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Production of mutations by streptonigrin in the ascomycete Ophiostoma multiannulatum
470
SHORT COMMUNICATIONS
Production of mutations by streptonigrin in the ascomycete Ophiostoma multiannulatum
The a n t i b i o t i c s t r e p t o n i g r i n has p r o v e d to be a powerful inducer of c h r o m o s o m a l a b e r r a t i o n s in h u m a n leukocytes in vitro ~ a n d in r o o t - t i p s of the b r o a d bean, I'icia faba2. R e g a r d i n g the t y p e of a b e r r a t i o n s p r o d u c e d a n d the m i t o t i c stages affected, the effect of s t r e p t o n i g r i n is more similar to t h a t of X - r a y s t h a n the effect of a n y o t h e r r a d i o m i m e t i c chemical so far i n v e s t i g a t e d 2. The p r e l i m i n a r y e x p e r i m e n t s briefly r e p o r t e d below show t h a t s t r e p t o n i g r i n is n o t only" a p o w e r f u l c h r o m o s o m e b r e a k i n g agent, b u t also is able to induce gene m u t a t i o n s . The e x p e r i m e n t a l m a t e r i a l was a h y p o x a n t h i n e - l e s s strain of the ascomycete Ophiosloma m~dEamudalum. T h e frequency of b a c k - m u t a t i o n s p r o t o t r o p h y was s t u d i e d a t 25 ° as a function of the d u r a t i o n of t r e a t m e n t with streptonigrin at two different concentrations, viz. 2. IO ~ M a n d 5" ~o s M. The m e t h o d s used in the backm u t a t i o n test have been r e p o r t e d earlier :). In the e x p e r i m e n t with the higher concentration, 2.5 mg of streptonigrin were
to
5.10"SM
100
1.0
50
05
h .
20
30
%
~
10
o
O >
\~
60
120
2.10-~M
U3 % 25
/
O~, Q_ m 20 Z O
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i
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~x 2'10-~ M
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0.2
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i
i
killing effect of 2-io
£ ' i g . 2. T h e m u t a g e n i c 21Izdalio:,z I?esea;.ch
.
4 ~[ a n d
5 " [o-S ~I s t r e p t o n i g r i n
effect of 2.Jr) ~ M and
( i 9 0 5 ) 47 ° .t71
5'1°
/
I ,I, O 30 60 DURATION OF TREATMENT IN MINUTES
O 30 60 120 DURATION O F T R E A T M E N T IN M I N U T E S l : i g . 1. T h e
/ I
<10
X
I
I
5 ~5
Ul I-. Z
/
in O.
s M streptonigrm
I
120
m~dtian~zdatum.
i n O.
mzdtiai, nzdatum.
SHORT COMMUNICATIONS
471
dissolved in i ml ethanol and 1/15 M phosphate buffer (pH 7.o) was added to bring t h e volume to 5 ° ml. Five ml of the solution were pipetted to a centrifuge tube containing washed log-phase cells. The tubes containing the suspension were placed in a w a t e r b a t h at 25 °. The control was 20/o ethanol in phosphate buffer. The t r e a t m e n t w a s stopped b y washing the cells twice in distilled water. The cells were immediately p l a t e d on minimal agar for the back-mutation test and after appropriate dilution on minimal agar plus adenine for the survival test. Mycelia were counted after incubation at 25 ° for a week. The results obtained are shown in Figs. I and 2. The mutagenic effect of streptonigrin in the strain used in these experiments is about as strong as t h a t of dimethvlsulfate or of 2537 A ultraviolet light but less than t h a t of N-nitroso-Nmethvlurethane at comparable survival values a, a. This investigation was supported by grant No. RG-93I 9 from the National Institutes of Health, U.S. Public Health Service, and by grants from the Swedish Natural S c i e n c e R e s e a r c h Council and the Swedish Cancer Society. We would like to thank Dr. T. McBRIDE, The John L. Smith Memorial for Cancer Research, Chas. Pfizer & Co., inc. for a gift of streptonigrin (work sponsored by the National Cancer Institute, National Institutes of Health, contract No. ph 43-64-5o).
Iustitule of Physiological Botany, University of Uppsala, Uppsala (Sweden)
G. ZETTERBERG
B. A. I{IHLMAN
I COHEN, ~l. M., "X,[. W. SFIAV,~ AND A. P. CRAIG, The effects of s t r e p t o n i g r i n on cultured h m n a n leukocytes. Pro& Natl. Acad. Sci. U.S., 5 ° 11963) 16-24. 2 K1HLMAN, B. A., Tile p r o d u c t i o n of chronlosonlal a b e r r a t i o n s by s t r e p t o n i g r i n in Vicia /aba. J~I~talio~* Research, i 11964) 54-62. 3 ZrTTERBERO, G., The m u t a g e n i c effect of 8-ethoxycaffein, caffein and dimethylsulfate in t h e O p h i o s t o n m b a c k - m u t a t i o n test. Hereditas, 46 1196o) 279 311. 4 ZETTERBERt;, G., On the specific mutagenic effect of N - n i t r o s o - N - m e t h y l u r e t h a n in O p h i o s t o m a . Hereditas, 48 (1962) 371-389 .
Received J u l y 2Ist, 1965 2~lutation Research 2 (1905) 470-471
O n the distribution of two-break exchanges on the satellited chromosomes of barley EVANS AND BIGGER 2 reported t h a t t h e distribution of radiation-induced chromatid interchanges in a cell is non-random in Vicia faba. The frequency of aberrations for a given chromosome was found not to be proportional to its length at metaphase and the distribution of aberrations was found to be associated with the distribution of heterochromatin. It was suggested t h a t more sites for aberrations were formed a t or near the heterochromatin, as a result of the chromosome strands coming close to each other b y chromocenter formation. The low frequency with which nucleolar 2~lutation Research 2 (1065) 471-475
SHORT COMMUNICATIONS
Production of mutations by streptonigrin in the ascomycete Ophiostoma multiannulatum
The a n t i b i o t i c s t r e p t o n i g r i n has p r o v e d to be a powerful inducer of c h r o m o s o m a l a b e r r a t i o n s in h u m a n leukocytes in vitro ~ a n d in r o o t - t i p s of the b r o a d bean, I'icia faba2. R e g a r d i n g the t y p e of a b e r r a t i o n s p r o d u c e d a n d the m i t o t i c stages affected, the effect of s t r e p t o n i g r i n is more similar to t h a t of X - r a y s t h a n the effect of a n y o t h e r r a d i o m i m e t i c chemical so far i n v e s t i g a t e d 2. The p r e l i m i n a r y e x p e r i m e n t s briefly r e p o r t e d below show t h a t s t r e p t o n i g r i n is n o t only" a p o w e r f u l c h r o m o s o m e b r e a k i n g agent, b u t also is able to induce gene m u t a t i o n s . The e x p e r i m e n t a l m a t e r i a l was a h y p o x a n t h i n e - l e s s strain of the ascomycete Ophiosloma m~dEamudalum. T h e frequency of b a c k - m u t a t i o n s p r o t o t r o p h y was s t u d i e d a t 25 ° as a function of the d u r a t i o n of t r e a t m e n t with streptonigrin at two different concentrations, viz. 2. IO ~ M a n d 5" ~o s M. The m e t h o d s used in the backm u t a t i o n test have been r e p o r t e d earlier :). In the e x p e r i m e n t with the higher concentration, 2.5 mg of streptonigrin were
to
5.10"SM
100
1.0
50
05
h .
20
30
%
~
10
o
O >
\~
60
120
2.10-~M
U3 % 25
/
O~, Q_ m 20 Z O
5 (/1 nO
/ I
X0
/
2
i! /
i
uJ 1
°js ,'//'
%
~x 2'10-~ M
U,J
a. 0.5
0.2
i
t
i
i
killing effect of 2-io
£ ' i g . 2. T h e m u t a g e n i c 21Izdalio:,z I?esea;.ch
.
4 ~[ a n d
5 " [o-S ~I s t r e p t o n i g r i n
effect of 2.Jr) ~ M and
( i 9 0 5 ) 47 ° .t71
5'1°
/
I ,I, O 30 60 DURATION OF TREATMENT IN MINUTES
O 30 60 120 DURATION O F T R E A T M E N T IN M I N U T E S l : i g . 1. T h e
/ I
<10
X
I
I
5 ~5
Ul I-. Z
/
in O.
s M streptonigrm
I
120
m~dtian~zdatum.
i n O.
mzdtiai, nzdatum.
SHORT COMMUNICATIONS
471
dissolved in i ml ethanol and 1/15 M phosphate buffer (pH 7.o) was added to bring t h e volume to 5 ° ml. Five ml of the solution were pipetted to a centrifuge tube containing washed log-phase cells. The tubes containing the suspension were placed in a w a t e r b a t h at 25 °. The control was 20/o ethanol in phosphate buffer. The t r e a t m e n t w a s stopped b y washing the cells twice in distilled water. The cells were immediately p l a t e d on minimal agar for the back-mutation test and after appropriate dilution on minimal agar plus adenine for the survival test. Mycelia were counted after incubation at 25 ° for a week. The results obtained are shown in Figs. I and 2. The mutagenic effect of streptonigrin in the strain used in these experiments is about as strong as t h a t of dimethvlsulfate or of 2537 A ultraviolet light but less than t h a t of N-nitroso-Nmethvlurethane at comparable survival values a, a. This investigation was supported by grant No. RG-93I 9 from the National Institutes of Health, U.S. Public Health Service, and by grants from the Swedish Natural S c i e n c e R e s e a r c h Council and the Swedish Cancer Society. We would like to thank Dr. T. McBRIDE, The John L. Smith Memorial for Cancer Research, Chas. Pfizer & Co., inc. for a gift of streptonigrin (work sponsored by the National Cancer Institute, National Institutes of Health, contract No. ph 43-64-5o).
Iustitule of Physiological Botany, University of Uppsala, Uppsala (Sweden)
G. ZETTERBERG
B. A. I{IHLMAN
I COHEN, ~l. M., "X,[. W. SFIAV,~ AND A. P. CRAIG, The effects of s t r e p t o n i g r i n on cultured h m n a n leukocytes. Pro& Natl. Acad. Sci. U.S., 5 ° 11963) 16-24. 2 K1HLMAN, B. A., Tile p r o d u c t i o n of chronlosonlal a b e r r a t i o n s by s t r e p t o n i g r i n in Vicia /aba. J~I~talio~* Research, i 11964) 54-62. 3 ZrTTERBERO, G., The m u t a g e n i c effect of 8-ethoxycaffein, caffein and dimethylsulfate in t h e O p h i o s t o n m b a c k - m u t a t i o n test. Hereditas, 46 1196o) 279 311. 4 ZETTERBERt;, G., On the specific mutagenic effect of N - n i t r o s o - N - m e t h y l u r e t h a n in O p h i o s t o m a . Hereditas, 48 (1962) 371-389 .
Received J u l y 2Ist, 1965 2~lutation Research 2 (1905) 470-471
O n the distribution of two-break exchanges on the satellited chromosomes of barley EVANS AND BIGGER 2 reported t h a t t h e distribution of radiation-induced chromatid interchanges in a cell is non-random in Vicia faba. The frequency of aberrations for a given chromosome was found not to be proportional to its length at metaphase and the distribution of aberrations was found to be associated with the distribution of heterochromatin. It was suggested t h a t more sites for aberrations were formed a t or near the heterochromatin, as a result of the chromosome strands coming close to each other b y chromocenter formation. The low frequency with which nucleolar 2~lutation Research 2 (1065) 471-475