DE SERRES, F. J., The utilization of leaky ad-3 m u t a n t s of Ne~rospora crassa 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 , Mutation Res., 3 (1966) 3-12. IV DE SERRES, F. J., H. E. BROCKMAN, W. E. BARNETT AND H. G. KOLMARK, Allelic complenlentatiou anlong n i t r o u s acid-induced ad-3 B m u t a n t s of Neurospora crassa, Mutation Res., 4 (1967) 415 424 • I I FISHER, C. R., D e t e r m i n a t i o n of the enzymatic functions controlled b y ad-3A and ad-3B loci in Neurospora crassa, Genetics, 56 (1967) 560 (Abstract). I2 JL?O\VLER, n . V., AND I. ZABIN, Co-linearity of fl-galactosidase w i t h its gene b y inlmunological detection of incomplete polypeptide chains, Science, 154 (1966) IO27-IO29. 13 15"REESE, F~., On the molecular e x p l a n a t i o n s of s p o n t a n e o u s and induced m u t a t i o n s , Brookhaven 5;ymposia in Biology, No. 12 (1959) 63-73.
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14 FREESE, E., E. BAUTZ AND E. B. FREESE, The chemical and m u t a g e n i c specificity of h y d r o x y l amine, Proc. Natl. Acad. Sci. (U.S.), 47 (1961) 845-855. 15 FREESE, E., E. BAUTZ-FREESE AND E. BAUTZ, H y d r o x y l a m i n e as a mutagenic and i n a c t i v a t i n g agent, J. Mol. Biol., 3 (196I) 133-143. 16 FREESE, E., AND E. B. FREESE, The o x y g e n effect on deoxyribonucleic acid inactivation b y h y d r o x y l a m i n e s , Biochemistry, 4 (1965) 2419 2433. 17 I~OLMARK, G., AND •. H. GILES, C o m p a r a t i v e studies on monoepoxides as inducers of reverse m u t a t i o n s in Neurospora, Genetics, 4 ° (1955) 890 902. 18 KRIEG, D. R., Specificity of chemical mutagenesis, Progr. Nucleic Acid Res., 2 (1963) I 2 5 - I 0 8 . 19 LITMAN, R. M., Genetic a n d chemical alterations in the t r a n s f o r m i n g D N A of JPneumococcus caused b y ultraviolet light and by n i t r o u s acid, J. Chim. Phys., 58 (1961) 997 lOO4. 20 MALLING, H. V., Identification of the genetic alteration in n i t r o u s acid-induced m u t a n t s of Neurospora crassa, Mutation Res., 2 (1965) 320-327. 21 MALLING, H. V., H y d r o x y l a m i n e as a mutagenic agent for Neurospora crassa, Mutation Res., 3 (1966) 470-476. 22 MALLING, H. V., Mutagenicity of m e t h y l h y d r o x y l a m i n e s in Neurospora crassa 3/lutation Res., 4 (1967) 265-274. 23 MALLING, H. V., AND F. J. DE SERRES, Relation b e t w e e n 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 n i t r o u s acid-induced ad-3B m u t a n t s of Neurospora crassa, Mutation Res., 4 (1967) 425-44 °. 24 MALLING, H. V., AND F. J. DE SERRES, N-Methyl-N'-nitroso-N-nitroguanidine (MNNG) as a mutagenic agent for Neurospora crassa, Genetics, 56 (1967) 575 (Abstract). 25 MINAGAWA, T., B. WAGNER AND B. STR#_USS, The nucleic acid c o n t e n t of Neurospora crassa, Arch. Biochem. Biophys., 80 (1959) 442-445 • 26 SARABHAI, z~k.S., A. D. STRETTON, S. BRENNER AND A. BOLLE, Co-linearity of the gene with the polypeptide chain, Nature, 2Ol (1964) 13-17. 27 SCHILDKRAUT, C. L., J. MARMUR AND P. DOTY, D e t e r m i n a t i o n of the base cmnposition of deoxyribonucleic acid from its b u o y a n t density in CsC1, J. Mol. Biol., 4 (1962) 430-443. 28 SEALE, T. W., Evidence for s u p e r s u p p r e s s o r s in Neurospora, Genetics, 56 (1967) 586 (Abstract). 29 STRACK, H. B., E. B. FREESE AND E. FREESE, Comparison of m u t a t i o n and inactivation rates induced in bacteriophage and t r a n s f o r m i n g D N A b y various mutagens, Mutation Res., i (i964) io-2I. 3 ° TESSMAN, I., H. ISHIWA AND S. ]~UMAR, Mutagenic effect of h y d r o x y l a m i n e in vivo, Science, 148 (1965) 5o7-5o8. 31 VIELMETTER, W., AND H. SCHUSTER, Die ]3asenspezifitXt bei d e r I n d u k t i o n von M u t a t i o n e n durch salpetrige SXure im P h a g e n T2, Z. Natur[orsch., I 5 b (196o) 304 31I. 32 WEIGERT, M. G., AND A. GAREN, Base composition of nonsense condons in E. coli, Evidence f r o m amino-acid s u b s t i t u t i o n s at a t r y p t o p h a n site in alkaline p h o s p h a t a s e , Nature, 206 (1965) 992-994. 33 YANOFSKY, C., Possible R N A code w o r d s for the eight a m i n o acids t h a t can o c c u p y one position in the t r y p t o p h a n s y n t h e t a s e A protein, Biochem. Biophys. Res. Commun., 18 (1965) 898-909.
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