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Ultraviolet photochemistry of DNA in mouse spermatozoa
Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
133
Ultraviolet photochemistry of D N A in mouse spermatozoa Introduction Ultraviolet light produces lethal and mutagenic effects on somatic and germ cells of animals 5,~,12,1s. To understand the mechanism of action of UV it is necessary to correlate these biological effects with the photochemical lesions produced. The UV photoproducts of DNA in somatic cells have been studied, the most abundant being the thymine-thymine dimer (T~) and the thymine-cytosine dimer (-~)17. There are, however, no reports on the photochemistry of DNA within animal germ cells. In the late spermatids and spermatozoa the DNA is bound to protamine TM, a highly basic protein, and assumes a condensed state when viewed through the electron microscope (refs. 6, 7). These changes might result in an alteration of the DNA secondary structure which can drastically change the photochemistrylL resulting in a variation in either the rate of production of specific photoproducts or the distribution of photoproducts. As an example, bacterial spores show a new photoproduct (spore photoproduct) which does not appear in germinated cells or in DNA in solution is. In this study we have compared the kinetics of production of TAT and "FC dimers in mouse spermatozoa to other cells and DNA in solution and have looked for other thymine-containing photoproducts. Materials and methods Mouse spermatozoa were labelled by injecting (C57BL × C3H) Fx hybrids (Cumberland View Farms, Clinton, Tenn.) intratesticularly with 20 #Ci of [3H~thymidine (about 20 Ci/mmole) per testis. 31 days later the mice were sacrificed and approx. IO' mature spermatozoa were obtained from the vas deferentia and cauda epididymi of each mouse 1°. The level of radioactivity was 1. 5 •lO -3 d.p.m./sperm. The sperm were washed and suspended in phosphate-buffered saline and kept at 4 °. Mouse L cells were labelled with II*C~thymidine in suspension culture I and DNA was extracted by means of a phenol-water procedure. The UV source consisted of 4 germicidal lamps with an output predominantly at 253. 7 nm. 3-ml samples of spermatozoa at 3" IOe cells per ml were placed in I-cmpathlength quartz cuvettes and stirred during irradiation. The incident UV dose rate under these conditions was approx. 3' IO4 erg/mm* per rain (ref. 4). The sample temperature during irradiation was approx. 14 °. The apparent absorbance at 253.7 nm of the sperm under these conditions was about I.O as measured in a Zeiss PMQ I I spectrophotometer. Most of this apparent absorbance was due to light scattering, hence the normally used dose correction for shielding due to absorption s proved to be unnecessary. That this was so was determined by irradiating labelled L cells at lO 4 per ml (apparent absorbance less than o.I at 253.7 nm) in the presence of different concentrations of sperm ( o - I . lO 7 sperm per ml). The percent of thymine present as dimer in the L cells after identical exposure times was unaffected by the presence of sperm up to a concentration of 5'IOe sperm per ml. Photoproducts in spermatozoa were assayed as done previously for mouse L cells. For comparison, L cell DNA was irradiated in the presence of 3"IO~ sperm/ml and photoproducts were assayed. E14C1Thymine-labelled spore product from UV-irradiated B. megaterium spores was obtained as a gift from R. S. STAFFORD,Oak Ridge National Laboratory. Mutation Res., 14 (1972.) 133-136
I34
SHORT COMMUNICATIONS
Io00
o)
-~
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°
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IO0
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C e n t i m e t e r s on C h r o m a t o g r a m
Fig. I. R a d i o a c t i v i t y d i s t r i b u t i o n on c h r o m a t o g r a m s of h y d r o l y s a t e s of [ a H ] t h y m i d i n e - l a b e l l e d mo use s p e r m : (a) u n i r r a d i a t e d sperm, (b) s p e r m i r r a d i a t e d w i t h o.8. lO 4 e r g / m m 2 of UV a t 253.7 A
nm. I, p o s i t i o n of m a r k e r t h y m i n e ; I1, p o s i t i o n of m a r k e r T U ; I I I , p o s i t i o n of m a r k e r T T ; S.P., p o s i t i o n of m a r k e r spore p r o d u c t : O, the origin of t h e c h r o m a t o g r a m ; S.F., t h e s o l v e n t front.
Results
Sperm labelled with E3H]thymidine were irradiated, hydrolyzed and chromatographed. Typical radiochromatograms are presented in Fig. I. Unirradiated sperm (Fig. Ia) showed a single peak of radioactivity, I, with an RF of 0.58 corresponding to thymine. UV irradiation produced two photoproducts, II and III, with Rv's of o.18 and 0.26, respectively, which correspond exactly with the position of marker thymineuracil dimers (q~lJ) and T>l" (see Fig. ib). Throughout the exposure range employed, ,
,
,
,
, .*'J
E "u
8
T"T
1,= •
|
0- 6
~4 ~2
.
.2 .
.
.4 .
.
.6
" / ~2
'
go
'
I 0
e r g / m m 2 . 10-4
Fig. 2. T h e f o r m a t i o n of TT a n d TC (the l a t t e r a n a l y s e d as TU) in m o u s e s p e r m a t o z o a (closed symbols) a n d mouse L cell D N A (open symbol) as a f u n c t i o n of UV e x p o s u r e a t 253.7 nm. M u t a t i o n R e s . , 14 (T972) X33--T36
SHORT COMMUNICATIONS
135
from 2.1o 3 to IOe erg/mm 2, these were the only two photoproducts detected. Negligible spore photoproduct was present. Peaks II and I I I were designated as TU and "FI", respectively. The former peak actually represents T'C dimer which was deaminated upon hydrolysis. The assignment of the latter peak as TT is confirmed by eluting it from chromatograms and re-irradiating in distilled water with the UV lamp. It was converted back to thymine with the same kinetics as marker T~'T.The possibility cannot be excluded that peak I I I contains some UT-adduct 14, but from the reversibility of I I I back to thymine the yield of this product must be less than lO% of TT. The kinetics of "F~" and "FC formation were measured by exposing identical samples of labelled sperm to different UV doses and determining the percentage of thymine present as " ~ or TTZ (Fig. 2). Both the kinetics and saturation yields of "1~" produced in mouse sperm were similar to previous results obtained with mouse L cells 1. The single point for f l " dimer production in L cell DNA also falls on the curve for sperm DNA in Fig. 2. The selective removal of "F~" by photoreactivation or intracellular repair processes was investigated. However, neither exposure to fluorescent light nor incubation in cell-culture medium at 34 ° for 24 h altered the percent of thymine present as dimer. This lack of photoreactivation is expected since no photoreactivating enzyme has been found in the cells of placental mammals 3. /x
Discussion The photochemistry of DNA in mouse spermatozoa was indistinguishable from that in mouse L cells or of DNA in solution. Furthermore, the lack of any detectable spore product indicates that sperm DNA is more like that of somatic cells than that of bacterial spores. Since the photochemistry, and particularly the rate of formation of dimers, is dependent on configuration, we conclude that the configuration of DNA in mouse spermatozoa is similar to that of DNA in solution, which is most likely in the B configuration 11. This conclusion is consistent with that determined for the DNA in several nonmammalian spermatozoa which have been shown by X-ray diffraction to be helical and probably in the B form 19. The observed differences between mammalian cells with respect to UV sensitivity*,2,17 might be due to alterations in either the production of photoproducts, the repair of the photochemical lesions or the ability to function with a given number of dimers in the DNA. Spermatozoa provide a test of the first possibility since the packaging of the DNA is so different. The results obtained in this study indicate that variations between mammalian cell types in sensitivity to lethal or mutagenie effects of UV irradiation is not likely a result of differences in photochemistry. Measurements of the biological effects of UV irradiation of mouse spermatozoa have been made using the results of artificial insemination as an assay 5. Although the lack of uniform irradiation of the sample in that study introduced some error in the dosimetry, the results indicated that IO4 erg/mm 2 to the sperm prevented implantation of the embryo, and lO 3 erg/mm 2 reduced the litter size to half. The latter value is about Io-fold higher than the Do dose of about lO 2 erg/mm 2 for mammalian ceils in culture I and m a y either indicate more efficient repair of UV damage in fertilized ova and early embryos than in somatic cells or selection for those sperm, at the level of fertilization, which received less than the nominal dose of radiation. Finally, thymine Mutation Res., 14 (1972) 133-136
136
SHORT COMMU NICATIONS
d i m e r s h a v e b e e n s h o w n t o b e p r e m u t a t i o n a l l e s i o n s in b a c t e r i o p h a g e 1~, T h e s e l e s i o n s a r e also p r o d u c e d in s p e r m a t o z o a a n d c o u l d b e r e s p o n s i b l e for t h e o b s e r v e d m u t a tionsS, 1~. H o w e v e r , t h i s h y p o t h e s i s r e m a i n s t o b e t e s t e d . W e t h a n k Mr. GORDON HUNTER a n d Mr. VINCENT W . S. ENG for e x c e l l e n t t e c h n i c a l a s s i s t a n c e , Dr. W . R. BRUCE for u s e f u l d i s c u s s i o n s , a n d t h e N a t i o n a l C a n c e r I n s t i t u t e a n d t h e M e d i c a l R e s e a r c h C o u n c i l of C a n a d a for f i n a n c i a l s u p p o r t .
Department of Medical Biophysics, University of Toronto, and Ontario Cancer Institute, Toronto (Canada)
M. L. MEISTRICH A. M. RAUTH
I CHIU, S., AND A. M. I~AUTH, A comparison of the sensitivity to ultraviolet light of mouse L cells and mouse bone marrow cells assayed in vitro, Radiation Res., 47 (1971) 11o-122. 2 CLEAVER, J. E., DNA repair and radiation sensitivity in human (xeroderma pigmentosum) cells, Int. J. Radiation Biol., 18 (197 o) 557-565 . 3 CooK, J. S., Photoreactivation in animal cells, in A. C. GIESE (Ed.), Photophysiology, Vol. 5, Academic Press, New York, 197 ° , pp. 191-233. 4 DOMON, M., B. BARTON, A. PORTE AND A. M. RAUTH, T h e i n t e r a c t i o n of caffeine w i t h ultraviolet-light-irradiated DNA, Int. J. Radiation Biol., 17 (197 o) 395-399. 5 EDWARDS, R. G., The experimental induction of gynogenesis in the mouse, 1I. Ultraviolet irradiation of the sperm, Proc. Roy. Soc., Ser. B, 146 (1957) 488-5o4. 6 FAWCETT, D. W., The structure of the mammalian spermatozoan, Intern. Rev. Cytol., 7 (1958) 195-234. 7 HOWATSON, A., AND W. R. BRUCE, in P. FAVARD (Ed.), Microscopie Electronique, Vol. 3, Soci6t6 fran~aise de Microscopie ]~lectronique, Paris, 197 o, pp. 639-64 o. 8 JOHNS, H. E., in K. KUSTIN (Ed.), Methods in Enzyrnology, Vol. 16, Fast Reactions, Academic Press, New York, 1969, pp. 253-316. 9 KAO, F. T., AND T. T. PUCK, Genetics of s o m a t i c m a m m a l i a n cells, IX. Q u a n t i t a t i o n of mutagenesis by physical and chemical agents, J. Cell. Physiol., 74 (1969) 245-257. IO LAM, D. M. K., AND W. R. BRUCE, T h e b i o s y n t h e s i s of p r o t a m i n e d u r i n g s p e r m a t o g e n e s i s of the mouse : extraction, partial characterization, and site of synthesis, J. Cell. Physiol., 78 (1971 ) 13-24. i i LUZZATI, V., The structure of DNA as determined by X-ray scattering techniques, in J. N. DAVlDSON ANt)W. E. COHN(Eds.), Progress in Nucleic Acid Research, Vol. i, Academic Press, New York, 1963 , pp. 347-368. 12 MACKENZIE, K., AND H. J. MULLER, Mutation effects of ultraviolet light in Drosophila, Proc. Roy. Soc., Ser. B, 129 (194 o) 491-517 . 13 MEISTRICH, M. L., AND R. G. SHULMAN, Mutagenic effect of sensitized irradiation of bacteriophage T 4, J. Mol. Biol., 46 (1969) 157-167. 14 PATRICK, M. H., Near-UV photolysis of pyrimidine heteroadducts in E. coli DNA, Photochem. Photobiol., II (197 o) 477-485. 15 PROOST, J., Action mutag~ne des UV sur les ovogonies de Drosophiles, I. Cindtique de la mutag6n~se, Mutation Res., 4 (1967) 473-'I89 16 RAHN, R. O., AND J. L. Hosszu, Influence of relative humidity on the photochemistry of DNA films, Biochim. Biophys. Acta, 19o (1969) 126-131. 17 RAUTH, A. M., Effects of ultraviolet light on established lines of mammalian cells, in M. EBERT AND A. HOWARD (Eds.), Current Topics in Radiation Research, Vol. 6, North-Holland, Amsterdam, 197 ° , pp. 195-245. I 8 STAFFORD, I~. S., AND J. E. DONNELLAN Jr., P h o t o c h e m i c a l evidence for c o n f o r m a t i o n c h a n g e s in DNA during germination of bacterial spores, Proc. Natl. Acad. Sci. (U.S.), 59 (1968) 822829. 19 WILKINS, M. H. F., Physical studies of the molecular structure of DNA and nucleoprotein, Cold Spring Harbor Symp. Quant. Biol., 21 (1956) 75-90.
R e c e i v e d A u g u s t I 7 t h , I97~
Mutation Res., 14 (1972 ) I33 136
133
Ultraviolet photochemistry of D N A in mouse spermatozoa Introduction Ultraviolet light produces lethal and mutagenic effects on somatic and germ cells of animals 5,~,12,1s. To understand the mechanism of action of UV it is necessary to correlate these biological effects with the photochemical lesions produced. The UV photoproducts of DNA in somatic cells have been studied, the most abundant being the thymine-thymine dimer (T~) and the thymine-cytosine dimer (-~)17. There are, however, no reports on the photochemistry of DNA within animal germ cells. In the late spermatids and spermatozoa the DNA is bound to protamine TM, a highly basic protein, and assumes a condensed state when viewed through the electron microscope (refs. 6, 7). These changes might result in an alteration of the DNA secondary structure which can drastically change the photochemistrylL resulting in a variation in either the rate of production of specific photoproducts or the distribution of photoproducts. As an example, bacterial spores show a new photoproduct (spore photoproduct) which does not appear in germinated cells or in DNA in solution is. In this study we have compared the kinetics of production of TAT and "FC dimers in mouse spermatozoa to other cells and DNA in solution and have looked for other thymine-containing photoproducts. Materials and methods Mouse spermatozoa were labelled by injecting (C57BL × C3H) Fx hybrids (Cumberland View Farms, Clinton, Tenn.) intratesticularly with 20 #Ci of [3H~thymidine (about 20 Ci/mmole) per testis. 31 days later the mice were sacrificed and approx. IO' mature spermatozoa were obtained from the vas deferentia and cauda epididymi of each mouse 1°. The level of radioactivity was 1. 5 •lO -3 d.p.m./sperm. The sperm were washed and suspended in phosphate-buffered saline and kept at 4 °. Mouse L cells were labelled with II*C~thymidine in suspension culture I and DNA was extracted by means of a phenol-water procedure. The UV source consisted of 4 germicidal lamps with an output predominantly at 253. 7 nm. 3-ml samples of spermatozoa at 3" IOe cells per ml were placed in I-cmpathlength quartz cuvettes and stirred during irradiation. The incident UV dose rate under these conditions was approx. 3' IO4 erg/mm* per rain (ref. 4). The sample temperature during irradiation was approx. 14 °. The apparent absorbance at 253.7 nm of the sperm under these conditions was about I.O as measured in a Zeiss PMQ I I spectrophotometer. Most of this apparent absorbance was due to light scattering, hence the normally used dose correction for shielding due to absorption s proved to be unnecessary. That this was so was determined by irradiating labelled L cells at lO 4 per ml (apparent absorbance less than o.I at 253.7 nm) in the presence of different concentrations of sperm ( o - I . lO 7 sperm per ml). The percent of thymine present as dimer in the L cells after identical exposure times was unaffected by the presence of sperm up to a concentration of 5'IOe sperm per ml. Photoproducts in spermatozoa were assayed as done previously for mouse L cells. For comparison, L cell DNA was irradiated in the presence of 3"IO~ sperm/ml and photoproducts were assayed. E14C1Thymine-labelled spore product from UV-irradiated B. megaterium spores was obtained as a gift from R. S. STAFFORD,Oak Ridge National Laboratory. Mutation Res., 14 (1972.) 133-136
I34
SHORT COMMUNICATIONS
Io00
o)
-~
'
I0O
°
!.
IO0
J
i
C e n t i m e t e r s on C h r o m a t o g r a m
Fig. I. R a d i o a c t i v i t y d i s t r i b u t i o n on c h r o m a t o g r a m s of h y d r o l y s a t e s of [ a H ] t h y m i d i n e - l a b e l l e d mo use s p e r m : (a) u n i r r a d i a t e d sperm, (b) s p e r m i r r a d i a t e d w i t h o.8. lO 4 e r g / m m 2 of UV a t 253.7 A
nm. I, p o s i t i o n of m a r k e r t h y m i n e ; I1, p o s i t i o n of m a r k e r T U ; I I I , p o s i t i o n of m a r k e r T T ; S.P., p o s i t i o n of m a r k e r spore p r o d u c t : O, the origin of t h e c h r o m a t o g r a m ; S.F., t h e s o l v e n t front.
Results
Sperm labelled with E3H]thymidine were irradiated, hydrolyzed and chromatographed. Typical radiochromatograms are presented in Fig. I. Unirradiated sperm (Fig. Ia) showed a single peak of radioactivity, I, with an RF of 0.58 corresponding to thymine. UV irradiation produced two photoproducts, II and III, with Rv's of o.18 and 0.26, respectively, which correspond exactly with the position of marker thymineuracil dimers (q~lJ) and T>l" (see Fig. ib). Throughout the exposure range employed, ,
,
,
,
, .*'J
E "u
8
T"T
1,= •
|
0- 6
~4 ~2
.
.2 .
.
.4 .
.
.6
" / ~2
'
go
'
I 0
e r g / m m 2 . 10-4
Fig. 2. T h e f o r m a t i o n of TT a n d TC (the l a t t e r a n a l y s e d as TU) in m o u s e s p e r m a t o z o a (closed symbols) a n d mouse L cell D N A (open symbol) as a f u n c t i o n of UV e x p o s u r e a t 253.7 nm. M u t a t i o n R e s . , 14 (T972) X33--T36
SHORT COMMUNICATIONS
135
from 2.1o 3 to IOe erg/mm 2, these were the only two photoproducts detected. Negligible spore photoproduct was present. Peaks II and I I I were designated as TU and "FI", respectively. The former peak actually represents T'C dimer which was deaminated upon hydrolysis. The assignment of the latter peak as TT is confirmed by eluting it from chromatograms and re-irradiating in distilled water with the UV lamp. It was converted back to thymine with the same kinetics as marker T~'T.The possibility cannot be excluded that peak I I I contains some UT-adduct 14, but from the reversibility of I I I back to thymine the yield of this product must be less than lO% of TT. The kinetics of "F~" and "FC formation were measured by exposing identical samples of labelled sperm to different UV doses and determining the percentage of thymine present as " ~ or TTZ (Fig. 2). Both the kinetics and saturation yields of "1~" produced in mouse sperm were similar to previous results obtained with mouse L cells 1. The single point for f l " dimer production in L cell DNA also falls on the curve for sperm DNA in Fig. 2. The selective removal of "F~" by photoreactivation or intracellular repair processes was investigated. However, neither exposure to fluorescent light nor incubation in cell-culture medium at 34 ° for 24 h altered the percent of thymine present as dimer. This lack of photoreactivation is expected since no photoreactivating enzyme has been found in the cells of placental mammals 3. /x
Discussion The photochemistry of DNA in mouse spermatozoa was indistinguishable from that in mouse L cells or of DNA in solution. Furthermore, the lack of any detectable spore product indicates that sperm DNA is more like that of somatic cells than that of bacterial spores. Since the photochemistry, and particularly the rate of formation of dimers, is dependent on configuration, we conclude that the configuration of DNA in mouse spermatozoa is similar to that of DNA in solution, which is most likely in the B configuration 11. This conclusion is consistent with that determined for the DNA in several nonmammalian spermatozoa which have been shown by X-ray diffraction to be helical and probably in the B form 19. The observed differences between mammalian cells with respect to UV sensitivity*,2,17 might be due to alterations in either the production of photoproducts, the repair of the photochemical lesions or the ability to function with a given number of dimers in the DNA. Spermatozoa provide a test of the first possibility since the packaging of the DNA is so different. The results obtained in this study indicate that variations between mammalian cell types in sensitivity to lethal or mutagenie effects of UV irradiation is not likely a result of differences in photochemistry. Measurements of the biological effects of UV irradiation of mouse spermatozoa have been made using the results of artificial insemination as an assay 5. Although the lack of uniform irradiation of the sample in that study introduced some error in the dosimetry, the results indicated that IO4 erg/mm 2 to the sperm prevented implantation of the embryo, and lO 3 erg/mm 2 reduced the litter size to half. The latter value is about Io-fold higher than the Do dose of about lO 2 erg/mm 2 for mammalian ceils in culture I and m a y either indicate more efficient repair of UV damage in fertilized ova and early embryos than in somatic cells or selection for those sperm, at the level of fertilization, which received less than the nominal dose of radiation. Finally, thymine Mutation Res., 14 (1972) 133-136
136
SHORT COMMU NICATIONS
d i m e r s h a v e b e e n s h o w n t o b e p r e m u t a t i o n a l l e s i o n s in b a c t e r i o p h a g e 1~, T h e s e l e s i o n s a r e also p r o d u c e d in s p e r m a t o z o a a n d c o u l d b e r e s p o n s i b l e for t h e o b s e r v e d m u t a tionsS, 1~. H o w e v e r , t h i s h y p o t h e s i s r e m a i n s t o b e t e s t e d . W e t h a n k Mr. GORDON HUNTER a n d Mr. VINCENT W . S. ENG for e x c e l l e n t t e c h n i c a l a s s i s t a n c e , Dr. W . R. BRUCE for u s e f u l d i s c u s s i o n s , a n d t h e N a t i o n a l C a n c e r I n s t i t u t e a n d t h e M e d i c a l R e s e a r c h C o u n c i l of C a n a d a for f i n a n c i a l s u p p o r t .
Department of Medical Biophysics, University of Toronto, and Ontario Cancer Institute, Toronto (Canada)
M. L. MEISTRICH A. M. RAUTH
I CHIU, S., AND A. M. I~AUTH, A comparison of the sensitivity to ultraviolet light of mouse L cells and mouse bone marrow cells assayed in vitro, Radiation Res., 47 (1971) 11o-122. 2 CLEAVER, J. E., DNA repair and radiation sensitivity in human (xeroderma pigmentosum) cells, Int. J. Radiation Biol., 18 (197 o) 557-565 . 3 CooK, J. S., Photoreactivation in animal cells, in A. C. GIESE (Ed.), Photophysiology, Vol. 5, Academic Press, New York, 197 ° , pp. 191-233. 4 DOMON, M., B. BARTON, A. PORTE AND A. M. RAUTH, T h e i n t e r a c t i o n of caffeine w i t h ultraviolet-light-irradiated DNA, Int. J. Radiation Biol., 17 (197 o) 395-399. 5 EDWARDS, R. G., The experimental induction of gynogenesis in the mouse, 1I. Ultraviolet irradiation of the sperm, Proc. Roy. Soc., Ser. B, 146 (1957) 488-5o4. 6 FAWCETT, D. W., The structure of the mammalian spermatozoan, Intern. Rev. Cytol., 7 (1958) 195-234. 7 HOWATSON, A., AND W. R. BRUCE, in P. FAVARD (Ed.), Microscopie Electronique, Vol. 3, Soci6t6 fran~aise de Microscopie ]~lectronique, Paris, 197 o, pp. 639-64 o. 8 JOHNS, H. E., in K. KUSTIN (Ed.), Methods in Enzyrnology, Vol. 16, Fast Reactions, Academic Press, New York, 1969, pp. 253-316. 9 KAO, F. T., AND T. T. PUCK, Genetics of s o m a t i c m a m m a l i a n cells, IX. Q u a n t i t a t i o n of mutagenesis by physical and chemical agents, J. Cell. Physiol., 74 (1969) 245-257. IO LAM, D. M. K., AND W. R. BRUCE, T h e b i o s y n t h e s i s of p r o t a m i n e d u r i n g s p e r m a t o g e n e s i s of the mouse : extraction, partial characterization, and site of synthesis, J. Cell. Physiol., 78 (1971 ) 13-24. i i LUZZATI, V., The structure of DNA as determined by X-ray scattering techniques, in J. N. DAVlDSON ANt)W. E. COHN(Eds.), Progress in Nucleic Acid Research, Vol. i, Academic Press, New York, 1963 , pp. 347-368. 12 MACKENZIE, K., AND H. J. MULLER, Mutation effects of ultraviolet light in Drosophila, Proc. Roy. Soc., Ser. B, 129 (194 o) 491-517 . 13 MEISTRICH, M. L., AND R. G. SHULMAN, Mutagenic effect of sensitized irradiation of bacteriophage T 4, J. Mol. Biol., 46 (1969) 157-167. 14 PATRICK, M. H., Near-UV photolysis of pyrimidine heteroadducts in E. coli DNA, Photochem. Photobiol., II (197 o) 477-485. 15 PROOST, J., Action mutag~ne des UV sur les ovogonies de Drosophiles, I. Cindtique de la mutag6n~se, Mutation Res., 4 (1967) 473-'I89 16 RAHN, R. O., AND J. L. Hosszu, Influence of relative humidity on the photochemistry of DNA films, Biochim. Biophys. Acta, 19o (1969) 126-131. 17 RAUTH, A. M., Effects of ultraviolet light on established lines of mammalian cells, in M. EBERT AND A. HOWARD (Eds.), Current Topics in Radiation Research, Vol. 6, North-Holland, Amsterdam, 197 ° , pp. 195-245. I 8 STAFFORD, I~. S., AND J. E. DONNELLAN Jr., P h o t o c h e m i c a l evidence for c o n f o r m a t i o n c h a n g e s in DNA during germination of bacterial spores, Proc. Natl. Acad. Sci. (U.S.), 59 (1968) 822829. 19 WILKINS, M. H. F., Physical studies of the molecular structure of DNA and nucleoprotein, Cold Spring Harbor Symp. Quant. Biol., 21 (1956) 75-90.
R e c e i v e d A u g u s t I 7 t h , I97~
Mutation Res., 14 (1972 ) I33 136