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Isolation and base composition of DNA's of primitive land plants I. Ferns and fern-allies
402
BIOCHIMICA ET BIOPHYSICA ACTA
8BA 97092
ISOLATION AND BASE COMPOSITION OF DNA's OF PRIMITIVE LAND PLANTS I. FERNS AND FERN-ALLIES
B E V E R L E Y R. G R E E N
Department o/Botany, University o/ British Columbia, Vancouver 8, B.C. (Canada) (Received July 27th, I97~)
SUMMARY
DNA was isolated from three genera of ferns (Angiopteris, Ophioglossum, Ciboteum) and three genera of fern-allies (Psilotum, Equisetum, Selaginella), using a homogenization medium designed to minimize action of phenoloxidases. The DNA base compositions of these evolutionarily diverse plants fell within the narrow range of 37-41% (G+C) as determined by buoyant density in CsC1, except for Selaginella emiliana and S. kraussiana which were 45 and 46 % (G+C). The latter also contained satellite DNA's. The overall range of % (G+C) was comparable to that of angiosperms. This suggests that the 400 million years since the time of divergence of land plants has been too short for large changes in the DNA base composition to have occurred.
INTRODUCTION
A number of plants living today have existed apparently unchanged for millions of years. Fossils of club mosses similar to modern Lycopodium and Selaginella have been found in Carboniferous strata 325 million years or more old 1, as have fossils of jointed plants closely related to the modern genus Equisetum. Psilotum, with its minute leaves and simple vascular system, is very similar to the most ancient land plants. Although these plants are probably not related to each other or to the ferns, they are referred to as the "fern-allies". Modern ferns also closely resemble Carboniferous fossils ~. Since the lines of evolution of these "living fossils" diverged from each other and from those of the ancestors of the angiosperms and gymnosperms hundreds of millions of years ago, it might be expected that this would be reflected in large differences in the DNA base compositions among these plants. DNA was therefore isolated from three genera of fern-allies and three widely separated genera of ferns, using a homogenization medium designed to minimize the effects of high tissue acidity and phenoloxidase action. Base compositions were determined by centrifugation in CsC18. Abbreviation: SSC, o.I5 l~I NaCI, o.oi 5 IV[ sodium citrate.
Biochim. Biophys. Acta, 254 (1971) 402-4o6
DNA's oF FERNS AND FERN-ALLIES
403
MATERIALS A N D METHODS
Plant material Selaginella kraussiana, S. era(liana, Angiopteris evecta, and Ciboteum splendens, originally from the collections of T.M.C. Taylor and V. J. Krajina, were obtained from the B o t a n y Department greenhouse. Psilotum nudum and Ophioglossum pendulum ssp. ]alcatum were collected in Hawaii by the author and identified b y Drs. C. H. Lamoureux and V. J. Krajina. Ophioglossum petiolatum was the gift of Dr. R. L. Peterson, Guelph University. Equisetum arvense was collected locally and identified b y Dr. K. Beamish. DNA isolation The homogenization medium consisted of i M NaCI, o.I M sodium diethyl dithiocarbamate, o.I 1~ Tris, 0.005 M EDTA (pH 9.0). Diethyl dithiocarbamate combines with quinones, inhibits o-diphenyl oxidases and prevents tannin formation 2. The high pH and buffer concentration counteract tissue acidity, and EDTA inactivates cation-dependent nucleases. Plant tissue was chilled, cut up with scissors or put through a meat grinder, then ground in a mortar with sand and enough cold homogenization medium to give a slurry. The homogenate was squeezed through four layers of cheesecloth and reextracted. Pooled extracts were made 1 % in sodium dodecyl sulfate and stirred with an equal volume of 9° % phenol at room temperature for 30 min. The aqueous phase was removed by centrifugation, and 2 vol. of 95 % ethanol added. If the DNA did not come out as fibres, the solution was left overnight at 4 ° to permit complete precipitation. The precipitate or fibres were washed with cold 67 % ethanol and dissolved in several ml of o.15 M NaCI, O.Ol5 M sodium citrate (SSC). The preparation was continued according to I~ARMUR4 with the addition of 50/~g/ml ~-amylase in the ribonuclease step, followed by several hours digestion with 5 °/~g/ml pronase (predigested) at 5o°. Depending on the amount of DNA present 5, it was either precipitated with ethanol or dialyzed extensively against SSC. DNA was also prepared from Equisetum with Kirby's homogenization medium e,7 which consisted of 0.3 M sodium diethyl dithiocarbamate and O.Ol5 M phenolphthalein diphosphate (pH 8.8). After the first ethanol precipitation the prepatation was continued as above. Base composition determination DNA base compositions were determined by centrifugation to equilibrium in CsCI according to SCHILDKRAUTet al. 8 with Micrococcus lysodeikticus DNA (p = 1.731 g/cm 3) as density reference. Repeated determinations on the same sample gave a standard deviation of less than :t:o.ooi g]cm a. Base compositions were also determined for P. nudum and O. pendulum from thermal denaturation profiles in SSC ~ or o.I × SSC1°, using a Gilford recording spectrophotometer. RESULTS Equisetum DNA prepared with both homogenization media gave bright yellow fibres at the first alcohol precipitation. The colour was not removed b y washing in
Block(re. Biophys. Acta, 254 (1971) 402-406
4o4
B . R . GREEN
67 % ethanol, but after ribonuclease treatment and deproteinization, it remained in the supernatant on subsequent alcohol precipitation, or was reduced in amount b y dialysis against o.I × SSC. On the other hand, when DNA was prepared from a crude nuclear fraction b y M.ARMUR'S 4 method, it was brown, and the colour could not be removed b y extensive deproteinization, repeated precipitation or dialysis. Including mercaptoethanol (20 raM) or polyvinylpyrrolidone (polyclar AT, 6 g/ioo g tissue) did not help. It therefore appears that the diethyl dithiocarbamate coming into contact during homogenization prevented the formation o strong bonds between polymerized phenolics and DNA. Yields of DNA were 1.5-3. 9 mg/ioo g fresh weight for Psilotum, Equisetum, and the Ophioglossums, and 0.2-0. 4 mg/Ioo g for the others. Equisetum extracted with Kirby's phenolphthalein diphosphate-containing medium 6,7 gave a slightly lower yield than with the medium reported here, but the two solutions were not compared on the same batch of tissue. Since Kirby's medium precipitates on storage at 4 °, it was not used further. Except for Selaginella kraussiana and S. emiliana, all the DNA's formed unimodal bands in CsC1 (Table I). S. kraussiana contained a second band at 1.713 g/cm 3. It was insensitive to x-amylase, pancreatic and Tl-ribonucleases and pronase, but disappeared on treatment with deoxyribonuclease. It was found in two preparations made as described above, but not in one where the tissue was disrupted in buffered IO % glycerol and crude nuclear and chloroplast fractions separated on a step gra-
TABLE I BUOYANT DENSITIES,
MELTING POINTS AND BASE COMPOSITIONS OF
DNA's
FROM FERNS AND FERN--
ALLIES
Division
Psilophyta
Family
% (G+C)
1.696
37
1.7o5 1.713 1.7o4 1.695, 1.711
46 54 45 36 , 52
1.698
39
1.697
38
1.698 1.697
39 38
1.7oo
41
Thermal denaturation Tm (°C)
% (G + C)
85.4" 68.o**
39 37
86.o*
4°
Psilotales
Psilotum nudum Lycopodophyta
CsCl gradients Buoyant density (g/cm 8)
Selaginellales
Selaginella kraussiana S. emiliana Arthrophyta
Equisetales
Pterophyta
Marattiales
Equisetum arvense A ngiopteris evecta Ophioglossales
Ophioglossum pendulum ssp. /alcatum
O. petiolatum Filicales
C iboteum s plendens
* D e t e r m i n e d in o.15 M NaCI, O.Ol5 M s o d i u m citrate 9. ** D e t e r m i n e d in o.oi 5 M NaC1, o.ooi5 M s o d i u m c i t r a t O °.
Biochim. Biophys. Acta, 254 (1971) 402-406
D N A ' s OF FERNS AND FERN-ALLIES
405
dient. In that case, both fractions yielded a single peak at 1.7o4-1.7o 5 g/cm 3. This suggests that the extra band might be mitochondrial DNA. The preparation from Selaginella emiliana contained three deoxyribonuclease-sensitive bands; the main band at 1.7o 4 g/cm 3 and two shoulders at 1.695 and 1.711 g/cm 3. They were not investigated further. Psilotum nudum and Ophioglossum pendulum DNA's showed normal monophasic thermal denaturation curves. Base compositions calculated from them ~,1° were consistent with those calculated from the buoyant densities (Table I), indicating that these two DNA's did not contain a large fraction of 5-methylcytosine or other odd bases n. The base compositions of the other plants, which were calculated only from buoyant densities, were therefole not corrected for the possible presence of 5-methylcytosine. Any which contain as much 5-methylcytosine as angiosperm DNA (4-6 %) would have to be corrected upward b y 2-3 %J~.
DISCUSSION
The range of DNA base compositions of the ferns and fern-allies repolted here is 37-46 % (G+C), or 3 7 - 4 1 % ( G + C ) if Selaginella is exluded. This is a surprisingly narrow range for a group of organisms which diverged from each other at least 400 million years ago 1. Furthermore, the range is v e I y similar to the range of 36-43 % ( G + C ) found b y BISWAS AND SARKAR3 for 61 species of angiosperms, with the exception of the family Gramineae which was 48-49 % (G+C). The range of 3 8 - 4 1 % ( G + C ) for ferns is consistent with 42 % (G+C) reported b y ISHIDA et al. TM for Pteridmm sp. The narrow range of DNA base compositions means that this parameter is not useful for determining phylogenetic relationships even between the most primitive and most modern vascular plants. However, the narrow range itself requires some explanation. Current estimates of the rate of base pair substitution derived from amino acid substitution in vertebrate 14-1e,19 and higher plant 18 proteins and from DNA sequence homologies 17 are of the order of lO -l° to lO -9 substitutions per base pair per year. If half of these resulted in a change of a (G+C) pair to an ( A + T ) pair or vice versa, and 400 million years is taken as the time of divergence of the primitive vascular plants 1, any given base pair would have had a probability of only 0.02-0.2 of being converted to the other kind. The narrow range of base compositions found for the vascular plants would therefore be due to the relatively short length of time of divergence from a common ancestor.
ACKNOWLEDGEMENTS
This study was supported b y the National Research Council of Canada (Grant A67-4688 ). I wish to t h a n k Drs. J. Levy and S. H. Zbarsky and the Canada Department of Agriculture for the use of equipment, and Dr. J a c k Maze for helpful discussions.
Biochim. Biophys. Acta, 254 (1971) 402-406
406
B.R.
GREEN
REFERENCES i 2 3 4 5 6 7 8 9 IO
Ii 12 13 14 15 16 17 18 19
H. P. ]3ANKS, Evolution and Plants o/the Past, W a d s w o r t h , B e l m o n t , 197 o. J. w . ANDERSON, Phytochemistry, 7 (1968) 1973. S. ]3. ]3ISWAS AND A. K. SARKAR, Phytochemistry, 9 (197 o) 2425. J. MARMUR, J. Mol. Biol., 3 (1961) 208. K. ]3URTON, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, Vol. 1213, A c a d e m i c Press, 1968, p. 163. K. S. KIRBY, Biochem. J., 66 (1957) 495. J. W . LYTTLETON AND G. ]3. PETERSEN, Biochim. Biophys. ,4cta, 80 (1964) 391. C. L. SCHILDKRAUT, J. I~ARMUR AND P. DOTY, j . Mol. Biol., 4 (1962) 43 o. M. MANDEL AND J. MARMUR, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, Vol. 1213, A c a d e m i c Press, 1968, p. 195. i . MANDEL, L. I G ~ m , J. ]3ERGENDAHL, M. L. DODSON AND E. SCHELTGEN, J. Bacteriol., IOI (197 ° ) 333J. T. O. KIRK, J. Mol. Biol., 28 (1967) 171. Nf. R. ISHIDA, T. KIKUCHI, T. IKUSHIMA, T. MATSUBARA AND ~NI. I~¢IIZUMA,,4nnu. Rep. Res. Reactor Inst. Kyoto Univ., 3 (I97 o) 5 I. J. T. O. KIRK, in .4 utonomy and Biogenesis o/ Mitochondria and Chloroplasts, Syrup. ,4 ust. ,4 cad. Sci., 1971, N o r t h Holland, A m s t e r d a m , p. 267. J. L. KING AND T. H. JUKES, Science, 164 (1969) 788. lVi. KIMURA, Nature, 217 (1968) 624. M. NEI, Nature, 221 (1969) 4 o. C. D. LAIRD, ]3. L. ~/[cCONAUGHY AND ]3. J. MCCARTHY, Nature, 224 (1969) 149. D. ]3OULTER, E. W. THOMPSON, J. A. M. RAMSHAW AND M. RICHARDSON, Nature, 228 (197 o) 552 • IV[. O. DAYHOFF, ,4tlas o/ Protein Sequence and Structure, N a t i o n a l B i o m e d i c a l R e s e a r c h F o u n d a t i o n , Silver Spring, Ohio, 1969, p. 7.
Biochim. Biophys. ,4cta, 254 (1971) 4 o 2 - 4 o 6
BIOCHIMICA ET BIOPHYSICA ACTA
8BA 97092
ISOLATION AND BASE COMPOSITION OF DNA's OF PRIMITIVE LAND PLANTS I. FERNS AND FERN-ALLIES
B E V E R L E Y R. G R E E N
Department o/Botany, University o/ British Columbia, Vancouver 8, B.C. (Canada) (Received July 27th, I97~)
SUMMARY
DNA was isolated from three genera of ferns (Angiopteris, Ophioglossum, Ciboteum) and three genera of fern-allies (Psilotum, Equisetum, Selaginella), using a homogenization medium designed to minimize action of phenoloxidases. The DNA base compositions of these evolutionarily diverse plants fell within the narrow range of 37-41% (G+C) as determined by buoyant density in CsC1, except for Selaginella emiliana and S. kraussiana which were 45 and 46 % (G+C). The latter also contained satellite DNA's. The overall range of % (G+C) was comparable to that of angiosperms. This suggests that the 400 million years since the time of divergence of land plants has been too short for large changes in the DNA base composition to have occurred.
INTRODUCTION
A number of plants living today have existed apparently unchanged for millions of years. Fossils of club mosses similar to modern Lycopodium and Selaginella have been found in Carboniferous strata 325 million years or more old 1, as have fossils of jointed plants closely related to the modern genus Equisetum. Psilotum, with its minute leaves and simple vascular system, is very similar to the most ancient land plants. Although these plants are probably not related to each other or to the ferns, they are referred to as the "fern-allies". Modern ferns also closely resemble Carboniferous fossils ~. Since the lines of evolution of these "living fossils" diverged from each other and from those of the ancestors of the angiosperms and gymnosperms hundreds of millions of years ago, it might be expected that this would be reflected in large differences in the DNA base compositions among these plants. DNA was therefore isolated from three genera of fern-allies and three widely separated genera of ferns, using a homogenization medium designed to minimize the effects of high tissue acidity and phenoloxidase action. Base compositions were determined by centrifugation in CsC18. Abbreviation: SSC, o.I5 l~I NaCI, o.oi 5 IV[ sodium citrate.
Biochim. Biophys. Acta, 254 (1971) 402-4o6
DNA's oF FERNS AND FERN-ALLIES
403
MATERIALS A N D METHODS
Plant material Selaginella kraussiana, S. era(liana, Angiopteris evecta, and Ciboteum splendens, originally from the collections of T.M.C. Taylor and V. J. Krajina, were obtained from the B o t a n y Department greenhouse. Psilotum nudum and Ophioglossum pendulum ssp. ]alcatum were collected in Hawaii by the author and identified b y Drs. C. H. Lamoureux and V. J. Krajina. Ophioglossum petiolatum was the gift of Dr. R. L. Peterson, Guelph University. Equisetum arvense was collected locally and identified b y Dr. K. Beamish. DNA isolation The homogenization medium consisted of i M NaCI, o.I M sodium diethyl dithiocarbamate, o.I 1~ Tris, 0.005 M EDTA (pH 9.0). Diethyl dithiocarbamate combines with quinones, inhibits o-diphenyl oxidases and prevents tannin formation 2. The high pH and buffer concentration counteract tissue acidity, and EDTA inactivates cation-dependent nucleases. Plant tissue was chilled, cut up with scissors or put through a meat grinder, then ground in a mortar with sand and enough cold homogenization medium to give a slurry. The homogenate was squeezed through four layers of cheesecloth and reextracted. Pooled extracts were made 1 % in sodium dodecyl sulfate and stirred with an equal volume of 9° % phenol at room temperature for 30 min. The aqueous phase was removed by centrifugation, and 2 vol. of 95 % ethanol added. If the DNA did not come out as fibres, the solution was left overnight at 4 ° to permit complete precipitation. The precipitate or fibres were washed with cold 67 % ethanol and dissolved in several ml of o.15 M NaCI, O.Ol5 M sodium citrate (SSC). The preparation was continued according to I~ARMUR4 with the addition of 50/~g/ml ~-amylase in the ribonuclease step, followed by several hours digestion with 5 °/~g/ml pronase (predigested) at 5o°. Depending on the amount of DNA present 5, it was either precipitated with ethanol or dialyzed extensively against SSC. DNA was also prepared from Equisetum with Kirby's homogenization medium e,7 which consisted of 0.3 M sodium diethyl dithiocarbamate and O.Ol5 M phenolphthalein diphosphate (pH 8.8). After the first ethanol precipitation the prepatation was continued as above. Base composition determination DNA base compositions were determined by centrifugation to equilibrium in CsCI according to SCHILDKRAUTet al. 8 with Micrococcus lysodeikticus DNA (p = 1.731 g/cm 3) as density reference. Repeated determinations on the same sample gave a standard deviation of less than :t:o.ooi g]cm a. Base compositions were also determined for P. nudum and O. pendulum from thermal denaturation profiles in SSC ~ or o.I × SSC1°, using a Gilford recording spectrophotometer. RESULTS Equisetum DNA prepared with both homogenization media gave bright yellow fibres at the first alcohol precipitation. The colour was not removed b y washing in
Block(re. Biophys. Acta, 254 (1971) 402-406
4o4
B . R . GREEN
67 % ethanol, but after ribonuclease treatment and deproteinization, it remained in the supernatant on subsequent alcohol precipitation, or was reduced in amount b y dialysis against o.I × SSC. On the other hand, when DNA was prepared from a crude nuclear fraction b y M.ARMUR'S 4 method, it was brown, and the colour could not be removed b y extensive deproteinization, repeated precipitation or dialysis. Including mercaptoethanol (20 raM) or polyvinylpyrrolidone (polyclar AT, 6 g/ioo g tissue) did not help. It therefore appears that the diethyl dithiocarbamate coming into contact during homogenization prevented the formation o strong bonds between polymerized phenolics and DNA. Yields of DNA were 1.5-3. 9 mg/ioo g fresh weight for Psilotum, Equisetum, and the Ophioglossums, and 0.2-0. 4 mg/Ioo g for the others. Equisetum extracted with Kirby's phenolphthalein diphosphate-containing medium 6,7 gave a slightly lower yield than with the medium reported here, but the two solutions were not compared on the same batch of tissue. Since Kirby's medium precipitates on storage at 4 °, it was not used further. Except for Selaginella kraussiana and S. emiliana, all the DNA's formed unimodal bands in CsC1 (Table I). S. kraussiana contained a second band at 1.713 g/cm 3. It was insensitive to x-amylase, pancreatic and Tl-ribonucleases and pronase, but disappeared on treatment with deoxyribonuclease. It was found in two preparations made as described above, but not in one where the tissue was disrupted in buffered IO % glycerol and crude nuclear and chloroplast fractions separated on a step gra-
TABLE I BUOYANT DENSITIES,
MELTING POINTS AND BASE COMPOSITIONS OF
DNA's
FROM FERNS AND FERN--
ALLIES
Division
Psilophyta
Family
% (G+C)
1.696
37
1.7o5 1.713 1.7o4 1.695, 1.711
46 54 45 36 , 52
1.698
39
1.697
38
1.698 1.697
39 38
1.7oo
41
Thermal denaturation Tm (°C)
% (G + C)
85.4" 68.o**
39 37
86.o*
4°
Psilotales
Psilotum nudum Lycopodophyta
CsCl gradients Buoyant density (g/cm 8)
Selaginellales
Selaginella kraussiana S. emiliana Arthrophyta
Equisetales
Pterophyta
Marattiales
Equisetum arvense A ngiopteris evecta Ophioglossales
Ophioglossum pendulum ssp. /alcatum
O. petiolatum Filicales
C iboteum s plendens
* D e t e r m i n e d in o.15 M NaCI, O.Ol5 M s o d i u m citrate 9. ** D e t e r m i n e d in o.oi 5 M NaC1, o.ooi5 M s o d i u m c i t r a t O °.
Biochim. Biophys. Acta, 254 (1971) 402-406
D N A ' s OF FERNS AND FERN-ALLIES
405
dient. In that case, both fractions yielded a single peak at 1.7o4-1.7o 5 g/cm 3. This suggests that the extra band might be mitochondrial DNA. The preparation from Selaginella emiliana contained three deoxyribonuclease-sensitive bands; the main band at 1.7o 4 g/cm 3 and two shoulders at 1.695 and 1.711 g/cm 3. They were not investigated further. Psilotum nudum and Ophioglossum pendulum DNA's showed normal monophasic thermal denaturation curves. Base compositions calculated from them ~,1° were consistent with those calculated from the buoyant densities (Table I), indicating that these two DNA's did not contain a large fraction of 5-methylcytosine or other odd bases n. The base compositions of the other plants, which were calculated only from buoyant densities, were therefole not corrected for the possible presence of 5-methylcytosine. Any which contain as much 5-methylcytosine as angiosperm DNA (4-6 %) would have to be corrected upward b y 2-3 %J~.
DISCUSSION
The range of DNA base compositions of the ferns and fern-allies repolted here is 37-46 % (G+C), or 3 7 - 4 1 % ( G + C ) if Selaginella is exluded. This is a surprisingly narrow range for a group of organisms which diverged from each other at least 400 million years ago 1. Furthermore, the range is v e I y similar to the range of 36-43 % ( G + C ) found b y BISWAS AND SARKAR3 for 61 species of angiosperms, with the exception of the family Gramineae which was 48-49 % (G+C). The range of 3 8 - 4 1 % ( G + C ) for ferns is consistent with 42 % (G+C) reported b y ISHIDA et al. TM for Pteridmm sp. The narrow range of DNA base compositions means that this parameter is not useful for determining phylogenetic relationships even between the most primitive and most modern vascular plants. However, the narrow range itself requires some explanation. Current estimates of the rate of base pair substitution derived from amino acid substitution in vertebrate 14-1e,19 and higher plant 18 proteins and from DNA sequence homologies 17 are of the order of lO -l° to lO -9 substitutions per base pair per year. If half of these resulted in a change of a (G+C) pair to an ( A + T ) pair or vice versa, and 400 million years is taken as the time of divergence of the primitive vascular plants 1, any given base pair would have had a probability of only 0.02-0.2 of being converted to the other kind. The narrow range of base compositions found for the vascular plants would therefore be due to the relatively short length of time of divergence from a common ancestor.
ACKNOWLEDGEMENTS
This study was supported b y the National Research Council of Canada (Grant A67-4688 ). I wish to t h a n k Drs. J. Levy and S. H. Zbarsky and the Canada Department of Agriculture for the use of equipment, and Dr. J a c k Maze for helpful discussions.
Biochim. Biophys. Acta, 254 (1971) 402-406
406
B.R.
GREEN
REFERENCES i 2 3 4 5 6 7 8 9 IO
Ii 12 13 14 15 16 17 18 19
H. P. ]3ANKS, Evolution and Plants o/the Past, W a d s w o r t h , B e l m o n t , 197 o. J. w . ANDERSON, Phytochemistry, 7 (1968) 1973. S. ]3. ]3ISWAS AND A. K. SARKAR, Phytochemistry, 9 (197 o) 2425. J. MARMUR, J. Mol. Biol., 3 (1961) 208. K. ]3URTON, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, Vol. 1213, A c a d e m i c Press, 1968, p. 163. K. S. KIRBY, Biochem. J., 66 (1957) 495. J. W . LYTTLETON AND G. ]3. PETERSEN, Biochim. Biophys. ,4cta, 80 (1964) 391. C. L. SCHILDKRAUT, J. I~ARMUR AND P. DOTY, j . Mol. Biol., 4 (1962) 43 o. M. MANDEL AND J. MARMUR, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, Vol. 1213, A c a d e m i c Press, 1968, p. 195. i . MANDEL, L. I G ~ m , J. ]3ERGENDAHL, M. L. DODSON AND E. SCHELTGEN, J. Bacteriol., IOI (197 ° ) 333J. T. O. KIRK, J. Mol. Biol., 28 (1967) 171. Nf. R. ISHIDA, T. KIKUCHI, T. IKUSHIMA, T. MATSUBARA AND ~NI. I~¢IIZUMA,,4nnu. Rep. Res. Reactor Inst. Kyoto Univ., 3 (I97 o) 5 I. J. T. O. KIRK, in .4 utonomy and Biogenesis o/ Mitochondria and Chloroplasts, Syrup. ,4 ust. ,4 cad. Sci., 1971, N o r t h Holland, A m s t e r d a m , p. 267. J. L. KING AND T. H. JUKES, Science, 164 (1969) 788. lVi. KIMURA, Nature, 217 (1968) 624. M. NEI, Nature, 221 (1969) 4 o. C. D. LAIRD, ]3. L. ~/[cCONAUGHY AND ]3. J. MCCARTHY, Nature, 224 (1969) 149. D. ]3OULTER, E. W. THOMPSON, J. A. M. RAMSHAW AND M. RICHARDSON, Nature, 228 (197 o) 552 • IV[. O. DAYHOFF, ,4tlas o/ Protein Sequence and Structure, N a t i o n a l B i o m e d i c a l R e s e a r c h F o u n d a t i o n , Silver Spring, Ohio, 1969, p. 7.
Biochim. Biophys. ,4cta, 254 (1971) 4 o 2 - 4 o 6