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UBC Theses and Dissertations

Studies on Acetabularia chloroplast DNA Muir, Bernice L. 1974

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STUDIES ON ACETABULABIA CHLOROPLAST DNA by BERNICE L. MUIR B.Sc., Uni v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Botany The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date A p r i l 8. 1974 i i ABSTRACT The phy s i c a l properties and renaturation k i n e t i c s of DNA extracted from i s o l a t e d chloroplasts of Acetabulavia meditewanea has been studied. 3 It has a buoyant density of 1.702 g/cm , which corresponds to a base com-po s i t i o n of 42.8% G+C. When melted i n SSC, Acetabulavia chloroplast DNA has a Tm of 86.7°, corresponding to a base composition of 43% G+C. The close agreement of the base compositions calculated from the buoyant den-s i t y and the melting temperature indicates the absence of unusual bases i n Acetdbularia meditewanea chloroplast DNA. In 0.1 x SSC, Acetabulavia chloroplast DNA melts with a T m of 70.7°, and the melting t r a n s i t i o n i s very broad. The breadth of the melting tran-s i t i o n suggests that t h i s DNA has a high degree of intramolecular hetero-geneity. A d i f f e r e n t i a l p l o t of the thermal t r a n s i t i o n of A. meditewanea chloroplast DNA supports t h i s conclusion. The buoyant den s i t i e s of DNA from b a c t e r i a l contaminants found i n Acetabulavia cultures d i f f e r e d from the buoyant density of the chloroplast DNA. In any case, the amount of b a c t e r i a l contamination was too low to account f o r any of the r e s u l t s obtained. 9 Renaturation experiments ind i c a t e a k i n e t i c complexity of 1.1 x 10 daltons from Acetabulavia meditewanea chloroplast DNA. As a r e s u l t of un-c e r t a i n t i e s i n the values of a l k a l i n e sedimentation c o e f f i c i e n t s , t h i s c a l -culated k i n e t i c complexity may be too low. The possible genetic information contained i n the chloroplast DNA of Acetabulavia meditewanea i s discussed. i i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 7 Materials 7 Treatment of cysts p r i o r to germination 7 C u l t i v a t i o n and treatment of algae ... 10 I s o l a t i o n of chloroplasts 10 Preparation of chloroplast DNA •• 12 Monitoring f o r b a c t e r i a l contamination.... 14 I s o l a t i o n of DNA from b a c t e r i a l contaminants.. 15 Cesium chloride density gradients • 16 Electron microscopy 16 Preparation of E. aoli DNA 16 Preparation of bacteriophage T^ DNA . 17 Thermal denaturation p r o f i l e s and determination of T .. 18 r m Renaturation of DNA • 18 Determination of a l k a l i n e sedimentation c o e f f i c i e n t s . . . 20 RESULTS . . . 22 Attempts to obtain axenic cultures of Aeetabulavia 22 B a c t e r i a l contamination i n chloroplast preparations.... 23 Electron microscopy •. 25 Buoyant density and T m of Aeetabulavia chloroplast DNA. 27 Renaturation and k i n e t i c complexity of Aeetabulavia chloroplast DNA 32 Sedimentation c o e f f i c i e n t s . . . . . . . . . 38 DISCUSSION 41 i v LIST OF FIGURES Page Figure 1. Electron micrograph of t h i n sections of Aeetabulavia meditevvanea chloroplasts 26 Figure 2. CsCl buoyant density c e n t r i f u g a t i o n of Aeetabulavia meditevvanea chloroplast DNA 28 Figure 3. Melting p r o f i l e s of Aeetabulavia meditevvanea chloroplast DNA and E. coli DNA 30 Figure 4. D i f f e r e n t i a l p l o t of the thermal t r a n s i t i o n of Aeetabulavia meditevvanea chloroplast DNA and E. coli DNA 31 Figure 5. Renaturation rate p l o t s for Aeetabulavia meditevvanea chloroplast DNA.... 33 Figure 6. Melting p r o f i l e s of renatured Aeetabulavia meditevvanea chloroplast DNA and renatured E. eoli DNA 35 V LIST OF TABLES Page Table I. B a c t e r i a l contaminants of chloroplast preparations. 24 Table I I . K i n e t i c complexities of DNA from d i f f e r e n t sources 36 Table I I I . A l k a l i n e sedimentation c o e f f i c i e n t s of sheared DNA samples 39 Table IV. V a r i a t i o n i n k i n e t i c complexity with d i f f e r e n t values of SPH13 40 20 ,w Table V. K i n e t i c complexities of chloroplast DNAs from various sources 44 v i ACKNOWLEDGMENTS I wish to thank Dr. B.R. Green for many h e l p f u l discussions throughout the course of t h i s work, and for guidance i n preparing t h i s t h e s i s . Technical assistance from Mrs. V a l e r i e Macdonald, Mrs. Margaret Ward and Mrs. L i v i a Beck was greatly appreciated. I also wish to thank the Canada Department of Ag r i c u l t u r e , Dr. S.H. Zbarsky of the Biochemistry Department, Dr. J . Levy and Dr. J . J . Stock of the Microbiology Department, Dr. H. Kasinsky of the Zoology Department and Dr. A. Bree of the Chemistry Department, U.B.C, for generously allowing me the use of equipment. Last, but not l e a s t , I wish to acknowledge the 51,570 Acetabulavia c e l l s that gave t h e i r l i v e s i n the cause of science. Without them, t h i s r e -search would not have been possible. INTRODUCTION T h e p u r p o s e o f t h i s r e s e a r c h w a s t o d e t e r m i n e t h e c o m p l e x i t y o f Aeetabulavia meditevvanea c h l o r o p l a s t DNA f r o m i t s r e n a t u r a t i o n k i n e t i c s . Aeetabulavia meditevvanea h a s b e e n u s e d e x t e n s i v e l y t o s t u d y n u c l e o -c y t o p l a s m i c i n t e r a c t i o n s a n d , t h u s , i t i s o f i n t e r e s t t o d e t e r m i n e s o m e -t h i n g a b o u t t h e g e n e t i c p o t e n t i a l o f t h e c h l o r o p l a s t DNA. A l s o , w i t h Aeetabulavia i t i s p o s s i b l e t o o b t a i n c h l o r o p l a s t DNA w h i c h i s c o m p l e t e l y f r e e f r o m c o n t a m i n a t i o n b y n u c l e a r DNA. F o r t h e s e r e a s o n s , Aeetabulavia meditevvanea w a s c h o s e n f o r t h i s r e s e a r c h . R e n a t u r a t i o n k i n e t i c s a r e u s e f u l f o r s t u d y i n g t h e c o m p l e x i t y a n d , t h e r e f o r e , t h e g e n e t i c p o t e n t i a l o f DNA f r o m a p a r t i c u l a r s o u r c e . DNA may b e d e n a t u r e d b y h e a t , a l k a l i o r v a r i o u s c h e m i c a l s , r e s u l t i n g i n c o m p l e t e s e p a r a t i o n o f t h e c o m p l e m e n t a r y s t r a n d s . U n d e r a p p r o p r i a t e c o n d i t i o n s o f t e m p e r a t u r e , i o n i c s t r e n g t h a n d p H , t h e s e p a r a t e d c o m p l e m e n t a r y s t r a n d s come b a c k t o g e t h e r i n r e g i s t e r , i . e . r e n a t u r e . T h e c o n d i t i o n s f o r r e n a t u r a -t i o n a n d t h e k i n e t i c s o f t h e r e a c t i o n h a v e b e e n s t u d i e d e x t e n s i v e l y b y v a r i o u s r e s e a r c h e r s (12,13,14,40,45,65,68,74) a n d may b e s u m m a r i z e d a s f o l l o w s : 1) T h e r e n a t u r a t i o n r e a c t i o n i s a s e c o n d o r d e r p r o c e s s 2) T h e s e c o n d - o r d e r r a t e c o n s t a n t f o r r e n a t u r a t i o n i s i n v e r s e l y p r o p o r t i o n a l t o t h e c o m p l e x i t y o f t h e DNA, w h e r e c o m p l e x i t y i s d e f i n e d a s t h e t o t a l n u m b e r o f DNA b a s e p a i r s i n n o n - r e p e a t -i n g s e q u e n c e s . 3) T h e o p t i m u m t e m p e r a t u r e f o r r e n a t u r a t i o n i s 20° t o 30°C b e l o w t h e m e l t i n g t e m p e r a t u r e ( T m ) o f t h e DNA. 2 4) The DNA renaturation rate i s dependent on i o n i c strength of the solvent for an e l e c t r o l y t e such as NaCl. 5) Decreasing the molecular weight of a given DNA r e s u l t s i n a decrease i n the rate of renaturation of the DNA. 6) The rate of renaturation increases s l i g h t l y with increasing GC content. 7) The rate of renaturation of DNA depends on solvent v i s c o s i t y . + 8) Within the pH range 5 to 9 i n 0.4 M Na the rate of renatura-t i o n i s e s s e n t i a l l y independent of pH. 9) Correct base sequence matching can occur to a greater extent when the DNA i s sheared to low molecular weight. 10) The thermal s t a b i l i t y of the DNA i s decreased about one degree f o r 1.5 per cent mispaired bases i n the renatured DNA. Wetmur and Davidson (74) have derived the following equation for deter-mining the k i n e t i c complexity of DNA. N n = 331N = 5 . 5 . x . l 0 8 ( S ? S 1 3 ) 1 , 2 5 D ZU ,w  k2 where N^ i s the complexity of the DNA i n daltons, i s the second order rate constant for renaturation of the DNA: and S?|P"^  i s the a l k a l i n e s e d i -20 ,w mentation c o e f f i c i e n t of the DNA. This equation i s v a l i d when the renatura-t i o n rate constant i s determined at [Na +J = 1.0 mole 1 ^ i n aqueous s o l u t i o n at a temperature near (T -25)°C. m Acetabulavia has been an important research organism for two reasons. Since the 1930's, this marine alga has been used extensively to study 3 nucleo-cytoplasmic i n t e r a c t i o n s . More recently, Aeetabulavia became the f i r s t plant i n which i t was possible to demonstrate the existence of DNA i n the chlo r o p l a s t s . With Aeetabulavia med'itevvanea, a s i n g l e c e l l may achieve a length of 3 to 5 cm i n a period of 3 months. During t h i s vegetative part of the l i f e cycle a s i n g l e , large nucleus i s located i n the r h i z o i d at the base of the c e l l . Growth of the c e l l culminates i n the formation of a repro-ductive cap, which takes about one month. The diameter of the s t a l k at t h i s time may be 0.3 to 0.5 mm, while the f u l l grown cap may a t t a i n a diameter of up to one cm. When the cap i s f u l l y grown, the large primary nucleus divides to form several thousand small, secondary n u c l e i . By means of cytoplasmic streaming, the secondary n u c l e i are transported from the r h i z o i d to the cap, where cysts are formed (33,48). Thus, Aeetabulavia can r e a d i l y be enucleated by excising the r h i z o i d before the c e l l forms a cap. I f immature Aeetabulavia c e l l s are cut to produce a nucleate, r h i z o i d portion and an anucleate s t a l k portion, not only can the nucleate r h i z o i d regenerate a complete new plant, but the anucleate s t a l k can survive for several weeks and continue to grow. In some cases the anucleate s t a l k w i l l even d i f f e r e n t i a t e a cap. This and other findings led Hammerling to postu-l a t e the existence of "morphogenetic substances" which are produced by the nucleus but may be present i n the cytoplasm for long periods of time (33). Ex c i s i o n and i n t e r s p e c i f i c g r a f t i n g experiments have shown that these "morphogenetic substances" induce cap formation and are necessary for d i f -f e r e n t i a t i o n of a cap with s p e c i e s - s p e c i f i c morphology (33). Within a si n g l e Aeetabulavia c e l l are several m i l l i o n c h l o r o p l a s t s . 4 The number of chloroplasts increases exponentially during normal growth of the c e l l , with the chloroplast doubling time being a l i t t l e longer than one week (55). The chloroplasts increase i n number i n anucleate fragments, also, but i n th i s case the doubling time i s about two weeks (55). The presence of DNA i n the chloroplasts of Acetdbularva was shown i n 1963 (4,27). This was the f i r s t instance i n which DNA could be d e f i n i t e l y a t tributed to the chloroplasts without the p o s s i b i l i t y of contamination by nuclear DNA. The presence of DNA i n the chloroplasts leads to speculation about i t s possible r o l e i n d i r e c t i n g the a c t i v i t i e s of the c e l l . Knowledge of the amount of DNA i n the chloroplasts and the presence or absence of repeated nucleotide sequences can give an i n d i c a t i o n of the genetic poten-t i a l of the chloroplast DNA. The s i t u a t i o n i s complicated by observations that the amount of DNA per chloroplast i s v a r i a b l e (74). Using several techniques, Woodcock and Bogorad (75) detected large, but v a r i a b l e , amounts of DNA i n only 35% of the chl o r o p l a s t s . S i m i l a r l y , Green and Burton (30) found DNA associated with only 20-40% of chloroplasts osmotically shocked by the Kleinschmidt technique and observed with the electron microscope. Green and Burton (30) a t t r i b u t e t h i s to an i n t r a c h l o r o p l a s t a l nuclease. In s p i t e of these obser-vations, however, estimates of the average amount of DNA per chloroplast can be made. Using fluorometric analyses, Gibor and Izawa (27) estimated the —16 amount of DNA to be 1 x 10 g per chloroplast. This i s comparable to the amount of DNA i n a v i r u s . Observation of ikcetabulaxrva chloroplast DNA with the e l e c t r o n microscope has shown, however, that at l e a s t some chloroplasts 5 contain much larger amounts of DNA (30,31,75). This suggests that the estimate of Gibor and Izawa i s too low. More recently, using a scaled down diphenylamine assay for DNA, Green determined that the average amount of DNA per chloroplast i s about 2.3 x 10 g (unpublished). Tbis i s com-parable to the amount of DNA i n the genomes of a number of b a c t e r i a l spe-c i e s , including Achromobacter anitratus, Streptococcus faecalis3 Diplococcus pneumoniae and Staphylococcus aureus (3). Gibor and Izawa estimated the amount of DNA per chloroplast from the protein to DNA r a t i o . Their calcu-l a t i o n of the amount of protein per chloroplast was based on the assumption that proteins comprise 20% of the organelles. Green did actual counts of the number of chloroplasts. Perhaps t h i s accounts f o r the discrepancy be-tween the two r e s u l t s . Determination of the k i n e t i c complexity of the chloroplast DNA should help to resolve t h i s . A p r e r e q u i s i t e for the DNA renaturation reaction i s that the DNA be double stranded. Evidence f o r the double strandedness of Acetabularia mediterranea chloroplast DNA comes from electron microscopy (31,73) and from buoyant density studies of chloroplast DNA which has been heat dena-tured (32). Thus, i t i s possible to study the renaturation k i n e t i c s of the chloroplast DNA. The formula of Wetmur and Davidson (74) can then be used to determine the k i n e t i c complexity of Acetabularia chloroplast DNA. Tbestudy the renaturation k i n e t i c s , i t i s necessary to obtain r e l a t i v e -l y pure chloroplast DNA. With Acetabularia, contamination by nuclear DNA can be ruled out by excising the r h i z o i d s , which contain the n u c l e i , and i s o l a t i n g chloroplasts only from the stalks of immature c e l l s . I t i s more d i f f i c u l t to ru l e out the p o s s i b i l i t y of contamination by mitochondrial DNA, but the amount of DNA i n a mitochondrion appears to be l e s s than i n a 6 chloroplast (75). Therefore, many mitochondria have to be present i n a chloroplast preparation i n order to contribute a s i g n i f i c a n t amount of DNA. Such an amount w i l l show up as a second UV absorbing band at p = 1.714 g/cm i n a cesium chloride density gradient (32). Heilporn and Limbosch (34) f i n d that the mitochondrial DNA renatures faster than the chloroplast DNA. Therefore, mitochondrial DNA, i f present, may be detected as a faster renaturing component. Axenic cultures of Acetabulavia are d i f f i c u l t to maintain, so that contamination with b a c t e r i a l DNA can be a problem. This can be minimized by treating the Acetabulavia c e l l s with a n t i b i o t i c s before use, and by selective techniques during the chloroplast i s o l a t i o n . The chloroplast pre-parations can be monitored to determine the amount of b a c t e r i a l contamination. I f necessary, the chloroplast DNA can be separated from b a c t e r i a l DNA by centrifuging i n a CsCl density gradient, provided that the b a c t e r i a l DNA has a different buoyant density than the chloroplast DNA. Unfortunately, there has been disagreement about the buoyant density of the chloroplast DNA as reported i n the l i t e r a t u r e (9, 26, 32, 34). In the process of t h i s 3 research, this problem was resolved. A buoyant density of 1.702 g/cm was found for Acetabulavia chloroplast DNA. This agrees quite closely with the value given by Green et a l . (32) and Heilporn and Limbosch (34). Whatever the amount of DNA i n the chloroplasts, i t i s not very great and, thus, a major d i f f i c u l t y of t h i s research i s to obtain large enough quantities of DNA to study the renaturation k i n e t i c s . The r e l a t i v e l y slow growth of Acetabulavia, and the fact that each c e l l has to be handled i n -d i v i d u a l l y to enucleate i t , are further l i m i t a t i o n s to preparing large amounts of Acetabulavia chloroplast DNA. 7 MATERIALS AND METHODS The following abbreviations are used: BSA, bovine serum albumin; EDTA, disodium ethylenediamine tetraacetate; SDS, sodium dodecyl sulphate; SSC, standard s a l i n e c i t r a t e (0.15 M NaCl plus 0.015 M sodium c i t r a t e , pH 7.0); TES, N - t r i s (hydroxymethyl)methyl-2-aminoethane s u l f o n i c acid; T r i s , t r i s (hydroxymethyl)aminomethane. Materials Reagent grade chemicals were used throughout. F i c o l l was purchased from Pharmacia (Uppsala, Sweden). Pentex brand BSA was obtained from Miles Laboratories. TES was purchased from the N u t r i t i o n a l Biochemical Company but contained large amounts of impurities. Therefore, l a t e r sup-p l i e s of TES were obtained from Calbiochem. Mallinckrodt phenol or Fisher l i q u i f i e d phenol were used. Pancreatic ribonuclease and lysozyme were pur-chased from Worthington Biochemical Corp., while pronase and a-amylase were obtained from Calbiochem. Ribonuclease T^ was purchased from the N u t r i t i o n a l Biochemical Company. Aquacide I I i s a product of Calbiochem. O p t i c a l grade CsCl was purchased from Schwarz Bioresearch Inc. Treatment of cysts p r i o r to germination: Acetabulavia cysts are r e s i s t a n t to k i l l i n g by some agents which w i l l k i l l growing c e l l s . Thus, i n an attempt to obtain axenic cultures, cysts were treated i n several ways. A l l solutions and equipment were s t e r i l i z e d before use and s t e r i l e technique was used throughout the procedures. Ripe caps which had been stored i n seawater i n the dark at 10° for several months were washed with seawater and cut with s c i s s o r s to release 8 the cysts. (In some cases, Shephard's a r t i f i c i a l seawater medium (57) was used i n place of seawater). To separate c e l l debris from cysts, the cut caps were f i l t e r e d on 123 y mesh b o l t i n g s i l k , washed with sea water and the cysts drawn through by gentle suction. The cysts s e t t l e d to the bottom of the f l a s k a f t e r one to two minutes and the seawater was decanted. Fresh seawater was added and the contents of the f l a s k shaken f o r one or two minutes. A f t e r the cysts s e t t l e d , the seawater was decanted. This washing procedure was repeated f i v e or s i x times. The cysts were then treated i n one of the following ways: 1) The cysts were placed i n a small volume of 1% SDS i n d i s t i l l e d water and the mixture agitated for one hour with a small magnetic s t i r r i n g bar. The cysts were then allowed to s e t t l e and the SDS s o l u t i o n was poured o f f . Following t h i s , the cysts were washed seven times with seawater, each time l e t t i n g the cysts s e t t l e and removing the seawater by decantation or by c a r e f u l l y withdrawing i t with a Pasteur pipette. Then the cysts were placed i n 2 ml of enriched seawater medium or a mixture of enriched seawater and Shephard's medium and l e f t i n the l i g h t at 20° to germinate. (Enriched seawater medium i s Shephard's medium i n which seawater with NaNO^ 0.04 g/1 and K^HPO^ 0.001 g/1 added, replaces the macronutrient s a l t s o l u t i o n . Micronutrient s a l t s , NaHCO^ and vitamins are added as for Shephard's medium). 2) The cysts were treated with SDS as above, then placed i n nutri e n t Shephard's medium (Shephard's medium with glucose 0.6 g/1 and tryptone 0.6 g/1 added) with p e n i c i l l i n 1 mg/ml, neomycin 100 yg/ml and strepto-mycin 100 yg/ml i n the dark at 20°. A f t e r two days, the cysts were 9 washed twice with seawater and placed i n enriched seawater i n the l i g h t to germinate. 3) The cysts (some with, and some without, p r i o r treatment with SDS as above), were treated with lysozyme 1 mg/ml i n 0.001 M T r i s , 0.001 M EDTA, pH 7.0, at room temperature for 20 minutes. They were washed twice with seawater, then treated with 1% SDS i n d i s t i l l e d water f or 15 minutes. A f t e r being washed 5 to 10 times with seawater, the cysts were placed i n 2 ml enriched seawater i n the l i g h t to germinate. In some cases the cysts were treated, as above, with a n t i b i o t i c s i n the dark f o r two days before being placed i n the l i g h t to germinate. 4) A f t e r treatment with SDS as above, the cysts were treated with 10% Argyrol i n seawater (27) f o r 20 minutes. Following t h i s , the cysts were c o l l e c t e d on 25 u mesh b o l t i n g s i l k (held i n a M i l l i p o r e f i l t e r apparatus), washed with 50 ml seawater, transferred to a test tube and washed three more times with seawater. Then they were put into 2 ml nutrien t Shephard's medium with p e n i c i l l i n 1 mg/ml and streptomycin 100 iyig/ml i n the dark f o r two days. Following t h i s , the cysts were washed three times with seawater and placed i n 2 ml Shephard's medium i n the l i g h t to germinate. 5) The cysts were treated with 10% Argyrol i n Shephard's medium for 20 minutes, c o l l e c t e d on 25 ;u'mesh b o l t i n g s i l k and washed with 150 ml Shephard's medium. They were then transferred to a tes t tube and washed f i v e times with Shephard's medium. Following t h i s , the cysts were treated with 1% SDS i n d i s t i l l e d water for 15 minutes, then washed seven times with Shephard's medium. F i n a l l y , the cysts were treated with a n t i -b i o t i c s as above before being l e f t i n the l i g h t to germinate. 10 C u l t i v a t i o n and treatment of algae Aeetabulavia meditevvanea were c u l t i v a t e d i n 500 ml Bellco f l a s k s containing 200 ml s t e r i l e Shephard's medium (57), with approximately 200 c e l l s per f l a s k . They were grown at 20°C and illuminated with 250-300 f t candles of fluorescent l i g h t for 12 hr/day. Some of the c e l l s were grown at a lower l i g h t i n t e n s i t y i n an attempt to i n h i b i t cap formation (48). For 2 to 5 days before use, c e l l s were treated with one or more of the following a n t i b i o t i c s , at the concentrations in d i c a t e d : p e n i c i l l i n 1 mg/ml, kanamycin 100 yg/ml, neomycin 100 yg/ml, streptomycin 100 yg/ml or chloramphenicol 10-50 yg/ml. When streptomycin or chloramphenicol were used, the c e l l s were placed i n the dark to prevent damage to the chloro-p l a s t s . C e l l s were also placed i n the dark to reduce the amount of starch i n the chloroplasts and thus prevent chloroplast breakage during the i s o l a -t i o n procedure. The usual regime was to wash the c e l l s two or three times and place them i n fresh medium with neomycin for two days. Then the c e l l s were washed twice and placed i n fresh medium with p e n i c i l l i n and kanamycin or streptomycin f o r two days i n the dark. The c e l l s were then used to pre-pare chloroplasts. I s o l a t i o n of Chloroplasts: S t e r i l e technique was used throughout. Equipment and glassware were s t e r i l i z e d i n an autoclave or oven. Solutions were s t e r i l i z e d by passage through a 0.22 y M i l l i p o r e f i l t e r . C e l l s two to f i v e cm i n length were used, p r i o r to cap formation. In a few cases, c e l l s with cap i n i t i a l s l e s s than 1.5 mm i n diameter were used. 11 The c e l l s were washed three times and each c e l l enucleated by c u t t i n g o f f the r h i z o i d along with 2 to 3 mm of the s t a l k , using iridectomy s c i s s o r s . In a few instances, the nucleate fragments were c o l l e c t e d and placed i n fresh medium to regenerate new s t a l k s . A f t e r a recovery period of one or two hours i n dim l i g h t , the sta l k s were c o l l e c t e d and used. In preliminary preparations, Shephard's method was used to i s o l a t e the chloro-p l a s t s (59). This was modified s l i g h t l y f o r l a t e r preparations. A l l operations were ca r r i e d out at 0-4°. Centrifuging was done i n the swinging bucket rotor of a S o r v a l l R2B centrifuge. Approximately 2 g wet weight of enucleated c e l l s were s c i s s o r minced i n 3 ml of a medium cons i s t i n g of 0.6 M mannitol, 0.1% BSA, 0.001 M EDTA, 0.1 M TES and 0.001 M d i t h i o t h r e i t o l , pH 7.8 (H medium). The s l u r r y was f i l t e r e d through 25 ymesh b o l t i n g s i l k under gentle suction and washed through with approximately 3 ml of a medium s i m i l a r to H but containing 0.01 M TES (W medium). C e l l w a l l debris was retained by the b o l t i n g s i l k , while the chloroplasts and cytoplasmic content passed through. The chloroplast suspension was layered onto 4 ml of W medium with 2% F i c o l l added (WF medium) and centrifuged f o r 15 min at 650 x g. The pale green supernatant was discarded. The chloroplast p e l l e t was suspended i n 4 ml W medium, l a y -ered over 4 ml WF medium and centrifuged at approximately 90 x g for 5 min-utes. The very small green and white p e l l e t was discarded and the green supernatant was passed through a 5 y Nucleopore f i l t e r to break up chloroplast aggregates and shear o f f tags of cytoplasm adhering to the ch l o r o p l a s t s . This step also removed a s i g n i f i c a n t number of b a c t e r i a . The f i l t r a t e con-t a i n i n g the chloroplasts was then centrifuged at 650 x g for 15 minutes and the pale green supernatant discarded. The chloroplast p e l l e t was 12 suspended i n 4 ml W medium, layered over 4 ml WF medium and centrifuged at 650 x g f o r 15 minutes. (This step was sometimes omitted). The pale green supernatant was discarded. The chloroplast p e l l e t was suspended i n 5 ml of a medium containing 0.6 M mannitol, 0.1% BSA, 0.025 M TES, 0.01 M KC1, 5 mM MgCl 2 and 0.5 mM KH^PO^, pH 7.8 (A medium) and centrifuged at 650 x g f o r 15 minutes. The very pale green or c l e a r supernatant was discarded and the chloroplast p e l l e t resuspended i n A medium. Depending on the amount of s t a r t i n g material, from one to four p e l l e t s were combined and suspended i n a f i n a l volume of 6.6 to 16.6 ml of A medium. A f t e r thorough mixing of the chl o r o p l a s t s , 0.6 ml was taken to assay f o r b a c t e r i a l contamination and the remaining chloroplast suspension was centrifuged at 650 x g for 15 minutes. The supernantant was discarded and the p e l l e t containing 90-95% i n t a c t chloroplasts, was used for the preparation of DNA. In a few preliminary preparations, instead of suspending the chloro-p l a s t s i n A medium i n the f i n a l steps, the chloroplast p e l l e t was suspended i n 4 ml W medium and layered over 4 ml 0.8 M sucrose i n 0.005 M EDTA, 0.01 M TES, 0.001 M d i t h i o t h r e i t o l , pH 7.8. The suspension was centrifuged at 650 x g for 15 minutes. Sometimes t h i s was not adequate to p e l l e t the chloroplasts, i n which case ce n t r i f u g a t i o n was repeated at 1000 x g. The chloroplast p e l l e t was then suspended i n 5 ml 0.4 M sucrose buffered as above, and 0.7 ml taken for contamination check. The nemaining suspension was centrifuged at 650 x g f o r 15 minutes and the chloroplast p e l l e t used for preparation of DNA. Preparation of chloroplast DNA In preliminary experiments, DNA was prepared by a s l i g h t modification of the phenol-pH9-RNases method of Miura (46). The chloroplast p e l l e t from 13 2-4 g wet weight of Acetabutavia c e l l s was suspended i n 2-3 ml tris-SDS-bu f f e r (0.1 M T r i s , 1% SDS, 0.1 M NaCl, pH 9.0) and l e f t on i c e for 10-15 min for l y s i s to occur. An equal volume of phenol saturated with tris-SDS bu f f e r was added and the mixture shaken i n the cold f or 20 min. Layers were separated by centrifugation and the c l e a r , upper, aqueous layer care-f u l l y pipetted o f f . Since there was not enough DNA to be p r e c i p i t a t e d with ethanol, the aqueous layer was dialyzed against 2 1 0.1 x SSC (3 changes) i n the cold for 24 to 48 hours. DNA from several preparations was pooled and stored i n the r e f r i g e r a t o r (4°) with a drop of chloroform. The volume of the pooled samples was reduced with Aquacide I I and the DNA s o l u t i o n i n -cubated at 37° with ribonuclease T^ 25-100 units/ml and pancreatic r i b o -nuclease 50 y§/ml for 30-45 min. Pronase (free of deoxyribonuclease by the method of Stern (62) 100 yg/ml was added and the s o l u t i o n incubated at 50° for one to two hours. An a d d i t i o n a l 50 yg pronase/ml was added and the i n -cubation continued for 2 to 3 hours. The s o l u t i o n was then dialysed against 2 1 SSC (2-3 changes) i n the cold and concentrated with Aquacide I I . The DNA s o l u t i o n was adjusted to a density of 1.700 g/cm by the addition of s o l i d CsCl and centrifuged at 42000 rpm i n the 50 T i rotor of a Beckman model L2B u l t r a c e n t r i f u g e f or 61 hr at 20°. The bottom of the centrifuge tube was punctured with a 27 gauge needle and 4 drop f r a c t i o n s were c o l l e c t e d . Each f r a c t i o n was d i l u t e d with 0.5 ml 0.1 x SSC and the OD^ ..^  monitored. 260nm 3 Peak f r a c t i o n s containing DNA of p = 1.702 g/cm were pooled and dialysed i n t o SSC i n the cold. In l a t e r experiments, a s l i g h t modification of Marmur's method (44) was used to prepare DNA from the chloroplasts. The chloroplast p e l l e t was suspended i n 1.25 ml 0.15 M Nad* 0.1 M EDTA, pH 8 and 0.1 ml 25% SDS was 14 added. The mixture was heated to 60° f o r 10 minutes and then c h i l l e d on i c e . Following the addition of 0.33 ml 5 M NaClO^, one volume of chloro-form: isoamyl alcohol (24:1 V/V) was added and the mixture shaken i n the cold for 20 minutes. The layers were separated by c e n t r i f u g a t i o n . The c l e a r , upper, aqueous layer containing the DNA was c a r e f u l l y withdrawn and dialysed against 2 1 SSC (2 changes) i n the cold i n the dark for 24 hours. A f t e r d i a l y s i s the DNA s o l u t i o n was stored i n the freezer (-20°). DNA from several preparations was thawed and pooled, then treated at 37° with pancreatic ribonuclease 100 yg/ml and ribonuclease T^ 25-50 units/ml for 30 minutes, and a-amylase 5-10 yg/ml for 15 minutes. Pronase 100 yg/ml was added and the incubation c a r r i e d out at 50°. A f t e r one to two hours, pronase 50 yg/ml was added and the incubation continued f o r 2 to 3-1/2 hours. The s o l u t i o n was c h i l l e d on i c e and then dialysed overnight against 2 1 SSC i n the cold i n the dark, concentrated with Aquacide I I , and d i a -lysed a further 6 to 18 hr against 2 1 SSC i n the cold i n the dark. Treat-ment with the ribonucleases, a-amylase and pronase, as above, was repeated and the DNA s o l u t i o n was again dialysed against SSC. A f t e r further reduc-t i o n of the volume with Aquacide I I , the DNA s o l u t i o n was c l a r i f i e d by c e n t r i f u g i n g at 18000 x g f o r 10 min. A small white p e l l e t was discarded and the c l e a r supernatant was dialysed i n t o SSC or 0.1 x SSC. Monitoring f o r b a c t e r i a l contamination To check the effectiveness of various treatments of cysts, aliquots of the f i n a l wash, along with a few cysts, were plated on nutrient Shephard's agar (nutrient Shephard's medium s o l i d i f i e d with 1.5% agar) with a soft agar overlay and incubated at 20°. A f t e r one week colonies were counted and contaminants, when present, were characterized. 15 Occasionally, i n d i v i d u a l Acetabulavia c e l l s were observed with a phase microscope to check for the presence of b a c t e r i a and other p r o t i s t s . At several times during the growth phase, Acetabulavia c e l l s were plated on nutrient Shephard's agar along with a small amount of the medium i n which they were growing. Plates were incubated at 20° and the nature and extent of b a c t e r i a l growth was noted. To check f o r contamination i n chloroplast preparations, aliquots of the f i n a l chloroplast suspension were added to nutrient Shephard's medium -1 -2 -3 to make f i n a l d i l u t i o n s of 10 , 10 and 10 . Five tubes of each d i l u -t i o n were incubated at 20° and the number of samples of each d i l u t i o n which became tur b i d a f t e r one week were counted. Most probable numbers of bac-t e r i a were estimated from the table given by C o l l i n s (17). In addition to t h i s , aliquots of each d i l u t i o n were plated on nutrient Shephard's agar. Colonies were counted and characterized a f t e r incubation at 20° for one week. Chloroplast numbers were determined by counting two aliquots of a 10 d i l u t i o n i n a hemacytometer. The r a t i o of ba c t e r i a to chloroplasts and thus the amount of DNA being contributed by b a c t e r i a could then be estimated. I s o l a t i o n of DNA from b a c t e r i a l contaminants Bacteria were grown i n nutrient Shephard's medium overnight with shaking at 25°. C e l l s were harvested by ce n t r i f u g a t i o n and DNA extracted according to the phenol-pH9-RNases method of Miura (46). Freezing and thawing was not necessary to lyse the c e l l s , but i n some cases pronase (free of deoxyribonuclease (62) 100 yg/ml was used to disrupt filaments. 16 Cesium chloride density gradients About 0.8 ml of the s o l u t i o n containing l-5ug DNA/ml was adjusted 3 to a density of 1.70 g/cm by the addition of s o l i d Q?C1. Micrococcus 3 lysodeikticus DNA (p = 1.731 g/cm ) was added as a density reference. The s o l u t i o n was centrifuged at 44,000 rpm i n a 12 mm Kel-F c e l l i n the AN-D rotor of a Spinco Model E at 20°. Photographs were taken a f t e r 20 hr and the negatives scanned with a Joyce-Loebl microdensitometer. Buoy-ant d e n s i t i e s were calculated according to Mandel et^ a l . (43) , without correction for pressure a f f e c t s . Electron microscopy Chloroplasts were fixed i n 2.5% glutaraldehyde i n sodium cacodylate b u f f e r , pH 6.8, and post-fixed i n 1% 0s0^ i n cacodylate b u f f e r . A f t e r de-hydrating with an ethanol s e r i e s , the chloroplasts were i n f i l t r a t e d with, and embedded i n , Spurcls medium (60) i n g e l a t i n capsules; cured i n a vacuum oven at 70° for 10 hours and t h i n sectioned. S i l v e r sections were c o l l e c t e d on 200 mesh copper grids and stained with uranyl acetate followed by lead c i t r a t e . Sections were examined with an H i t a c h i HU 11-A or HS-7S ele c t r o n microscope. Preparation of E. coli DNA E. coli K 12 was obtained from Dr. R. Warren of the Microbiology De-partment, U.B.C. C e l l s were grown i n nutrient broth (Difco) with 5% yeast extract (D i f c o ) , pH 7.5, at 30° or 37° with shaking and harvested by c e n t r i -fugation. DNA was extracted by the method of Miura (46) , with several addi-t i o n a l p u r i f i c a t i o n steps. Following treatment with pancreatic ribonuclease 50 yg/ml and ribonuclease T, 25 units/ml at 37° f o r 30 minutes, the DNA 17 i n SSC was incubated with pronase (deoxyribonuclease free) 100 yg/ml at 50° f o r one to two hr. Pronase 50 yg/ml was added and the treatment continued for 2 to 3 hours. The s o l u t i o n was c h i l l e d and 2 volumes of cold ethanol added. DNA f i b e r s were c o l l e c t e d on a glass rod, washed i n ethanol and dissolved i n 0.1 M sodium acetate b u f f e r , pH 6.0. The s o l u t i o n was ad-justed to 0.13 M sodium acetate by addition of 3 M sodium acetate and one volume of ethoxyethanol was added to p r e c i p i t a t e the DNA. This step separ-ates the DNA from ribonucleotides (35). Kirby's two-phase method, as des-cribed by Bellamy and Ralph (7), was used to remove polysaccharides. The DNA was dissolved i n 0.025 M Tr i s - H C l b u f f e r , pH 8.1, containing 0.025 M NaCl. One volume cold 2.5 M phosphate b u f f e r , pH 8, and one volume cold 2-methoxyethanol were added and the mixture shaken vigorously f o r 3 minutes at 4°. A f t e r centrifuging at 12,000 x g for 5 minutes, the c l e a r upper l a y -er was c a r e f u l l y withdrawn and two volumes of cold ethanol added to i t . The DNA p r e c i p i t a t e was dissolved i n 0.1 x SSC and the s o l u t i o n stored i n the r e f r i g e r a t o r (4°) with a drop of chloroform. Preparation of bacteriophage T4 DNA P u r i f i e d phage T4 p a r t i c l e s were a g i f t of Dr. R. M i l l e r of the Micro-biology department, U.B.C. The phage p a r t i c l e s i n 0.5 ml of 0.01 M T r i s , 0.15 M NaCl, pH 7.5, were shaken with an equal volume of water saturated phenol for 15 min at 4°. The mixture was centrifuged at 2000 x g for 10 min to separate the l a y e r s . The c l e a r upper aqueous layer was c a r e f u l l y pipetted o f f and dialysed against 2 1 of SSC (2 changes) i n the cold for 24 hrs. The DNA s o l u t i o n was then stored i n the r e f r i g e r a t o r (4°) with a drop of chloroform. 18 Thermal denaturation p r o f i l e s and determination of Tm Solutions of DNA i n SSC or 0.1 x SSC were degassed by evacuation i n a vacuum dessicator. S p e c t r o s i l semi-micro cuvettes f i t t e d with t e f l o n stoppers were used and the QJ^Onm automatically recorded with a G i l f o r d 2400 spectrophotometer. The temperature i n the chamber was increased r a -p i d l y to about 50° and them at a rate of 1° per 4 min u n t i l the onset of melting. A f t e r that point, the temperature was increased 1° per 10 min u n t i l maximum hyperchromicity was reached. Absorbance measurements were corrected f o r thermal expansion of the solvent and the Tm determined from the pl o t of r e l a t i v e absorbance (A,j,/A2^o) versus temperature (42) . A sam-ple of E. coli- K 12 DNA was included as a reference during each experiment. Renaturation of DNA The DNA samples were dialysed i n t o the appropriate solvent and an aliquot of the d i a l y s i s s o l u t i o n was used^for the absorbance reference. DNA was sheared by passage through a 27 gauge needle, using a 2 ml syringe and applying maximum pressure by hand. DNA solutions at concentrations of 6 to 20 yg/ml were degassed by evacuation i n a vacuum dessicator. The r e -naturation reaction was followed o p t i c a l l y , with the DNA solutions contained i n S p e c t r o s i l semi-micro cuvettes f i t t e d with t e f l o n stoppers and the ab-sorbance at 260 nm being measured i n a G i l f o r d 2400 automatic recording spectrophotometer. In any experiment, two or three samples were run simul-taneously. In one experiment, a f t e r determining the 0^260 of the native DNA, the DNA was denatured by adding one part 1.0' M NaOH to 8 parts of the DNA sol u t i o n i n 4.5 x SSC. A f t e r 10 minutes at room temperature the 0D 9 f t f ) of 19 the denatured DNA was read. The s o l u t i o n was then n e u t r a l i z e d by adding one part 2 M NaH^PO^, mixed w e l l , and absorbance readings begun immediately while the s o l u t i o n e q u i l i b r a t e d to the renaturation temperature. DNA was renatured at 70 + 1.0°C. This i s (Tm-28?° for E. coll DNA and (Tm-22)° for — m T4 DNA at t h i s i o n i c strength (74). The Tm f o r Acetabulavia chloroplast DNA has not been determined i n a solvent of t h i s i o n i c strength, but from the base compositions the Tm of the chloroplast DNA should be between those of E. coll DNA and T4 DNA. In another experiment, DNA i n SSC was melted as described above. The temperature was then lowered to 6 3 + 0 . 5 ° and the DNA allowed to renature. In other experiments, a f t e r determining the absorbance of the native DNA, the DNA i n 0.1 x SSC was heated on a b o i l i n g water bath for 12 to 15 minutes. Using a warm pip e t t e , 1.625 ml hot denatured DNA was added to 0.175 ml 20 x SSC ( f i n a l concentration 2 x SSC) i n a cuvette e q u i l i b r a t e d to 65°. The solutions were mixed and absorbance readings begun immediately. Since the temperature recorded on the chart d i f f e r e d s l i g h t l y from the actual temperature of the solutions i n the cuvettes, an a d d i t i o n a l cuvette contain-ing 2 x SSC was included. The temperature of t h i s s o l u t i o n was monitored with a Tele-Thermometer (Yellow Springs Instrument Company) and used to c a l i b r a t e the temperature recorded on the chart. Absorbance values were corrected as necessary f o r changes i n the volume of the DNA s o l u t i o n . Corrections f or thermal expansion of the solvent were also made. The chloroplast DNA s o l u t i o n , i n p a r t i c u l a r , contained a contaminant of unknown nature which had a considerable absorbance at 260 nm. For t h i s 20 reason, the concentration of DNA i n each sample was determined by a micro assay using Burton's modification of the diphenylamine assay (15). The percentage of the 0^250 w n i c n w a s due t o DNA w a s then calculated, and allaabsorbance readings corrected according to t h i s value. The following equations given by Wetmureand Davidson (74) were then used to determine the : A„ - A k„ P_ and P_ = 1.47 x 1 0 _ 4 A mole 1 _ 1 6 °° = 2 T 1 T 0 0 A - A 2 00 where A i s the absorbance of native DNA; 00 A i s the maximum absorbance of denatured DNA; o A i s the absorbance of the p a r t i a l l y renatured DNA at time t; t i s the time, i n seconds, P^ i s the t o t a l DNA phosphate concentration; and i s the second order renaturation rate constant. Since the rate of renaturation of the DNA i s influenced by the i o n i c strength of the solvent, a l l values were normalized to a solvent monova-lent cation concentration of 1.0 M, using values of r e l a t i v e r e a s s o c i a t i o n rates given by B r i t t e n (11). Base composition of the DNA also a f f e c t s i t s renaturation rate, therefore values for Aeetabulavia chloroplast DNA and T4 DNA were normalized to 50% G+C, using data given by Wetmur and Davidson (39, 74). Determination of a l k a l i n e sedimentation c o e f f i c i e n t s Band v e l o c i t y sedimentation was used to determine the sedimentation c o e f f i c i e n t of denatured DNA (63,69). Sedimentation was c a r r i e d out at 28,000 or 30,000 rpm i n the AN-D rotor of a Spinco Model E u l t r a c e n t r i f u g e , 21 at a temperature of 22° to 25°. Photographs were taken at 8 min or 16 min i n t e r v a l s and the negatives scanned with a Joyce-Leobl microdensito-meter. About 25 ,yil of a DNA s o l u t i o n i n 0.1 x SSC ( 0 D 2 6 Q -0.4) was used i n the sample well of a 12 mm Kel-F band forming centerpiece. In some cases 0.5 ml of 0.9 M NaCl, 0.1 M NaOH was used as the bulk s o l u t i o n and the a l k a l i n e sedimentation c o e f f i c i e n t (S??"^) determined d i r e c t l y . In other 20 ,w cases, the DNA was heat denatured on a b o i l i n g water bath and quick cooled on i c e before being loaded into the sample w e l l . The bulk s o l u t i o n was 1.0 M NaCl, 0.05 M sodium c i t r a t e and the a l k a l i n e sedimentation c o e f f i c i e n t was stipulated from the following r e l a t i o n s h i p derived from equations given by Studier (63): log S ^ 1 3 = 0.400 (log SPH7,denatured _ l Q g 0 Q 1 Q 5 ) + l o g 0 . 0 5 2 8 , w 0.549 ' W 22 RESULTS Attempts to obtain axenic cultures of Aeetabulavia Washing Aeetabulavia cysts with seawater, alone, was important i n reducing the amount of b a c t e r i a l contamination. P l a t i n g of 0.1 ml of medium used for the f i r s t wash resulted i n heavy, confluent white growth of b a c t e r i a . A f t e r the f i f t h wash, 0.1 ml of washing medium gave r i s e to 300-400 b a c t e r i a l colonies. Treatment of cysts with SDS was very e f f e c -t i v e against b a c t e r i a which produced white colonies, but not as e f f e c t i v e against the contaminants which gave r i s e to yellow colonies. Although axenic cultures did not r e s u l t , the degree of contamination was s l i g h t , with 0.1 ml of the f i n a l washing medium giving r i s e to 5-10 b a c t e r i a l colonies. Lysozyme was not as e f f e c t i v e as SDS i n eliminating the white contaminants, but was more e f f e c t i v e against the yellow contaminants. Treatment of cysts with lysozyme followed by SDS was more e f f e c t i v e than either of these agents alone, although completely axenic cultures s t i l l were not obtained. A n t i b i o t i c treatment was h e l p f u l i n reducing the number of b a c t e r i a , but was not s u f f i c i e n t to produce axenic c u l t u r e s . Argyrol was the most e f f e c t i v e agent for eliminating b a c t e r i a l conta-minants but i t was also the most damaging to Aeetabulavia cysts. Cysts treatednt'with argyrol a f t e r being treated with SDS f a i l e d to germinate, and died. When cysts were treated with argyrol alone, or when SDS treatment followed the treatment, with argyrol, however, the cysts survived and ger-minated. This suggests that cysts which have been previously treated with SDS may be more permeable to argyrol and that argyrol i s toxi c to the cysts i f i t penetrates them. Some axenic cultures of Aeetabulavia were obtained 23 by t r e a t i n g the cysts with a r g y r o l , but i n most cases contamination was a c c i d e n t a l l y introduced-: into the cultures before the Acetabulavia c e l l s were f u l l y grown. When Acetabulavia cultures did remain axenic, the c e l l s did not appear to grow as w e l l as non-axenic cultures. Whether t h i s was due to a lack of some metabolite which i s supplied by the b a c t e r i a i n I | contaminated cultures, or whether r e s i d u a l traces of Ag from the argyrol i n h i b i t e d growth of the Acetabulavia c e l l s , was not investigated. At one point, h a l f of the stock cultures of Acetabulavia became con-taminated with Nanachloves. Probably the cysts were contamined with Nana-chloves before germination. I t was observed that the Acetabulavia c e l l s a r i s i n g from cysts which had been treated with SDS did not have any 'Nanachloves growing i n with them, but a l l the cultures a r i s i n g from Acetabulavia cysts which had not been treated with SDS were contaminated with Nanachloves. B a c t e r i a l contamination i n chloroplast preparations Several types of b a c t e r i a l contaminants were present i n the chloroplast preparations. These are l i s t e d i n table I, together with the buoyant den-s i t i e s of t h e i r DNA. The f i r s t four types of b a c t e r i a l i s t e d i n the table were the ones most commonly found i n chloroplast preparations. Since the bjuoyant de n s i t i e s of DNA from these contaminants d i f f e r from the buoyant density of Acetabulavia chloroplast DNA, i t was possible to separate chloro-p l a s t DNA from b a c t e r i a l DNA by means of a preparative CsCl gradient. A l -though t h i s was done i n preliminary experiments, the amount of b a c t e r i a l contamination was low enough that t h i s was not considered necessary with l a t e r preparations. 24 Table I: B a c t e r i a l contaminants of chloroplast preparations Macroscopic Microscopic Gram Buoyant Appearance Appearance Reaction Density a) shiny, white, smooth, domed colony with e n t i r e margin b) creamy-white, shiny, domed colony with e n t i r e margin c) small shiny, gold-en-yellow, f l a t colony. Colonies have "fried-egg" appearance on o l d plates d) very small, shiny, golden-yellow colony motile rod negative e) shiny, yellow, f l a t colony with i r r e g u -l a r margin; hydroly-zes agar f) shiny, creamy-white domed colony with i r r e g u l a r margin g) d i f f u s e white, f l a t colony with i r r e g u l a r margin motile; short, f a t rods non-motile; long, t h i n rods non-motile; long, filamentous rods, s t r a i g h t and curved;; form net-^ works. Networks broken up by l y -sozyme but c e l l s not lysed non-motile; long filamentous rods p o s i t i v e p o s i t i v e ? negative p o s i t i v e non-motile rod oc- negative curring s i n g l y and i n short chains non-motile rod oc- negative curring s i n g l y , paired and i n short chains 1.716 g/cm" 1.743 g/cm" 1.718 g/cm" 1.699 g/cm" 1.698 g/cm 1.681 g/cm3 (polysacchar-ide?) 1.697 g/cm3 1.715 g/cm" 1.716 g/cm h) shiny, orange, f l a t colony with e n t i r e margin nonnmojfcilei?., long p o s i t i v e rods, some i n chains curved (filaments?) 1.706 g/cm" 25 The degree of contamination i n chloroplast preparations ranged from one bacterium per 6500 chloroplasts to one bacterium per 107,000 chloro-p l a s t s . I f a chloroplast contains the same amount of DNA as a bacterium, the b a c t e r i a may have contributed 0.001% to 0.015% of the DNA i n the f i n a l preparation. Assuming that a chloroplast contains one tenth the amount of DNA i n a bacterium, at most the amount of b a c t e r i a l DNA i n the f i n a l pre-paration could be 0.15%. Electron Microscopy Sections of i s o l a t e d chloroplasts observed with the electron micro-scope showed mostly i n t a c t c h l o r o p l a s t s , although i n many cases there was bulging of the membranes ( f i g . 1). This bulging may have been caused by osmotic changes during the f i x a t i o n of the chloroplasts. No b a c t e r i a l pro-f i l e s were seen. For chloroplasts which were sedimented through a buffered sucrose so-l u t i o n , one mitochondrial p r o f i l e was seen f or every 5 chloroplast p r o f i l e s . Fewer mitochondria were observed when the chloroplasts were sedimented through A medium during the i s o l a t i o n procedure. In t h i s case, one mito-chondrial p r o f i l e per ten chloroplast p r o f i l e s was seen. For t h i s reason, except for a few preliminary preparations, A medium was used rather than sucrose s o l u t i o n during the f i n a l steps of the chloroplast i s o l a t i o n . Although only one mitochondrial p r o f i l e was seen per 10 chloroplast p r o f i l e s , the actual r a t i o of mitochondria to chloroplasts was probably much higher than t h i s . Since a chloroplast i s larger than a mitochondrion, the p r o b a b i l i t y of sectioning through a chloroplast i s greater than the p r o b a b i l i t y of sectioning through a mitochondrion. The mean volume of an 26 Figure 1: Thin sections of AcetcLbulavia medttevranea chloroplasts fixed i n glutaraldehyde and post-fixed OsO^. Sections were stained with uranyl acetate followed by lead c i t r a t e . Bar = 1 y(x 10,000.) 27 3 Acetabulavia chloroplast i s 8 ,p (55) . Assuming an e l l i p s o i d shape for a mitochondrion, and by measuring the diameters of mitochondrial p r o f i l e s observed with the electron microscope, the mean volume of an Acetabulavia 3 mitochondrion i s calculated to be approximately 0.4 u . Assuming that an equal number of chloroplasts and mitochondria are randomly d i s t r i b u t e d i n a given volume of embedding medium (this may not be a v a l i d assumption), then the chances of sectioning through a chloroplast are 20 times greater than the changes of sectioning through a mitochondrion. Thus, the actual r a t i o could be as high as two mitochondria^for every ch l o r o p l a s t . The amount of DNA i n an Acetabulavia mitochondrion i s not known, but i t appears to be le s s than i s i n a chloroplast (75). From buoyant density studies of the cytoplasmic DNAs of Acetabulavia, there appears to be about 10 times more chloroplast DNA than mitochondrial DNA (32). Assuming, then, that a mitochondrion contains one tenth the amount of DNA that i s i n a chloroplast, mitochondrial DNA could contribute as much as 20% of the DNA i n the f i n a l preparation. In DNA from p u r i f i e d c h l o r o p l a s t s , however, there i s no measurable amount of DNA at the buoyant density of mitochondrial DNA ( f i g . 2), p a 1.714 g/cm3 (32). Buoyant density and Tm of Acetabulavia chloroplast DNA Acetabulavia chloroplast DNA from two d i f f e r e n t preparations was cen-t r i f u g e d i n an a n a l y t i c a l CsCl density gradient. In both cases, a si n g l e 3 peak with a buoyant density of 1.702 g/cm was observed ( f i g . 2). This i s d i f f e r e n t from the buoyant density of the DNA of any of the b a c t e r i a l con-taminants (table I ) . Using the r e l a t i o n s h i p given by Schildkraut et a l . 3 (51), p = 1.702 g/cm corresponds to a base composition of 42.8% G+C. A 28 Figure 2: Densitometer tracings of u l t r a v i o l e t photographs of Aeetabulavia meditevvanea chloroplast DNA a f t e r ana-l y t i c a l density-gradient c e n t r i f u g a t i o n i n CsCl. 3 Micrococcus lysodeikticus DNA (p = 1.731 g/cm ) was used as density reference. 29 buoyant density of 1.702 g/cm3 p r e d i c t s a Tm of 87.0°C i n SSC and 71.2° i n 0.1 x SSC, using the following equations given by Mandel e± al_ (41): Tm (SSC) = 418.2 (p- 1.494) Tm (SSC/10) = 512.2 (p-1.563) In one attempt to determine the Tm i n SSC, the curve began to l e v e l a f t e r a hyperchromic increase of about 31%, but this was followed by a sudden hyperchromic r i s e above 95° ( f i g . 3). This sudden increase i n hy-perchromicity may have been due to evaporation of the solvent, since about 1.5 ml of sample was contained i n a cuvette which has a capacity of 1.8 ml. Another possible explanation i s that t h i s sudden increase was an a r t i f a c t produced by a gas bubble. Taking 31.5% as the maximum hyperchromicity, the Tm i n SSC of the Aeetabulavia chloroplast DNA i s 86.7°, based on a Tm = 90.5 for E. coli K 12 DNA.(41). This i s i n good agreement with the pre-dicted value of 87.0°. Aeetabulavia chloroplast DNA had 22.8% hyperchromicity i n 0.1 x SSC, with a Tm of 70.7° ( f i g . 3) based on a Tm = 75.7° for E. coli K 12 DNA (41). This i s 0.6° lower than predicted, but t h i s may be due to the low molecular weight of the DNA (20,21). The melting t r a n s i t i o n was not smooth. This, also, i s possibly due to the fact that t h i s DNA was sheared to a low mole-cular weight (about 7.5 x 10^ daltons). I f there i s a high degree of i n t r a -molecular heterogeneity i n Aeetabulavia chloroplast DNA, then shearing of the DNA could produce fragments of varying base compositions. A d i f f e r e n t i a l p l o t of the thermal t r a n s i t i o n of Aeetabulavia chloroplast DNA shows m u l t i -ple peaks ( f i g . 4), which i s also i n d i c a t i v e of intramolecular heterogeneity (23). 30 Figure 3: Melting curves of native DNA. (•••) Acetabulavia meditewanea chloroplast DNA melted i n 0.1 x SSC; (ooo) Acetabulavia meditewanea chloroplast DNA melted i n SSC; ( A — A ) E. coli DNA melted i n SSC. 3 0 a U i 1-3 in 1-2 CM 1-1 VO 3 0 4 0 50 60 70 TEMPERATURE (°C) 8 0 90 31 Figure 4: D i f f e r e n t i a l melting p l o t of native DNA i n 0.1 x SSC ( o — o ) Aoetabularia meditewanea chloroplast DNA; (•=-•) E. coll DNA. 3 1 a 32 Acetabulavia chloroplast DNA shows a very broad t r a n s i t i o n width for melting. The t r a n s i t i o n width (°C between 17% and 83% of t o t a l hy-perchromicity) of melting i n SSC i s 8.2° and i n 0.1 x SSC i s 9.7°. This could be p a r t l y due to the low molecular weight of the DNA (20,21), but may also be i n d i c a t i v e of a high degree of intramolecular heterogeneity (42). Renaturation and k i n e t i c complexity of Acetabulavia chloroplast DNA The rate p l o t s for the renaturation of Acetabulavia chloroplast DNA are given i n f i g u r e 5. There appears to be a small amount of a f a s t re-naturing component, representing about 3% of the t o t a l DNA. This i s pro-bably due to mitochondrial DNA. In one experiment ( f i g . 5b) there was a considerable l a g before the reaction proceeded. This i s d i f f i c u l t to ex-p l a i n , but may be an a r t i f a c t due to i n s u f f i c i e n t mixing of the DNA s o l u -ti o n i n 0.1 x SSC with the concentrated (20x) SSC s o l u t i o n . Also, the concentration of DNA i n t h i s sample was very low (about 6 yg/ml), and t h i s may be part of the reason for t h i s l a g . In the f i r s t renaturation experiment, the DNA was a l k a l i denatured. No hyperchromicity of the Aeeta&Uikavia chloroplast DNA was observed. When the n e u t r a l i z e d sample was subsequently run i n an a n a l y t i c a l CsCl density gradient, no band was detected, suggesting tbat the DNA had been degraded. It i s very u n l i k e l y that this a l k a l i l a b i l i t y i s due to the presence of un-usual bases i n Acetabulavia chloroplast DNA, since the melting behavior of the chloroplast DNA and i t s buoyant density i n CsCl c o r r e l a t e w e l l (41,42). The problem of a l k a l i l a b i l i t y has been encountered with mitochondrial DNA (10). I t i s probable that free r a d i c a l s are generated during the DNA 33 Figure 5: Renaturation rate p l o t s for Acetabulavia meditewanea chloroplast DNA. (a) Experiment 2: DNA concentration approximately 15 yg/ml; renatured i n SSC at 63 + 0.5°; k^ = 5.90 1 mole ''"sec ^ (b) Experiment 3: DNA concentration approximately 6 yg/ml; renatured i n 2 x SSC at 65 + 0.5°; k^ = 5.00 1 mole ''"sec (c) Experiment 4: DNA concentration approximately 11 yg/ml; renatured i n 2 x SSC at 65 + 0.5 ; k 2 = 7.62 1 mole sec k^ values corrected for i o n i c strength of solvent and norma-l i z e d to 50% G+C 3 3 a a) ^ 1 1 1 r 1-0jr 1 1 1 1 — • • — i 1 1 — 0 1000 2000 3000 4000 5000 6000 7000 (50min) dOOmin) T IME(sec) 34 i s o l a t i o n procedure (10) and these attack the N-glycosidic bond (49). This r e s u l t s i n base elimination and subsequent chain breakage at pH > 7 (49). Therefore, i n subsequent experiments, heat denaturation was used rather than a l k a l i n e denaturation. In experiment 4, a f t e r the DNA had been renatured f o r 34 hours (E. col-i DNA 88% renatured; Aoetahularia chloroplast DNA 59% renatured) i t was quickly cooled and dialysed into 0.1 x SSC i n the cold. Following t h i s , the DNA was melted. There was some loss of O.D. for the chloroplast DNA sample, even a f t e r correcting f o r the volume change due to d i a l y s i s . I t i s probable that some degradation of the DNA occurred during the renatura-t i o n reaction and subsequently small fragments were l o s t during d i a l y s i s . The melting curves f o r the renatured DNA samples are shown i n figure 6. The renatured AoetabulcLvta chloroplast DNA has a Tm of 70.0°, which i s 0.7° lower than the Tm of native chloroplast DNA. Renatured E. ooli DNA also has a Tm which i s lowered by 0.7°.. This lowering of the Tm indicates about 1% mismatched base pai r s i n the renatured DNA (40). The renaturation rate constants, a l k a l i n e sedimentation c o e f f i c i e n t s and k i n e t i c complexities of a number of DNA samples are presented i n table 9 I I . The k i n e t i c complexity of E. coli DNA should be 2.5 x 10 daltons, i n agreement with i t s a n a l y t i c a l complexity (74). The values obtained i n some of these experiments, however, are lower than t h i s . The most probably ex-planation i s that the values for the a l k a l i n e sedimentation c o e f f i c i e n t s are i n e r r o r . Sedimentation c o e f f i c i e n t s were determined a f t e r the DNA had been used for the renaturation react i o n . A f t e r t h i s prolonged exposure to a high temperature there would have been some degradation of the DNA (22) , r e s u l t i n g 35 Figure 6: Thermal t r a n s i t i o n p r o f i l e s of native and renatured DNA melted i n 0.1 x SSC. (a) Acetabulavia meditewanea chloroplast DNA; (•••) native DNA, Tm = 70.7°; (ooo) renatured DNA, Tm = 70.0°. (b) E. coli DNA; (•••) native DNA, Tm = 75.7°; (ooo) renatured DNA, Tm = 75.0°. 1-3, 1-2 h in CNJ 1-1r-1-Oh 5a o oo o o ~ o ooo' O 0 oo o o o • • • • mo a 30 40 50 60 70 80 T E M P E R A T U R E (°C) 30 40 50 60 70 80 T E M P E R A T U R E (°C) Table I I . K i n e t i c complexities of DNA from d i f f e r e n t sources Exp. No. Source of DNA ,-1 -1 (mole 1 ) (1 mole sec meas ,pH13 320,w sec 1) meas. before a f t e r calculated K i n e t i c Complexity (daltons) 1 1 33 5 2 bacteriophage T4 E. coli E. coli E. coli E. coli E. coli Aeetabulavia meditevvanea chloroplasts Aeetabulavia chloroplasts Aeetabulavia chloroplasts 3.01x10 -5 3.57x10 -5 5.73x10 -5 3.92x10 -5 4.54x10 §5 6.00x10 -5 4.39x10 -5 1.82x10 -5 3.10x10 -5 197 11.60 13.57 5.31 6.88 6.05 5.90 5.00 7.62 28 13.4 24 14.9 13 10 24 13 15.7 13 19.5 11 20,w 1.5 x 10 1.4 x 10 2.5 x 10' 1.0 x 10< 2.6 x 10' 1.0 x 10^ 2.5 x 10' 1.4 x 10j 2.5 x 10' 9 2.3 x 10' 8.7 x 10 2.3 x 10' 8 1.7 x 10^ 4.5 x 10' 8.1 x 10 1.4 x \V 8 * pH13 9 S„„ corrected to obtain a k i n e t i c complexity of 2.5 x 10 daltons for E. coli DNA Co ON 3 7 i n lower values f o r the sedimentation c o e f f i c i e n t s . In one experiment with E. coli DNA, an aliquot of DNA for determination of the sedimentation c o e f f i c i e n t was taken at the s t a r t of the experiment, immediately a f t e r 9 the DNA had been heat denatured. In t h i s case, a value of 2 . 3 x 1 0 daltons was obtained f o r the k i n e t i c complexity (table I I , experiment 5 ) . In another experiment (table I I , experiment 2 ) , a value of S^Q"^ 3 = 1 3 was determined on a sample of DNA a f t e r i t had been used i n the renaturation reaction. With another sample of sheared DNA from the same batch, but which had not been heat denatured and renatured, S?^ F"3 = 2 8 . Using 1 3 as 2 0 ,w 9 the a l k a l i n e sedimentation c o e f f i c i e n t , a k i n e t i c complexity of 1 . 0 x 1 0 daltons i s calculated for E. aoli DNA, but using S^ ?"*"3 = 2 8 , the k i n e t i c 2 0 ,w 9 complexity of E. aoli DNA i s 2 . 6 x 1 0 daltons. Therefore, the values of the sedimentation c o e f f i c i e n t s were corrected to obtain a k i n e t i c complexi-ty of 2 . 5 x 1 0 daltons for E. coli DNA. A corresponding c o r r e c t i o n was made for the sedimentation c o e f f i c i e n t s of the Acetabulavia chloroplast DNA. This correction assumes that the chloroplast DNA was degraded to the same degree as the E. coli DNA. This may not be a v a l i d assumption. Indeed, a look at the corrected values given i n table II would suggest that t h i s assumption i s not v a l i d . One would expect the DNA i n experiment 4 to be the most degraded, since i t was exposed to a high temperature f o r the long-est period of time, yet i t has the smallest co r r e c t i o n . On the other hand, the c o r r e c t i o n applied i n experiment 3 i s probably too large. An attempt was made to determine a correction f a c t o r f o r the a l k a l i n e sedimentation c o e f f i c i e n t s by shearing b a c t e r i a l DNA under the same conditions as had been used to shear the chloroplast DNA (see below). 38 The k i n e t i c complexity of Acetabulavia meditewanea chloroplast DNA g i s at l e a s t 8.1 x 10 daltons, and i s probably greater than t h i s . The average r e s u l t of three separate experiments gives a k i n e t i c complexity 9 of 1.1 + 0.4 x 10 daltons. This agrees c l o s e l y with an a n a l y t i c a l com-9 -15 p l e x i t y of 1.4 x 10 daltons (2.3 x 10 g) of DNA as determined by Green (Unpublished). Thus, i t appears probable that the chloroplast DNA e x i s t s as one unique sequence, with no repeated segments. At most, there could be two copies of the unique sequence. Sedimentation c o e f f i c i e n t s I f the method of shearing produced the same s i z e of DNA fragments f a i r l y reproducibly, then any DNA sample, could be taken and sheared under the same conditions as were used f o r shearing the DNA i n the renaturation experiments. The a l k a l i n e sedimentation c o e f f i c i e n t s could then be deter-mined f or these samples and the values obtained could be used i n c a l c u l a t -ing the k i n e t i c complexities. This was done, using p u r i f i e d DNA, which was extracted from one of the b a c t e r i a l contaminants. Solutions of the DNA were adjusted to the concentrations used i n the renaturation experiments. Three aliquots of each concentration of DNA were taken and each aliq u o t sheared once or twice with a 27 gauge needle, depending on the conditions used previously. The DNA samples were concentrated with Aquacide I I , d i a -lyzed into 0.1 x SSC, and then the aVlkaline sedimentation c o e f f i c i e n t s were determined. The r e s u l t s of these experiments are presented i n table I I I . I t can be seen that within the concentration range used, the concen-t r a t i o n of DNA had l i t t l e e f f e c t on the s i z e of the DNA fragments produced 39 T a b l e I I I . A l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s o f s h e a r e d DNA S a m p l e s DNA c o n c e n t r a t i o n ( y g / m l ) 6.5 8 1 0 1 2 1 5 s h e a r e d : x 2 x l x 2 x l x l q p H 1 3 b 2 0 , w 2 5 . 6 2 5 . 8 2 6 . 2 2 6 . 6 2 5 . 4 2 3 . 8 2 4 . 2 2 5 . 3 2 6 . 4 2 7 . 2 2 5 . 0 3 1 . 7 2 6 . 6 2 9 . 2 2 5 . 5 m e a n : 2 8 . 7 + 1 . 1 2 2 7 . 2 + 3 . 9 2 • 26 + 0 . 2 1 27 .4+1.56 2 6 . 0 + 1 . 0 1 b y s h e a r i n g w i t h a 27 g a u g e n e e d l e . T h e r e w a s some v a r i a t i o n i n t h e s e d i -m e n t a t i o n c o e f f i c i e n t s o b t a i n e d f o r d i f f e r e n t DNA s a m p l e s a t t h e same c o n -c e n t r a t i o n , p a r t i c u l a r l y w h e n t h e DNA w a s s h e a r e d o n l y o n c e . T h e s e d i f f e r -e n c e s i n S ? ? ^ " 3 f o r d i f f e r e n t s a m p l e s o f DNA c a n l e a d t o c o n s i d e r a b l e 20,w v a r i a t i o n i n t h e c a l c u l a t e d v a l u e o f t h e k i n e t i c c o m p l e x i t y , a s s h o w n i n t a b l e I V u s i n g t h e k 2 d e t e r m i n e d f o r E. ooli DNA i n e x p e r i m e n t o n e . I n e a c h c a s e , t h e v a l u e f o r t h e k i n e t i c c o m p l e x i t y i s t o o h i g h . T h e s e d i m e n -t a t i o n c o e f f i c i e n t s d e t e r m i n e d i n t h e s e e x p e r i m e n t s a r e g e n e r a l l y h i g h e r t h a n t h e v a l u e s m e a s u r e d f o r t h e DNA s a m p l e s u s e d i n t h e r e n a t u r a t i o n e x -p e r i m e n t s . T h i s g i v e s s u p p o r t t o t h e i d e a t h a t some DNA d e g r a d a t i o n o c -c u r r e d d u r i n g t h e r e n a t u r a t i o n e x p e r i m e n t s . On t h e o t h e r h a n d , b o t h t h e E. ooli DNA a n d t h e c h l o r o p l a s t DNA ( p a r t i c u l a r l y t h e c h l o r o p l a s t DNA) u s e d f o r t h e r e n a t u r a t i o n e x p e r i m e n t s wer:e h a n d l e d m o r e e x t e n s i v e l y t h a n t h i s 40 Table IV. V a r i a t i o n i n k i n e t i c complexity with d i f f e r e n t values of S^ ,-20 ,w  Source DNA ^20^w k i n e t i c complexity of DNA concen- ' (daltons) t r a t i o n 12 yg/ml 11.6 1 mole 14.9 1.4 X 10 9 s e c ~ l 24.0 2.5 X 109 12 yg/ml 11.6 1 mole 26.4^ 2.8 X 10 9 s e c ~ l 27.4^ 2.9 X 10 9 29.2^ 3.2 X 10 9 * data taken from experiment 1. taken from table I I I . b a c t e r i a l DNA and, therefore, possibly had a lower molecular weight be-fore shearing with the needle. Since the sedimentation c o e f f i c i e n t s determined i n these experiments give k i n e t i c complexities for E. coli DNA which are too large, i t was not possible to use a c o r r e c t i o n factor based on them. 41 DISCUSSION Aeetabulavia meditevvanea chloroplast DNA has a buoyant density 3 i n CsCl of 1.702 g/cm . This agrees quite c l o s e l y to the value of 3 1.704 g/cm reported by Green et a l . (32) and Heilporn and Limbosch (34), and indicates a basedcomposition of 42.8% G+C. The Tm of Aeetabulavia chloroplast DNA i n SSC i s 86.7°. This gives a value of 43% G+C for the base composition, as calculated from equation 5 of Mandel et_ al_. (41) using _E. c o l i K 12 DNA as the standard. The close agreement of the base compositions calculated from the buoyant density and the melting tempera-ture indicates the absence of any unusual bases i n Aeetabulavia meditevva-nea chloroplast DNA (36, 41, 42). Chromatographic data of Gibor supports this conclusion. Aeetabulavia chloroplast DNA which was hydrolyzed enzy-m a t i c a l l y and then chromatographed showed only deoxyadenosine, thymidine, deoxycytosine and deoxyguanosine (26). Aeetabulavia meditevvanea chloroplast DNA melts with a Tm of 70.7° i n 0.1 x SSC. This i s 0.6° lower than expected for a DNA of 43% G+C content. This lowering of the Tm i s probably due to the low molecular weight of the DNA (20,20)). The melting t r a n s i t i o n of Aeetabulavia chloro-p l a s t DNA i s very broad. This i s p a r t l y due to the low molecular weight of the DNA (20,21) but could also be i n d i c a t i v e of a high degree of i n t r a -molecular heterogeneity (42). The observed unevenness of the melting t r a n s i t i o n , e s p e c i a l l y noticeable when the DNA was melted i n 0.1 x SSC, may also be i n d i c a t i v e of a high degree of intramolecular heterogeneity. This uneven melting t r a n s i t i o n i s s i m i l a r to that reported for the mito-chondrial DNA of Dvosophila melanogastev (47). Polan et a l . (47) a t t r i b u t e 42 the m u l t i t r a n s i t i o n a l behavior of D. melanogastev mitochondrial DNA to differences i n base composition i n various portions of the molecule. A d i f f e r e n t i a l p l o t of the melting t r a n s i t i o n of A. meditewanea chloroplast DNA shows mul t i p l e peaks, which supports the conclusion that t h i s DNA has a high degree of intramolecular heterogeneity. The melting curves of chloroplast DNAs from Chlovella pyrenoidosa (6), Chlamydomonas reinhavdtii (5,72) and Euglena gracilis (65) show considerable intramolecular hetero-geneity i n these DNAs as w e l l . The k i n e t i c complexity of Acetabulavia chloroplast DNA, based on the 9 average value obtained i n three separate experiments i s 1.1 + 0.4 x 10 g daltons, with the l e a s t value being 8.1 x 10 daltons (see table I I ) . E. coli DNA which was renatured at the same time as the chloroplast DNA, under the same conditions, has a k i n e t i c complexity lower than the l i t e r a -9 ture value of 2.5 x 10 daltons (74). This i s probably due to errors i n the values of the a l k a l i n e sedimentation c o e f f i c i e n t s (see Results). Therefore, the k i n e t i c complexity of Acetabulavia meditewanea chloroplast 9 DNA i s probably greater than 1.1 x 10 daltons. This would indi c a t e that —16 7 the amount of DNA per chloroplast i s more than the 1 x 10 g (6x10 d a l -tons) estimated by Gibor and Izawa (27). I f the a n a l y t i c a l complexity of 9 _15 1.4 x 10 daltons (2.3 x 10 g) determined by Green (unpublished) i s co r r e c t , then at most there could be two copies of the DNA nucleotide se-g quence, based on a k i n e t i c complexity of 8.1 x 10 daltons. I f a f r a c t i o n of the DNA i s highly r e p e t i t i o u s , t h i s would have been evident as a separ-ate k i n e t i c component i n the renaturation reaction (12, 13, 14, 74). A very small amount (approximately 3% of the t o t a l DNA) of a f a s t e r renatur-ing component was observed but t h i s i s most l i k e l y due to mitochondrial 43 g DNA. Since 8.1 x 10 daltons represents a minimum value, and the average 9 value for the k i n e t i c complexity (1.1 + 0.4 x 10 daltons) i s probably too low due to errors i n the values of the sedimentation c o e f f i c i e n t s , i t seems l i k e l y that there i s no nucleotide sequence r e p e t i t i o n i n Aeetabulavia meditevvanea chloroplast DNA. On the other hand, since 2.3 x 10 "'"^ g represents an average amount of DNA per c h l o r o p l a s t , i t i s p o s s i -ble that while some chloroplasts contain one copy of the genome, others may contain two or more copies. Other chloroplasts may not contain DNA i f , during some chloroplast d i v i s i o n s , a l l of the DNA goes to one daughter chloroplast and none to the other. This would be compatible with the ob-servations of Woodcock and Bogorad (75) that no DNA could be detected i n 65-80% of c h l o r o p l a s t s , using four d i f f e r e n t methods; and that i n those chloroplasts with detectable DNA the amount present was v a r i a b l e . The k i n e t i c complexities of chloroplast DNAs from a number of sources are shown i n table V. The k i n e t i c complexity of Aeetabulavia meditevvanea chloroplast DNA i s larger than the complexities of other chloroplast DNAs which have been investigated by renaturation k i n e t i c s . The larger k i n e t i c component of kidney bean chloroplast DNA i s the only value that approaches the k i n e t i c complexity of Aeetabulavia chloroplast DNA. Crandall's r e s u l t s have never been published, however, and there i s some question as to the p u r i t y of the DNA used i n h i s experiments (M.D. C h i l t o n , personal communica-ti o n to B.R. Green). This throws doubt on the v a l i d i t y of h i s high value for the k i n e t i c complexity of kidney bean chloroplast DNA. Thus, the k i -n e t i c complexity of Aeetabulavia meditevvanea chloroplast DNA seems unus-u a l l y l a r g e — b u t then, Aeetabulavia i s an unusual organism. The f a c t that Aeetabulavia can survive f o r long periods following enucleation makes one 44 Table V: K i n e t i c complexities of chloroplast DNAs from various sources Organism Chloroplast DNA k i n e t i c com- A n a l y t i c a l p l e x i t y complexity (daltons) (daltons) Reference Aeetabulavia meditevvanea Ch lanry domonas veinhavdtii Chlovella pyvenoidosa Phaseolus vulgaris (kidney bean) Lettuce 1.1 + 0.4 x 10' 8 Euglena gvaoilis 1.8 x 10 1.94 x 10 1-10 x 10 6 2 x 10 8 3 x 10 1.3 x 10 E 7.4 x 10 £ 7.5 x 10* 3 x 10 6 1.2 x 10 8 8 1.4 x 10' 5.16 x 10' 7.2 x 10 2 x 10' 8 Stutz (64) Bastia et^ a l . (5) Wells and Sager (72) Bayen and Rode (6) Crandall (19) Wells and B i r n s t i e l (71) Pea Tobacco 9.5 x 10 1.14 x 10 8 Kolodner and Tewari (38) Tewari and Wildman (67) Suspect that t h i s a b i l i t y may be related to the amount of genetic informa-t i o n i n the chloroplasts. I t hardly seems s u r p r i s i n g that the chloroplast DNA of a uninucleate c e l l as large as Aeetabulavia has a greater unique sequence length than the few chloroplast DNAs of other organisms which have been studied to date. 45 Proponents of the symbiotic theory of the o r i g i n of chloroplasts suggest that there was a reduction i n the amount of genetic information i n the chloroplast as t h i s information was transferred from the organelle-symbiont to the nuclear genome during the course of time, and the chloro-p l a s t became an i n t e g r a l part of the c e l l (61). I f t h i s i s the case, then perhaps Acetabulavia chloroplasts have not become integrated into the c e l l to the same degree as chloroplasts of other plants that have been studied. Bayan and Rode (6) suggest that since most chloroplast DNAs studied are quite s i m i l a r i n terms of k i n e t i c complexity and heterogeneity, t h i s i s din agreement with the theory that chloroplasts of d i f f e r e n t organ-isms might have had a common o r i g i n . I t i s possible, however, that chloro-p l a s t s arose more than once, rather than as a r e s u l t of a s i n g l e evolution-ary event. Two k i n e t i c components have been observed for the chloroplast DNA' from some sources (6, 19, 71, 72), but for Acetabulavia meditewanea chloroplast DNA there i s only one component. A very minor, f a s t e r renatur-ing component was observed, but this probably r e s u l t s from contamination by mitochondrial DNA. I t i s i n t e r e s t i n g to note that with Chlamydomonas veinhavdtii chloroplast DNA, Wells and Sager (72) observe two k i n e t i c com-ponents, but Bastia et a l . (5) report only the slower renaturing component. Perhaps t h i s difference r e f l e c t s differences i n techniques used for i s o l a t -ing the DNA and carrying out the renaturation reaction. The k i n e t i c complexity of Acetabulavia meditewanea chloroplast DNA 9 ( l i l +0.4 x 10 daltons) i s comparable to the k i n e t i c complexities of DNAs from a number of f r e e - l i v i n g micro-organisms, such as Hemophilus influenzae (1.01 + 0.18 x 10 9 daltons), H. aegyptius (1.17 + 0.11 x 10 9 daltons), 46 9 Vasteurella muVbooi&a (1.13 + 0.16 x 10 daltons), Staphylococcus albus (1.12 + 0.02 x 10 9 daltons), Neisseria catarrhalisi 1.04 + 0.16 x 10 9 9 daltons) (3) and Acholeplasma laidlawii (1.10 x 10 daltons) (2). A 9 6 genome s i z e of 1.1 x 10 daltons represents 3.3 x 10 nucleotides, or 1.7 x 10 nucleotide p a i r s . This amount of DNA could code f or a con s i -derable number of proteins, i n addition to coding for ribosomal ENA and transfe r RNA. Experimental evidence indicates that chloroplast ribosomal RNA and possibly some transfer RNAs are synthesized i n the chloroplasts of A. mediterranea and A. cliftonii (8, 24, 25, 53). Incorporation of precur-sors i n t o RNA by i s o l a t e d chloroplasts i s i n h i b i t e d by darkness, actino-mycin and deoxyribonuclease (8), i n d i c a t i n g that t h i s RNA i s transcribed from chloroplast DNA. Farber's studies (24,25) of RNA metabolism i n whole c e l l s and c e l l fragments of Acetabularia mediterranea support the findings of Berger (8). The ribosomal RNA fr a c t i o n s found i n Acetabularia 3 together represent a chain of about 5 x 10 ribonucleotides (52) . These 3 would be coded by 5 x 10 deoxyribonucleotide p a i r s . 3 At l e a s t 1.6 x 10 DNA base pairs are required to code for transf e r RNA, assuming 20 d i f f e r e n t species of chloroplast tRNA each with 80 nu-cl e o t i d e s . This leaves 1,693,400 deoxyribonucleotide p a i r s (or l e s s i f the ribosomal RNAs and transfer RNAs are formed from l a r g e r precursor molecules) f o r regulatory and s t r u c t u r a l genes. The amount of DNA which i s involved i n the regulation of gene a c t i v i t y i s not known. I f t h i s i s ignored, then an approximate maximum number of proteins which can be coded by t h i s amount of DNA can be estimated. I f the ribosomal proteins are coded by the chloroplast DNA (although recent evidence suggests that some 47 of them may be under nuclear c o n t r o l — s e e below), they may account f o r 4 4 approximately 1.1 x 10 amino acids (52) or 3.3 x 10 nucleotide p a i r s . This leaves 16.6 x 10^ deoxyribonucleotide p a i r s , which could code for approximately 5.5 x 10^ amino acids. Assuming an average p r o t e i n chain length of 300 amino acids (70), then the chloroplast DNA might code f o r as many as 1800 proteins. T h e o r e t i c a l l y , then, the chloroplast DNA could code for most, i f not a l l , of the chloroplast proteins. What i s the experimental evidence to suggest which proteins, i f any, are coded by the chloroplast DNA? Goffeau and Brachet (29) studied the incorporation of radioactive amino acids i n t o proteins by chloroplasts i s o l a t e d from anucleate f r a g -ments of Aeetabulavia. From the e f f e c t s of a number of i n h i b i t o r s on t h i s process, these researchers concluded that the synthesis of chloro-p l a s t proteins i s dependent on chloroplast DNA. Further studies showed that 60% of the t o t a l r a d i o - a c t i v i t y i s associated with the chloroplast membranes following incorporation of radioactive amino acids into proteins by i s o l a t e d chloroplasts (28). Using autoradiography, Shephard (56) compared the e f f e c t s of enuclea-t i o n and actinomycin D on the incorporation of n u c l e i c a c i d and protein precursors by Aeetabulavia chloroplasts. He found that thymidine was s t i l l incorporated i n t o chloroplast DNA one week a f t e r enucleation. Periods up to 5 weeks a f t e r enucleation had l i t t l e e f f e c t on the a b i l i t y of the chloroplasts to incorporate ur id in e i n t o ribonuclease removable material. Treatment with actinomycin, however, resulted i n the almost complete ab-sence of l a b e l l e d uridine from the p l a s t i d RNA of normal as w e l l as anucleate c e l l s . Incorporation of leucine by the chloroplasts was affected 48 very l i t t l e by enucleation, but treatment with actinomycin greatly reduced leucine incorporation i n normal and anucleate plants. Shephard concluded that r e p l i c a t i o n of the chloroplast DNA i s independent of the nucleus. He also suggested the evidence i s strong that synthesis of p l a s t i d RNA and protein i s under the d i r e c t i o n of p l a s t i d DNA. Craig and Gibor (18) found that photosynthetic a c t i v i t y decreased i n whole and anucleate Acetabutavia c e l l s which had been maintained i n the dark. Upon r e i l l u m i n a t i o n , however, both whole and anucleate c e l l s regained photosynthetic a c t i v i t y . Treatment with puromycin t o t a l l y i n -h i b i t e d recovery of photosynthetic a c t i v i t y , i n d i c a t i n g that p r o t e i n syn-thesis i s necessary for this recovery. During treatment with actinomycin D, some recovery of a c t i v i t y was observed during the e a r l y stages of r e i l l u m i n a t i o n but f u l l reattainment of photosynthetic a c t i v i t y did not occur i n whole or anucleate c e l l s . This suggests that t r a n s c r i p t i o n of chloroplast DNA i s required during the l i g h t induced recovery of photo-synthetic a c t i v i t y . These conclusions c o n f l i c t with evidence presented by Schweiger and others which demonstrates nuclear control over some of the chloroplast proteins. Schweiger et_ al_. (54) studied the isozyme patterns of malic dehydrogenase by acrylamide gel d i s c electrophoresis, and found that they were species s p e c i f i c i n four species of Dasycladaceae (Acetabularia). Following enucleation, the positions and r e l a t i v e concentrations of the d i f f e r e n t isozyme bands remained constant. I n t e r s p e c i f i c nuclear trans-plants, however, changed the isozyme patterns of the r e c i p i e n t species to that of the nucleus donor species. Thus, i t appears that malic dehydrogenase, an enzyme associated with the chloroplasts, i s c o n t r o l l e d by the nucleus. 49 I n a s i m i l a r s e t o f e x p e r i m e n t s , R e u t e r a n d S c h w e i g e r ( 5 0 ) d e m o n s t r a t e d t h a t l a c t i c d e h y d r o g e n a s e , a l s o a s s o c i a t e d w i t h t h e c h l o r o p l a s t s , i s c o d e d b y t h e c e l l n u c l e u s . , M a l i c d e h y d r o g e n a s e a n d l a c t i c d e h y d r o g e n a s e a r e n o t e n z y m e s w h i c h o n e e x p e c t s t o f i n d a s s o c i a t e d w i t h c h l o r o p l a s t s , b u t r a t h e r w i t h m i t o c h o n d r i a . I t i s q u i t e p o s s i b l e t h a t t h e c h l o r o p l a s t p r e p a r a t i o n s u s e d i n t h e s e e x p e r i m e n t s w e r e c o n t a m i n a t e d w i t h m i t o c h o n -d r i a . ( 5 3 ) . A l s o u s i n g e l e c t r o p h o r e s i s , A p e l a n d S c h w e i g e r ( 1 ) s t u d i e d t h e p a t -t e r n s o f a m e m b r a n e p r o t e i n f r a c t i o n f r o m A. calyculus a n d A. meditewanea c h l o r o p l a s t s . T h e p r o t e i n p a t t e r n s w e r e c o m p o s e d o f 8 p e a k s common f o r b o t h s p e c i e s a n d 3 p e a k s w h i c h w e r e s p e c i e s s p e c i f i c . S i x w e e k s a f t e r i n t e r s p e c i f i c n u c l e a r t r a n s p l a n t a t i o n o r i m p l a n t a t i o n t h e p r o t e i n p a t t e r n c h a n g e d t o t h a t o f t h e n u c l e u s d o n o r s p e c i e s . T h u s , t h e s p e c i e s s p e c i f i c c h l o r o p l a s t p r o t e i n s a r e n u c l e u s d e p e n d e n t a n d a p p e a r t o b e c o d e d b y t h e n u c l e a r DNA. T h i s d o e s n o t r u l e o u t t h e p o s s i b i l i t y t h a t t h e p e a k s w h i c h a r e common t o b o t h s p e c i e s a r e d u e t o p r o t e i n s w h i c h a r e c o d e d b y c h l o r o -p l a s t DNA. A n o t h e r p o s s i b i l i t y i s t h a t t h e p r o t e i n s w h i c h a r e c o d e d b y t h e n u c l e u s c o n s t i t u t e o u t e r m e m b r a n e p r o t e i n s , w h i l e t h e i n n e r c h l o r o -p l a s t m e m b r a n e c o m p o n e n t s a r e c o d e d b y c h l o r o p l a s t DNA. T h e t e c h n i q u e s u s e d w o u l d n o t s e p a r a t e i n n e r c h l o r o p l a s t m e m b r a n e s a n d o u t e r c h l o r o p l a s t m e m b r a n e s . T h e i n c o r p o r a t i o n o f r a d i o a c t i v e a m i n o a c i d s i n t o t h e m e m b r a n e p r o t e i n f r a c t i o n o f Acetabulavia meditewanea w a s a l s o s t u d i e d b y t h e s e r e s e a r c h e r s . T h e e l e c t r o p h o r e t i c p a t t e r n o f t h e l a b e l l e d c h l o r o p l a s t p r o -t e i n s c o n s i s t e d o f a t l e a s t 3 p e a k s . B o t h c y c l o h e x i m i d e a n d c h l o r a m p h e n i -c o l a f f e c t e d t h e i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o p e a k s 1 a n d 2, w h i l e 50 cycloheximide i n h i b i t e d the incorporation into peak 3. Isolated chloro-p l a s t s incorporated amino acids only into the f i r s t two peaks. This i n -dicates that some of the proteins i n peaks 1 and 2 are probably synthe-sized on chloroplast ribosomes whereas peak 3 proteins are synthesized on cytoplasmic ribosomes. More recently, Kloppstech and Schweiger (37) have used the same techniques to demonstrate species s p e c i f i c differences i n the e l e c t r o p h o r e t i c patterns of chloroplast ribosomal proteins from A. meditevvanea, A. eliftonii and A. evenulata. Again, following i n t e r s p e c i f i c nuclear im-plants and transplants, the protein patterns of the host species change to those of the nucleus donor species. The r e s u l t s i n d i c a t e that those chloroplast ribosomal proteins which are s p e c i e s - s p e c i f i c are coded by the nuclear genome. They do not r u l e out the p o s s i b i l i t y that the pro-t e i n bands which are the same i n the electrophoretic patterns f o r a l l 3 species may be coded by chloroplast DNA. Ceron and Johnson (16) have studied the control of protein synthesis during the development of Aeetabulavia. Using zone electrophoresis, they analyzed proteins from the soluble, c h l o r o p l a s t i c and. c e l l membrane f r a c -tions of a x e n i c a l l y grown Aeetabulavia evenulata. C e l l s from various de-14 velopmental stages were l a b e l l e d with C leucine. Throughout develop-ment, the electrophoretic patterns of proteins from the soluble f r a c t i o n remained constant with respect to the number of bands, but the r e l a t i v e rates of synthesis of several proteins changed. Proteins from the membrane f r a c t i o n , however, showed changes i n both the number of bands i n the electrophoretic pattern and i n the r e l a t i v e synthetic rates of various protein species. A f t e r the c e l l s were enucleated, there was an i n i t i a l 51 decrease i n the o v e r a l l rate of synthesis of proteins from the soluble f r a c t i o n . This returned to normal l e v e l s by 6 weeks post enucleation. Enucleation did not a f f e c t the electrophoretic pattern of membrane f r a c -t i o n proteins. Analysis of soluble proteins from p u r i f i e d chloroplasts a f t e r t h e i r l a b e l l i n g i n normal and anucleate c e l l s , showed that both the s t a i n i n g and autoradiographic patterns were e s s e n t i a l l y unchanged even 4 weeks a f t e r enucleation. When i s o l a t e d chloroplasts were incuba-ted and l a b e l l e d in vitro some of the components of the chloroplast pro-t e i n pattern were synthesized. From these r e s u l t s i t appears that syn-thesis of most of the protein components of the soluble and membrane f r a c -tions i s not under immediate con t r o l of the nucleus. Since i t i s u n l i k e l y that a l l these proteins are coded by chloroplast DNA, the genetic messages for at l e a s t some of them must be i n the form of lon g - l i v e d messenger RNA from the nucleus. As these researchers point out, however, attempts to i s o l a t e messenger RNA from Acetabularia have been unsuccessful. Again, the r e s u l t s suggest that at l e a s t some of the chloroplast proteins are coded by chloroplast genes. Despite the lack of evidence to show that s p e c i f i c proteins are coded by chloroplast DNA, and despite evidence that some proteins associated with chloroplasts are under nuclear c o n t r o l , i t seems unreasonable that 1.1.x 9 10 daltons of DNA could e x i s t i n a chloroplast and not code for anything. Is i t , perhaps, possible that most, i f hot a l l , of the chloroplast proteins are i n f a c t coded by chloroplast DNA, but that the expression of chloro-p l a s t genes i s under the control of the nucleus? Or perhaps some proteins may be coded i n the nucleus as well as i n the chloroplast DNA. Goffeau (28) suggested t h i s when he stated: 52 " T h e f a c t t h a t t h e i n c o r p o r a t i o n b y i s o l a t e d c h l o r o -p l a s t s i s n o t d e p e n d e n t u p o n t h e t i m e o f e n u c l e a t i o n e x c l u d e s t h e p a r t i c i p a t i o n o f a s t a b l e mRNA o f n u c l e a r o r i g i n i n o u r in vitro s y s t e m : i f s u c h w e r e t h e c a s e o n e w o u l d h a v e e x p e c t e d a d e c a y o f t h e a c t i v i t y a f t e r l o n g p e r i o d s o f e n u c l e a t i o n . T h i s c o n c l u s i o n d o e s n o t n e c e s s a r i l y m e a n , t h a t in vivo, t h e g e n e s i s a n d r e p l i -c a t i o n o f c h l o r o p l a s t s d o e s n o t r e q u i r e t h e p a r t i c i p a -t i o n o f c y t o p l a s m i c r i i b o s o m e s a n d o f n u c l e u s - c o d e d m R N A . . . H o w e v e r , i t s e e m s c l e a r , t h a t a f t e r i s o l a t i o n t h e c h l o r o p l a s t s d i s p l a y o n l y t h e i r own i n d e p e n d e n t p r o t e i n s y n t h e s i s c a p a c i t i e s . I n o t h e r w o r d s , a l l t h e p r o t e i n s w h i c h a r e l a b e l l e d i n o u r in vitro s y s t e m m u s t b e u n d e r t h e c o n t r o l o f c h l o r o p l a s t DNA a n d n e c e s -s a r i l y s y n t h e s i z e d i n s i d e t h e c h l o r o p l a s t s " . I f some p r o t e i n s a r e c o d e d b y g e n e s i n t h e n u c l e u s a s w e l l a s i n t h e c h l o r o p l a s t , t h e n u c l e a r g e n e s may b e t r a n s c r i b e d i n w h o l e c e l l s , w h i l e t h e c h l o r o p l a s t g e n e s a r e r e p r e s s e d . F o l l o w i n g e n u c l e a t i o n , h ow-e v e r , t h e c h l o r o p l a s t g e n e s may b e c o m e a c t i v e . S u c h a p h e n o m e n o n c o u l d e x p l a i n t h e o b s e r v a t i o n s o f C e r o n a n d J o h n s o n ( 1 6 ) t h a t f o l l o w i n g e n u -c l e a t i o n , t h e o v e r a l l r a t e o f s y n t h e s i s o f p r o t e i n s f r o m t h e s o l u b l e f r a c t i o n s h o w e d a n i n i t i a l d e c r e a s e , b u t r e t u r n e d t o n o r m a l l e v e l s b y 6 w e e k s p o s t - e n u c l e a t i o n . On t h e o t h e r h a n d , t h e m e m b r a n e f r a c t i o n p r o -t e i n s w h i c h s h o w c h a n g e s d u r i n g d e v e l o p m e n t , b u t a r e u n a f f e c t e d b y e n u -c l e a t i o n , may b e t r a n s l a t e d f r o m l o n g l i v e d m e s s e n g e r RNA t r a n s c r i b e d i n t h e n u c l e u s . I t i s a l s o p o s s i b l e , o f c o u r s e , t h a t t h e r e i s n o d u p l i c a t i o n o f c h l o r o p l a s t g e n e s i n t h e n u c l e a r g enome. P e r h a p s some c h l o r o p l a s t p r o -t e i n s a r e c o d e d b y t t h e n u c l e a r DNA, w h i l e o t h e r s a r e c o d e d b y t h e c h l o r o -p l a s t DNA. Z e t s c h e ( 7 6 ) e x a m i n e d t h e c h l o r o p h y l l c o n t e n t o f n u c l e a t e a n d a n u c l e a t e c e l l s o f Aoetabularia.mediterranea a n d f o u n d t h a t i t i n c r e a s e s d u r i n g a b o u t 4 w e e k s a f t e r e n u c l e a t i o n . F r o m t h e e f f e c t s o f v a r i o u s i n -h i b i t o r s o f RNA a n d p r o t e i n s y n t h e s i s o n t h e i n c r e a s e i n c h l o r o p h y l l c o n -t e n t , h e c o n c l u d e d t h a t p r o t e i n s y n t h e s i s i s n e c e s s a r y f o r c h l o r o p h y l l 53 formation or s t a b i l i z a t i o n . Furthermore, Zetsche concluded that the chl o r o p h y l l content of the c e l l s i s con t r o l l e d by chloroplast DNA as wel l as the nuclear genome. I t would be of i n t e r e s t to study the DNA: DNA h y b r i d i z a t i o n between the nuclear and chloroplast DNA to determine the extent to which they contain base sequences i n common, i f at a l l . As already mentioned, another a l t e r n a t i v e i s that a l l of the chloro-p l a s t proteins are coded by the chloroplast genome, but the t r a n s c r i p t i o n of chloroplast DNA i s under nuclear control i n i n t a c t plants. Control of chloroplast gene expression might be mediated by small molecules, the metabolism of which i s under d i r e c t nuclear c o n t r o l . Shephard and Bidwell (58) have found that i s o l a t e d chloroplasts from Acetabulavia meditewanea carry out normal photosynthesis, as w e l l as the biosynthesis of proteins, pigments, l i p i d s and nucl e i c acids from inorganic precursors, at the same rates as i n t a c t c e l l s . They suggest that the membrane surrounding the chloroplasts i s highly s e l e c t i v e and that in vivo control of chloroplast a c t i v i t y may be mediated by transport mechanisms which govern the rate of entry of some molecules, such as HCO^, and the release of photosynthetic products. Their studies also suggest that indole a c e t i c acid may mediate a control mechanism i n i n t a c t c e l l s . Obviously, much more research i s required i f we are to understand the complex nuclear-cytoplasmic i n t e r a c t i o n s i n Acetabulavia. Although Acetabulavia chloroplasts exhibit autonomous behavior in vitvo, the i n t e r -actions between the nucleus, the chloroplasts and other c e l l u l a r components must be of some benefit to the organism, otherwise there i s no reason to believe that Acetabulavia meditewanea would have evolved i n t o the complex 54 c e l l that i t i s . To conclude, Acetabulavia meditewanea chloroplast DNA has a 9 k i n e t i c complexity of at l e a s t 1.1 + 0.4 x 10 daltons. T h e o r e t i c a l l y , t h i s amount of DNA could code for many, i f not a l l , of the chloroplast proteins. At the present time, however, there i s no conclusive evidence to i n d i c a t e s p e c i f i c a l l y which proteins are coded by the chloroplast genome. 55 BIBLIOGRAPHY 1. Apel, Klaus, and Hans-Georg Schweiger. 1972. Nuclear Dependency of Chloroplast Proteins in Aeetabulavia. Eur. J . Biochem. 25: 229-238. 2. Askaa, Gerd, C. Christiansen and H. ErntfJ.. 1973. Bovine Mycoplasmas: Genome Size and Base Composition of DNA. J . General Microbiology 75: 283-286. 3. Bak, A. Leth, C. Christiansen and A. Stenderup. 1970. Bacterial Genome Sizes Determined by DNA Renaturation Studies. J . General Microbiology. 64: 377-380. 4. Baltus, E. and J . Brachet. 1963. Presence of deoxyribonucleic acid in the chloroplasts of Aeetabulavia meditevvanea. Biochim. Biophys. Acta. 76: 490-492. 5. Bastia, Deepak, Kwen-Sheng Chiang, Hewson Swift, and Peter Siersma. 1971. Heterogeneity, Complexity and Repetition of the Chloroplast DNA of Chlamydomonas veinhavdtii. Proc. Nat. Acad. Sci. (U.S.A.). 68: 1157-1161. 6. Bayen, Marcel and Andre Rode. 1973. Heterogeneity and Complexity of Chlovella Chloroplastic DNA. Eur. J . Biochem. 39: 413-420. 7. Bellamy, A.R. and R.K. Ralph. 1968. Recovery and Purification of Nucleic Acids by Means of Cetyltrimethylammonium Bromide, p. 156-160. In: L. Grossman and K. Moldave [ed.] Methods in Enzymology (XII) Nucleic Acids (B) Academic Press, New York and London. 8. Berger, Sigrid. 1967. RNA-Synthesis in Aeetabulavia.II.RNA-Synthe-s i s . i n Isolated Chloroplasts. Protoplasma 64: 13-25. 9. Berger, S. and H.G. Schweiger. 1969. Synthesis of chloroplast DNA in Aeetabulavia. Physiol. Chem. Physics. 1: 280-292. 10. Borst, P. 1972. Mitochondrial Nucleic Acids. Annual Review of Bio-chemistry. 41: 333-376. 11. Britten, Roy J . 1970. Repeated DNA and Transcription p. 187-216. In: E.W. Hanly^dJProblems in Biology: RNA i n Development. Univ. of Utah Press, Salt Lake City. 12i Britten, R . J . and D.E. Kohne. 1966. Nucleotide sequence repetition in DNA. Carnegie Institution Yearbook 65: 78-106. 56 13. B r i t t e n , R.J. and D.E. Kohne. 1967. Repeated nucleotide sequences. Carnegie I n s t i t u t i o n Yearbook 66: 73-88. 14. B r i t t e n , R.J. and D.E. Kohne. 1968. Repeated Sequences i n DNA. Science 161: 529-540. 15. Burton, K. 1956. Study of the Conditions and Mechanism of the Diphenylamine Reaction for the Colorimetric Estimation of Deoxy-ri b o n u c l e i c Acid. Biochem. J . 62: 315-323. 16. Ceron, Gabriel, and E. Marshall Johnson. 1971. Control of p r o t e i n synthesis during the development of Acetabulavia. J . Embryol. exp. Morph 26: 323-338. 17. C o l l i n s , CH. 1964. M i c r o b i o l o g i c a l Methods. Butterworths, London, 330 p. 18. Craig, I.W. and A. Gibor. 1970. Biosynthesis of proteins involved with photosynthefeie a c t i v i t y i n enucleated Acdtabulavia sp. Biochim. Biophys. Acta. 217: 488-495. 19. Crandall, G. Douglas. 1971. A comparative study of the DNA extrac-ted from chloroplast and nuclear f r a c t i o n s of Phaseolus vulgavis L. leaves. Ph.D. Thesis, Indiana U n i v e r s i t y . 20. Crothers, D.M., N e v i l l e , R. Kallenbach and B.H. Zimm. 1965. The Melting T r a n s i t i o n of Low-Molecular-Weight DNA: Theory and Experi-ment. J. Mol. B i o l . 11: 802-820. 21. DeLey, J . 1969. Compositional Nucleotide D i s t r i b u t i o n and the Theo-r e t i c a l P r e d i c t i o n of Homology i n B a c t e r i a l DNA. J . Theoret. B i o l . 22: 89-116. 22. Eigner, Joseph, Helga Boedtker and George Michaels. 1961. The ther-mal degradation of n u c l e i c acids. Biochim. Biophys. Acta. 51: 165-168. 23. Falkow, S. and D.B. Cowie. 1967. Intramolecular heterogeneity of the DNA of temperate bacteriophages. Carnegie I n s t i t u t i o n Year-book 66: 88-95. 24. Farber, Florence E. 1969. Studies on RNA metabolism i n Acetabulavia meditewanea. I. The i s o l a t i o n of RNA and l a b e l l i n g studies on RNA of whole plants and plant fragments. Biochim. Biophys. Acta 174: 1-11. t 57 25. Farber, Florence E. 1969. Studies on RNA metabolism i n Aceta-bulavia meditewanea. I I . The l o c a l i z a t i o n and s t a b i l i t y of RNA species; the e f f e c t s on RNA metabolism of dark periods and actinomycin D. Biochim. Biophys. Acta 174: 12-22. 26. Gibor, A. 1967. DNA Synthesis i n Chloroplasts, p.321-328. In T.W. Goodwin [ed.] Biochemistry of Chloroplasts, Vol. I I . Aca-demic Press. New York and London. 27. Gibor, A. and M. Izawa. 1963. The DNA content of the chloroplasts of Acetabulavia. Proc. Nat. Acad. S c i . (U.S.A.) 50: 1164-1169. 28. Goffeau, A. 1969. 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Mandel, M. , C L . Schildkraut, and J . Marmur. 1968. Use of CsCl Density Gradient Analysis for Determining the Guanine plus Cytosine Content of DNA. p. 184-195. In: L. Grossman and K. Moldave [ed.] Methods i n Enzymology (XII) Nucleic Acids (B). Academic Press. New York and London. 44. Marmur, J . 1961. Procedure f o r the I s o l a t i o n of Deoxyribonucleic Acid from Micro-organisms. J . Mol. B i o l . 3: 208-218. 45. Marmur, J u l i u s , and Paul Doty. 1961. Thermal Renaturation of Deoxy-ri b o n u c l e i c Acids. J . Mol. B i o l . 3: 585-594. 46. Miura, K i n - i c h i r o . 1967. Preparation of B a c t e r i a l DNA by the Phenol-pH9-RNases Method, p. 543-545. In: L. Grossman and K. Moldave [ed.] Methods i n Enzymology (XII) Nucleic Acids (A). Academic Press. New York and London. 47. Polan, MaryLake, Susan Friedman, Joseph G. G a l l , and Walter Gehring. 1973. I s o l a t i o n and ch a r a c t e r i z a t i o n of mitochondrial DNA from Drosophila melanogaster. J . C e l l B i o l . 56: 580-589. 59 48'.. P u i s e u x - D a o , S. 1 9 7 0 . A c e t a b u l a r i a a n d C e l l B i o l o g y . S p r i n g e r -V e r l a g . New Y o r k I n c . 1 6 2 p . 4 9 . R h a e s e , H a n s - J u r g e n a n d E r n s t F r e e s e . 1 9 6 8 . C h e m i c a l a n a l y s i s o f DNA a l t e r a t i o n s . I . B a s e l i b e r a t i o n a n d b a c k b o n e b r e a k a g e o f DNA a n d o l i g o d e o x y a d e n y l i c a c i d i n d u c e d b y h y d r o g e n p e r o x i d e a n d h y d r o x y l a m i n e . B i o c h i m . B i o p h y s . A c t a 1 5 5 : 4 7 6 - 4 9 0 . 5 0 . R e u t e r , W. a n d H.G. S c h w e i g e r . 1 9 6 9 . K e r n k o n t r o l l i e r t e L a c t a t -d e h y d r o g e n a s e i n Acetabulavia [ i n G e r m a n , E n g l i s h s ummary] . P r o t o p l a s m a 6 8 : 3 5 7 - 3 6 8 . 5 1 . S c h i l d k r a u t , C a r l L . , J u l i u s M a r m u r a n d P a u l D o t y . 1 9 6 2 . D e t e r -m i n a t i o n o f t h e B a s e C o m p o s i t i o n o f D e o x y r i b o n u c l e i c A c i d f r o m i t s B u o y a n t D e n s i t y i n C s C l . J . M o l . B i o l . 4: 4 3 0 - 4 4 3 . 5 2 . S c h w e i g e r , H.G. 1 9 6 9 . C e l l B i o l o g y o f A c e t a b u l a r i a . C u r r e n t T o p i c s i n M i c r o b i o l o g y a n d I m m u n o l o g y 5 0 : 1 - 3 6 . 5 3 . S c h w e i g e r , H.G., W.L. D i l l a r d , A. G i b o r a n d S i g r i d B e r g e r . 1 9 6 7 . R N A - S y n t h e s i s i n Acetabulavia I . R N A - S y n t h e s i s i n E n u c l e a t e d C e l l s . P r o t o p l a s m a 6 4 : 1 - 1 2 . 5 4 . S c h w e i g e r , H.G., R.W.P. M a s t e r a n d G. W e r z . 1 9 6 7 . N u c l e a r C o n t r o l o f a C y t o p l a s m i c E n z y m e i n Acetabulavia. N a t u r e 2 1 6 : 5 5 4 - 5 5 7 . 5 5 . S h e r p h a r d , D.C. 1 9 6 5 . C h l o r o p l a s t m u l t i p l i c a t i o n a n d g r o w t h i n t h e u n i c e l l u l a r a l g a Acetabulavia meditewanea. E x p e r i m e n t a l C e l l R e s e a r c h 3 7 : 9 3 - 1 1 0 . 5 6 . S h e p h a r d , D.C. 1 9 6 5 . A n a u t o r a d i o g r a p h i c c o m p a r i s o n o f t h e e f -f e c t s o f e n u c l e a t i o n a n d a c t i n o m y c i n D o n t h e i n c o r p o r a t i o n o f n u c l e i c a c i d a n d p r o t e i n p r e c u r s o r s b y Acetabulavia c h l o r o p l a s t s . B i o c h i m . B i o p h y s . A c t a . 1 0 8 : 6 3 5 - 6 4 3 . 5 7 . S h e p h a r d , D.C. 1 9 7 0 . A x e n i c C u l t u r e o f Acetabulavia i n a S y n t h e t i c M e d i u m , p . 4 9 - 6 9 . I n : D.M. P r e s c o t t [ed.] M e t h o d s i n C e l l P h y s i o l o g y , V o l . 4. A c a d e m i c P r e s s , New Y o r k a n d L o n d o n . 5 8 . S h e p h a r d , D.C. a n d R.G.S. B i d w e l l . 1 9 7 3 . 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K i n e t i c and Spectrophotome-t r i c Studies on the Renaturation of Deoxyribonucleic Acid. Biochem. J. 109: 543-557. 69. Vinograd, Jerome, Robert Bruner, Rebecca Kent and Jean Weigle. 1963. Band-centrifugation of macromolecules and viruses i n self-generat-ing density gradients. Proc. Nat. Acad. S c i . (U.S.A.) 49: 902-910. 70. Watson, James D. 1970. Molecular Biology of the Gene, second e d i t i o n , W.A. Benjamin, Inc., New York 662 p. 71. Wells, Richard, and Max B i r n s t i e l . 1969. K i n e t i c Complexity of C h l o r o p l a s t a l Deoxyribonucleic Acid and Mitochondrial Deoxyribo-n u c l e i c Acid from Higher Plants. Biochem. J. 112: 777-786. 72. Wells, Richard, and Ruth Sager. 1971. Denaturation and the Renatur-ation K i n e t i c s of Chloroplast DNA from Chlamydomonas reinhardi. J. Mol. B i o l . 58: 611-622. 73. Werz, G., and G. K e l l n e r . 1968. Molecular C h a r a c t e r i s t i c s of Chloroplast DNA of Acetdbularia^ C e l l s . J . U l t r a s t r u c t u r e Research. 24: 109-115. 61 Wetmur, James G., and Norman Davidson. 1968. Kinetics of Renatura-tion of DNA. J. Mol. B i o l . 31: 349-370. Woodcock, Christopher L.F., and Lawrence Bogorad. 1970. Evidence for v a r i a t i o n i n the quantity of DNA among pla s t i d s of Aeetabulavia. J. C e l l B i o l . 44': 361-375. Zetsche, K. 1969. Die Wirkung von RNA-und Proteinsyntheseinhibitoren auf den Chlorophyllgehalt kernhaltiger und kernloser Acetabularien. [in German, English summary] Planta (Berl.) 89: 284-298. 

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