The kinetic complexity of Acetabularia chloroplast DNA

67

Biochimica et Biophysica Acta, 521 ( 1 9 7 8 ) 6 7 - - 7 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 9 9 2 9 9

THE KINETIC COMPLEXITY OF A C E T A B U L A R I A C H L O R O P L A S T DNA

U S H A P A D M A N A B H A N a n d B E V E R L E Y R. G R E E N

Botany Department, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada) ( R e c e i v e d May 1 8 t h , 1978)

Summary The kinetic complexity of Acetabularia cliftonii chloroplast DNA is 1.52 +0 . 2 6 . 109 daltons, compared to 0.2 • 109 daltons for Chlamydomonas chloroplast DNA. There is an average of three genomes per chloroplast. The unusually large size of the Acetabularia genome may reflect the ancient evolutionary history o f this organism.

Introduction

Acetabularia is a genus of large, uninucleate, single-celled green algae, which has been extensively studied because of some unusual characteristics. Without benefit o f cell or nuclear division, a mature cell can differentiate an umbrellashaped reproductive structure, or cap, at the apical end [1]. If the single nucleus, conveniently located in the rhizoid, is surgically removed before initiation of cap formation, the cell will continue to grow at the same rate as a nucleate cell for up to five or six weeks. Some cells will even differentiate a cap which is indistinguishable from the cap of a normal nucleate cell. The ability to get along without a nucleus extends to the subcellular level. The chloroplasts in an enucleated cell continue to increase in number with cell growth [2] and the a m o u n t o f non-nuclear DNA per cell continues to increase for several weeks [3]. Chloroplasts continue to incorporate precursors of DNA, R N A and protein [2,4]. These unusual features suggested that perhaps the chloroplasts are as unique as the organism, and might have a greater degree of genetic a u t o n o m y than chloroplasts of other algae. We therefore used the renaturation kinetics approach of Wetmur and Davidson [5] to determine the approximate size o f the chloroplast genome, and to compare it with the chloro-

Abbreviations: SSC, 0.15 M NaCI/0.015 M s o d i u m citrate; 0.1 × SSC: 15 mM NaCI/1.5 mM sodium citrate.

68 plast genome size previously determined for another green alga, Chlamydomonas [6--8]. Methods

A cetabularia cultures The original stocks of Acetabularia (Polyphysa) cliftonii were obtained from Dr. A. Gibor. Cysts were treated with 10% Argyrol (Crookes-Barnes) and 1% sodium d o d e c y l sulfate before germination to get rid of contaminants. Cells were grown in artificial sea water according to the method of Shephard [9]. The A. cliftonii cultures contained no detectable bacteria, b u t were treated with 1 mg/ml penicillin for several days before use. Two or three days prior to chloroplast isolation, the cells were placed in continuous darkness to destarch the chloroplasts. No cell which had initiated cap formation was used, to ensure that the nucleus of each cell would be removed with the rhizoid. DNA isolation Chloroplasts were isolated from enucleated cells as previously described [10]. Each preparation was routinely assayed for bacteria b y the Most Probably N u m b e r m e t h o d in enriched sea water [10]. In most cases, the DNA was isolated from detergent-lysed chloroplasts on an ethidium bromide/CsC1 gradient [10--12]. The main band fraction was extracted with isoamyl alcohol, dialysed against 0.1 X SSC in the dark, treated with pancreatic and T1 ribonucleases, then with pronase, and dialysed against SSC. The chloroplast DNA used in Expt. 2 was made b y Marmur's method [13] with the addition of a pronase digestion and dialysis instead of precipitation. Total cell DNA was isolated from Chlamydomonas reinhardii (wild-type) b y the m e t h o d of Wells and Sager [7], and the nuclear and chloroplast DNAs separated from each other b y t w o cycles of CsC1 centrifugation at 41 000 rev./min for 70 h in a 50 Ti rotor. DNA was isolated from E. coli B-23 b y Marmur's m e t h o d [13]. The E. coli K-12 DNA was the generous gift of Dr. Bela Sivak. Analytical ultracentrifugation B u o y a n t densities in CsC1 were determined according to the m e t h o d of Schfldkraut et al. [14] using Micrococcus lysodeikticus DNA (1.731 g/cm 3) as density reference. Sedimentation rates of sheared DNA were determined according to the m e t h o d of Studier [15] at pH 7 in 1.0 M NaC1, 0.05 M sodium citrate, and converted to 8 ~ , ¢ s for convenience. It was necessary to determine the sedimentation coefficient for each preparation since the value obtained varied from one preparation to the next even when the DNA was sheared under identical conditions. This may be due to the presence of single strand breaks resulting from extensive handling of a dilute sample. Renaturation kinetics Dialysed DNA samples at a concentration of 5--10 #g/ml in 0.1 X SSC were sheared by 2 passages through a 27-gauge needle at 4°C. The DNA was denaturated b y heating in a boiling water bath for 12 min. An aliquot was removed

69 and q u i c k ~ o o l e d for determination of the sedimentation coefficient. The denaturated DNA was transferred quickly to Teflon-stoppered semi-micro cuvettes which had been equilibrated at the renaturation ten~perature (66 or 70°C) and contained the appropriate volume of concentrated saline-citrate to give a final concentration of 2 × SSC. Renaturation was followed at 260 nm in a Gilford 2400 spectrophotometer. The maximum hyperchromicities were determined b y remelting the partly renaturated DNA at the end of the renaturation period. The absolute a m o u n t o f DNA in each sample was determined b y the diphenylamine m e t h o d as previously described [11]. It was necessary to do all the operations on the same sample because of the logistic difficulties involved in obtaining enough DNA. For example, Expt. 2 was done on a total o f 10 #g o f chloroplast DNA which was the yield from 5 chloroplast preparations involving the individual enuclation of approximately 10 000 cells. Renaturation rates were plotted as described b y Wetmur and Davidson [5] and the renaturation rate constant k2 determined from the slope. The k2 values were corrected to 1.0 M Na ÷ using Britten's tables [16] and to 50% G + C b y the method of Laird [17]. A sample of E. coli DNA was renatured as a standard in each experiment, and the corrected k2 values were normalized to give a kinetic complexity of 2.5 • 109 daltons for the E. coli DNA [5,18]. Results Acetabularia chloroplast DNA prepared b y the ethidium bromide/CsC1 m e t h o d formed a single band at 1.706 g / c • 3 when rerun in CsC1 w i t h o u t

1.8 1.7 1.6 II < 1.5 I < 1A

J I

1.3 1.2 1.1

1.695 1.706

1.731

p(g/¢m 2)

1.0

i

10

3'0

5'0

7'0

9'0

' 110

i~0

' 150

T I M E (rnin)

Fig. 1. A n a l y t i c a l CsCI gradients of isolated chloroplast DNA. Upper c u r v e : C. reinhardii. L o w e r c u r v e : A. cliftonii. T h e p e a k a t 1 . 7 3 1 g / c m 3 is M. Z y s o d e i k t i c u s r e f e r e n c e D N A . Fig. 2. Renaturation rate p l o t s . • A, C h l a m y d o m o n a s c h l o r o p l a s t D N A ( E x p t . 1); • m, E. coli DNA (Expt. 4); o o, A c e t a b u l a r i a c h l o r o p l a s t D N A ( E x p t . 4) -1, A c e t a b u l a r i a chloroplast D N A ( E x p t . 2). C h l o r o p l a s t D N A 5 I~g/ml; E. coil D N A 11 /~g/mL

70 ethidium (Fig. 1). It contained a negligible amount of the minicircular DNA of b u o y a n t density 1.712 g/cm 3, which is a minor c o m p o n e n t of the total chloroplast DNA [10]. The Chlamydomonas chloroplast DNA banded at 1.695 g/cm 3 and was completely free of nuclear DNA, which has a b u o y a n t density of 1.722 g/cm 3 [7]. Several renaturation rate plots are shown in Fig. 2. It can be seen that the Acetabularia chloroplast DNA renatured very slowly compared to Chlamydomonas chloroplast DNA at the same concentration. Its rate of renaturation was more comparable to that of E. coli DNA. Some preparations of Acetabularia chloroplast DNA renaturated as a single c o m p o n e n t (closed circles), while others showed an additional fast-renaturing c o m p o n e n t (open circles). However, the slope of the main c o m p o n e n t was the same in both experiments. The Chlamydomonas chloroplast DNA also had a rapid-renaturing component, in agreement with the published work of other [7,8]. It is not clear to us, or to the other workers, whether this represents snapback, a methodological artifact (difficulties with the recording apparatus in the first few minutes of the experiment), or a real c o m p o n e n t of chloroplast DNA. Since it was not present in all preparations, no a t t e m p t was made to determine its kinetic complexity. The rate constants, sedimentation coefficients, and kinetic complexities calculated b y the procedure of Wetmur and Davidson [5] are given in Table I. The kinetic complexities of the chloroplast DNAs were normalized to give a value of 2.5 • 109 daltons [5,18] for their E. coli controls. In most cases, values obtained experimentally for E. coli were between 2.1 and 2 . 6 . 1 0 9 daltons. The major c o m p o n e n t of Chlamydomonas chloroplast DNA had a kinetic complexity of 2.08 • 108 daltons, also in good agreement with published values [6--8]. This shows that reliable data can be obtained by this method at DNA concentrations of 5--10 pg/ml. To check the fidelity of duplex formation, DNA which had renatured for 4 h was remelted in 2 × SSC. The Acetabularia chloroplast DNA melted at 39.5 ° C, 0.7C ° lower than the calculated Tm of 92.8°C [19]. The E. coli DNA melted at 93.5°C, 0.5C ° lower than the predicted value. This indicates that there is only a small percentage of mismatching in the reformed duplexes [20]. TABLE I RENATURATION PLAST DNAs

K I N E T I C S O F A. C L I F T O N I I A N D C H L A M Y D O M O N A S

REINHARDH

CHLORO-

S h e a r e d , h e a t - d e n a t u r e d D N A w a s r e n a t u r e d i n 2 X S S C a t 6 6 o r 7 0 ° C , a n d t h e s e c o n d - o r d e r rate c o n s t a n t ( k 2 ) c o r r e c t e d t o 1 . 0 M N a + [ 1 6 ] . T h e c h l o r o p l a s t D N A s w e r e also c o r r e c t e d t o 5 0 % G + C [ 1 7 ] . N D, n o r m a l i z e d t o 2 . 5 • 1 0 9 f o r E. coli [ 5 , 1 8 ] .

Expt. No.

DNA

k2

~pH 13 ~20,w

1

Chlamydomonas chloroplast E. coli ( K - 1 2 ) A. cliftonii chloroplast E. coil ( B - 2 3 ) A. cliftonii chloroplast E. coli ( B - 2 3 ) A . cliftonli c h l o r o p l a s t E. coil ( K - 1 2 )

38.38 4.16 3.787 2.545 4.808 3.349 5.436 3.520

8.22 10.11 9.77 10.35 7.75 7.71 7.49 9.34

2 3 4

ND

2.08 • 108 1.57 • 109 1.75 • 109 1.23 • 109

71 The 3 experiments on A. cliftonii chloroplast DNA gave an average kinetic complexity o f 1.52 + 0.26 • 109 daltons. This is more than seven times larger than the Chlarnydomonas chloroplast genome, as determined b y us (Table I) and b y others [6--8]. It is approx. 60% of the size of the E. coli genome. This large kinetic complexity is not due to nuclear contamination, since the cells were enucleated and individually checked before chloroplasts were prepared from them. Neither is it due to bacterial contamination, since most o f the DNA was prepared from axenic cells, and in the other preparations the level of contamination o f the purified chloroplasts was t o o low to have contributed a significant a m o u n t of DNA to the total. Discussion The data in Table I show that the kinetic complexity of A. cliftonii chloroplast DNA is 7--8 times larger than Chlamydomonas chloroplast DNA. Similar results have been obtained for another species of Acetabularia, A. mediterranea [12]. This unexpected result raises the question of the accuracy of the kinetic complexity m e t h o d for determining genome sizes. In general, kinetic complexities o f bacterial DNAs agree rather well with the analytical complexities determined b y m i c r o s p e c t r o p h o t o m e t r y or chemical analysis [5,18]. However, there is some evidence that this m e t h o d may overestimate the genome sizes of algal chloroplast DNAs. Although three groups of workers [6--8] have obtained kinetic complexities of 1.9 and 2.0 • l 0 s daltons for Chlamydomonas chloroplast DNA (a remarkable degree of unanimity in the Chlamydomonas field), Behn and Herrmann (personal communication) found 63-pm circles in Chlamydomonas DNA preparations, which would give a kinetic complexity of only 1.3 • 108 daltons [21]. In apparent agreement with this, restriction fragments of purified Chlamydomonas chloroplast DNA add up to a maximum of 1.2 • 108 daltons [22], b u t these estimates could be low due to incomplete resolution or loss of small fragments during electrophoresis. Although there is no disagreement b e t w e e n kinetic complexity and electron microscopic measurem e n t for higher plant chloroplast DNAs [23,24], the possibility that the renaturation kinetics m e t h o d may over estimate genome size by as much as a factor of two should be kept in mind. This would probably be due to the fact t h a t the kinetic complexity is very sensitive to small changes in ~'~20, ¢vH13 W (Equation 18 of Wetmur and Davidson [5]), and all shearing methods produce a range of fragment sizes. Unfortunately, it is n o t possible to compare the kinetic complexity with the molecular length of Acetabularia chloroplast DNA, since intact molecules have n o t been obtained b y any of the methods tried. The largest molecules seen in chloroplast lysates were 200 ~zm long, and there was no clearly defined mean length [10]. This is hardly surprising, since a single molecule of 1.5 • 109 daltons would be 750 p m long and would be extremely sensitive to shear forces. A. cliftonii chloroplasts contain an average of 7.9 • 10 -is g of DNA each [12], compared to 8.6 • 10 -~s g and 4.8 • 10 -Is g for the single large cup-shaped chloroplasts o f Chlarnydomonas and Chlorella [26,25]. In other words, the analytical complexity of 4.7 • 109 daltons is close to that o f o t h e r green algal chloroplasts. However, with a kinetic complexity of 1 . 5 . 1 0 9 daltons, there

72 should be only three copies of the genome per chloroplast, in contrast to 26-52 copies in Chlamydomonas [6,7] and about 20 copies in Chlorella [25]. It is worth pointing o u t that if the three copies were not uniformly distributed among chloroplasts, it would explain Woodcock and Bogorad's finding that a large proportion of Acetabularia chloroplasts contained no detectable DNA at all [27]. It is clear that the Acetabularia chloroplast genome is different from other algal genomes, b o t h in its complexity and in the number of copies per chloroplast. Whether or not the renaturation kinetics m e t h o d overestimates the genome size, the kinetic complexity of Acetabularia chloroplast DNA is still much larger than that of Chlamydomonas chloroplast DNA. However, it is not at all clear whether this large genetic potential is expressed. Current evidence suggests that at least four chloroplast ribosomal proteins [28] and probably some thylakoid membrane proteins [29] are coded for b y the nuclear genome and apparently synthesized on cytoplasmic ribosomes. Isolated chloroplasts incorporate labeled amino acids into a number of membrane proteins, b u t into only a few of the more than 50 soluble proteins (Green, B.R., unpublished). It therefore appears that Acetabularia chloroplasts do n o t have complete genetic or biochemical a u t o n o m y . The Dasycladaceae, to which Acetabularia belongs, has an ancient fossil record, with recognizable members from strata more than 500 million years old [30]. Perhaps Acetabularia and its relatives have been as conservative on the molecular level as on the morphological level. Whether chloroplasts originated from photosynthetic endosymbionts, or b y compartmentalization of single cells, there might well have been a time when they conrained more genetic information than they do now. It is tempting to speculate that Acetabularia is a 'living fossil' which has not had its chloroplast DNA pruned d o w n b y selection or b y transfer to the nucleus [31].

Acknowledgements We should like to thank Livia Beck and Margaret Ward for cheerful technical assistance, B.L. Muir for helpful discussions and Dr. J.J. Stock for use of his spectrophotometer. This work was supported b y the National Research Council o f Canada.

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