Characterization of a blue-green algal genome

J. Mol. Biol. (1977) 110, 341-361

Characterization of a Blue-green Algal Genome THOI~IAS i~. ROBERTS, L r ~

C. KLOTZ A_~D ALFRED R. LOEBLICH III

Departments of Biochemistry and Molecular Biology, and Biology Harvard University, Cambridge, Mass. 02138, U.S.A. (Received 27 May 1976, and in revised form 11 October 1976) The following properties of the genomic DNA of the unicellular blue-green alga Agmenel/um quadrulvl/catum have been determined: (1) buoyant density in neutral CsC1 (1"7012 g/cm3); (2) thermal denaturation profile (Tin ~ 89"2~ (3) kinetic complexity (2.2 • 109 to 2.8 • 109 daltons); (4) quantity per cell (8 • 109 to 13 • 109 daltons); and (5) molecular weight (3.9 • 109). These experiments indicate that blue-green algal DNA is similar to that of bacteria in the following ways: (1) there is little base composition heterogeneity present; (2) repeated sequences are below the level of detection; and (3) the size of the chromosomal DNA is most likely equal to the kinetic complexity. Our studies do suggest, however, that Agmenellum unlike common bacteria, may have a basal DNA content of two or more chromosomes per cell.

1. I n t r o d u c t i o n Blue-green algae, or eyanophytes, could well be called the "other" prokaryotes, relatively neglected b y molecular biologists studyhlg bacteria. Yet, the blue-greens are important organisms for a number of reasons. In the perspective of evolution, blue-greens assume a key position among all groups as the putative source of the first significant amounts of molecular oxygen in the earth's atmosphere. In historical times the blue-green algae, due to their ability to fix nitrogen, have played a vital role in the cultivation of rice and, hence, in the nutrition of the human race (De, 1939; Singh, 1961). I t is the capacity of certain filamentous cyanophytes to fix atmospheric nitrogen that has recently led to increased interest in these organisms among molecular biologists. Finally, these algae play an important role in the theories of the blossoming field of molecular evolution. Thus, on the one hand, the blue-greens possess molecular mechanisms for oxidative metabolism not far removed from those of the mitochondria of eukaryotes (though not so closely related as those of the purple non-sulfur bacteria (Dickerson et al., 1976)); while, on the other hand, the blue-green algal photosynthetic apparatus is so similar to that of plant choloroplasts that it was suggested quite early on t h a t chloroplasts m a y have arisen as endosymbiotic blue-greens (Buetow, 1976; Mereschkowsky, 1905; Tabita eta/., 1974,1976). For excellent general reviews of the blue-green algae see Wolk (1973), Carr & Whitton (1973) and Fogg et al. (1973). Unfortunately, the DNA of the blue-greens is comparatively poorly understood. The buoyant density in cesium chloride gradients has been determined for a large number of blue-green algae, and thermal denaturation profiles have been reported for several species (Craig et al., 1969; Edelman et al., 1967; K a y e et al., 1967; Stanier e~a/., 341

342

T. M. R O B E R T S , L. C. K L O T Z A N D A. R. L O E B L I C H I I I

1971). A q u a l i t a t i v e s t u d y o f t h e r e n a t u r a t i o n of t h e D N A o f t h e species Anacystis nidulans a n d a Lyngbya species has shown t h a t t h e D N A from t h e s e species r e n a t u r e s m u c h m o r e slowly t h a n t h a t of c h l o r o p l a s t D N A w i t h a r a t e r o u g h l y c o m p a r a b l e t o t h a t o f b a c t e r i a l D N A ( K u n g et al., 1971). R e c e n t l y , r e n a t u r a t i o n r a t e c o n s t a n t s for t w o species of blue-greens h a v e b e e n m e a s u r e d b y H e r d m a n & Carr (1974) using o p t i c a l d e n s i t y a t 260 nm. T h e y show none o f t h e i r d a t a , m e r e l y r e p o r t i n g t h a t 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 , /r has a v a l u e t h a t r a n g e s b e t w e e n 2.62 1 m o l - 1 s - ~ a n d 2.85 1 tool -~ s -1 r e l a t i v e t o Escherichia coli D N A a t 2-70 1 mo1-1 s -1. This c o r r e s p o n d s t o a g e n o m e size of 2.2 • l 0 s to 2.5 • 109 d a l t o n s ( u s i n g t h e i r a s s u m p t i o n t h a t E. coli has a g e n o m e size of 2.4 • 109 daltons). T h e y also r e p o r t no r e p e a t e d D N A ; however, t h e y were u n a b l e to follow t h e first e i g h t m i n u t e s o f r e n a t u r a t i o n , w o r k i n g u n d e r c o n d i t i o n s where r e n a t u r a t i o n was followed for some 35 to 45 m i n u t e s . I n tlfis p a p e r we e x a m i n e in some d e t a i l t h e genomic D N A of a p a r t i c u l a r b l u e - g r e e n alga, Agmenellum quadruplicatum. (A s u b s e q u e n t p a p e r ( R o b e r t s & K o t h s , 1976) c h a r a c t e r i z e s t h e supercoiled circular molecules w h i c h m a k e u p a p p r o x i m a t e l y 2 t o 5 % of t h e t o t a l D N A in Agmenellum.) W e r e p o r t here (1) t h e k i n e t i c c o m p l e x i t y o f t h e D N A as d e t e r m i n e d b y b o t h o p t i c a l h y p e r c h r o m i c i t y m e a s u r e m e n t s a t 260 n m a n d h y d r o x y a p a t i t e c h r o m a t o g r a p h y , (2) t h e D N A p e r cell m e a s u r e d u n d e r t w o different g r o w t h c o n d i t i o n s via t w o i n d e p e n d e n t m e t h o d s of m e a s u r e m e n t , a n d (3) t h e m o l e c u l a r weight of t h e c h r o m o s o m a l D N A as d e t e r m i n e d b y viscoelastic r e t a r d a tion time measurements.

2. Experimental Procedure (a) Cells and culture methods A. quadruplicatum, strain P R 6 was a gift from C. van Baalen. Recently strain P R 6 has been shown to belong to the genus Synechoeoccus (Stanier et al., 1971). Cells were grown at 38~ under cool-white fluorescent light in t h e modified sea-water m e d i u m of Loeblich (1975). Large quantities of cells for D N A isolation were grown in 3.5-gal bottles (narrow mouth, large capacity, P y r e x 1595) with bubbled air. Cells used in DNA/cell determination and for molecular weight measurements were raised in 100 ml of medium in 500-ml Nephelo culture flasks using r o t a r y shaking. Light at an intensity of 135 foot-candles gave a doubling time of 20 h =k 1 h, while illumination at 210 foot-candles with bubbling of 5% CO2 balanced air reduced the doubling time to 7.0 h q- 0.5 h. Growth was followed b y measurement of optical density at 620 nm. Contamination was monitored both b y microscopic examination (either of cultures grown into stationary-phase or of cultures grovcn up in medium enriched with proteose-peptone and glucose) and b y plating out the culture on agar-solidified bacterial growth medium. Tests for contamination were negative. (c) DNA isolation D N A was isolated b y a modification of the MUP method (Britten et al., 1970). E a r l y stationary-phase cells from 20 to 30 1 of medium were harvested b y centrifugation in a Sharples centrifuge, resuspended in 30 ml 0.10 M-EDTA, 0"15 M-NaC1 (pH 8.4), and t r e a t e d with lysozyme for 30 min at room temperature. The cell suspension was brought to 8 M in urea, 1% (w/v) sodium dodecyl sulfate and 1 M-NaC104, and e x t r a c t e d once with a 24 : 1 mixture of chloroform and isoamyl alcohol. The aqueous phase was separated b y eentrifugation, brought to 0-12 M in phosphate, re-extracted with the chloroform/isoamyl alcohol mixture, a n d applied to 15 ml of h y d r o x y a p a t i t e . The h y d r o x y a p a t i t e with D N A bound was washed extensively, first with a solution of 0.12 M-phosphate buffer (pH 6-8), 8.0 ~ urea a n d then with 0.12 m-phosphate buffer (pH 6.8). After all cell pigments h a d been removed from the h y d r o x y a p a t i t e , the D N A was eluted with 3 washes of 5 ml

CHARACTERIZATION

OF BLUE-GREEN

ALGAL

GENOME

343

0"6 m-phosphate buffer (pH 6-8). The resulting D N A was quite pure as revealed by its optical spectrum (o.D.2oo/O.D.2s0 = 1"80, O.D.280/O.D-~3O ----2"1) and its hyper-chromi~ity. D N A was isolated from E . cell strain C600 by the m e t h o d of i~Iarmur (1961). (c) T h e r m a l d e n a t u r a t i o n s a n d r e n a t u r a t i o n s Thermal denatm'ations were conducted on a Gilford model 2000 recording speetrophotom et er with calibrated 5th channel t e m p e r a t u r e recording accessory. A n E . cell D N A control was run simultaneously in all melts and all Tm values are reported relative to an E . cell D N A Tm of 90"0~ F o r renaturation experiments D N A samples in 0.12 M-phosphate buffer (pH 6.8) were boiled for 5 min and then quickly cooled to the renaturation temperature. Renattu'ation was monitored either by continuously following the loss of optical hyperchromicity at 260 n m on the Gilford 2000 or by measuring the percentage of D N A in portions bound to an h y d r o x y a p a t i t e column (Britten & Koime, 1968). The raw data for percentage bound to the cohtmn were corrected for the small a m o u n t of D N A which bound to the column at the zero time point (Davidson et al., 1973). Although a m a x i m u m of 92% 4` 3% of the D N A botmd h y d r o x y a p a t i t e at the completion of the renaturation, no correction for this less t h a n optimal binding was made in the calculation of C0t112~. H y d r o x y a p a t i t e columns yielded 95% 4- 5% of the applied DNA. F o r experiments where optical hyperchromicity at 260 n m was continuously monitored, semi-micro cuvettes of 1-cm p a t h length were used, and an E . cell D N A control was run simultaneously. O.D.~o was taken as the O.D.260 of sheared native D N A measured at the t em p er at u r e of renaturation, while O.D.o was ta k e n as the o.D.26o of the same D N A at 100~ The renaturation rate constant was calculated from the initial slope of the curve obtained by graphing (O.D ~.- - O.D. oo)/(O.D.o -- O.D. oo) v e r s u s time (t), using the formula k2 = slope/Co, where Co is the initial concentration of D N A nucleotides. R a t e constants for b o t h optical hyperchromicity experiments and h y d r o x y a p a t i t e work were corrected for length (L) variations in t h e reacting strands using the square-root of L dependence determined by W e t m u r & Davidson (1968). Strand lengths were determined by alkaline sedimentation velocity measurement (Studier, 1965). The curved reciprocal second-order plots obtained in the optical hyperchromicity measurements were linearized by plotting the data using the equation p[(O.D,

t -- O.D.

oo ) ( O . D "0 - - O . D .

oo ) - 1 - -

(l __p)]-x __ 1 = K~Cot,

where p is obtained by multiplying 1~Co times the product of the inverse of the slope and the intercept of a plot of the square-root of the inverse of the rate of decrease in concentration v e r s u s time (for details see R a u (1975) and R a u & Klotz (unpublished results)). The graphed form of this equation is almost the same as t h a t of the purely empirical relation obtained by Morrow (1974) to characterize renaturation kinetics monitored by single-strand specific nuclease (see also Smith et al. (1975) and Br i t t en & Davidson (1976)). (d) lllolecular weigl~t m e a s u r e m e n t s Cells were harvested by a 10-min centrifugation at 10,000 g at 4~ and resuspended in 10 ml of B A buffer (Klotz & Zimm, 1972b). Solid lysozyme was added to 0.5 mg/ml, and the m i x t u r e was incubated 1 to 2 h at room temperature. Next, ceils were centrifuged as before and resuspended in 10 ml of H E T buffer (Kavenoff & Zimm, 1973). After sitting at 4~ overnight, the cells were ready for loading into the retardimeter. This incubation at 4~ was essential to proper lysis. The molecular weight of the D N A in the resulting lysates was not affected by the length of storage time at 4~ in I-IET buffer for periods up to several weeks. To form a lysate 2 solutions were required: (1) a solution consisting of 3.0 ml of 6.0 ~-GuC1, 0.5 ml of 16"66% (w/v) Sarcosyl (Ciba-Giegy), 2.25 g urea; (2) 1 ml of t h e cell suspension consisting of a suitable dilution of the stock cell suspension with H E T buffer. The cells were diluted to an O.D.62o of 0"4 to 1'5 units where 109 cells/ml had an O.D.e20 of 6 • 0"7 Lmits. The solutions above were cooled to 0"0~ on ice; then t h e cells were pipetted into the urea/GuC1/detergent solution; and the resulting solution was m i x ed rapidly by inversion and poured into the sample chamber of the retardimeter. The chamber was sealed and incubated at 50~ for 20 h, and measurements made in the m a n n e r described by Klotz & Zimm (1972a,b). t For definition, see Brltten & Kohne (1968).

344

T.M.

ROBERTS,

L. C. K L O T Z

AND A. R. LOEBLICH

III

(e) D N A p e r oell measurement8 D i a m i n o b e n z o i e a c i d m e a s u r e m e n t s w e r e p e r f o r m e d as d e s c r i b e d b y A l l e n et al. (1975). D N A / c e l l e s t i m a t i o n s u s i n g t h e m o d e l E u l t r a c e n t r i f u g e w e r e m a d e as follows. T o 3.0 m l o f 6 M-GuC1, a n d 0.5 m l o f 16"66% Sarcosyl, w a s a d d e d 1.0 m l o f cells i n 1 ~ - T r i s ( p t t 9.0) a t a k n o w n cell c o n c e n t r a t i o n . Cells w e r e c o u n t e d i n a P e t r o f f - H a u s s e r b a c t e r i a c o u n t e r . Cells w e r e p r e p a r e d for lysis i n t h e s a m e m a n n e r as t h o s e u s e d i n m o l e c u l a r w e i g h t m e a s u r e m e n t s . T h e r e s u l t i n g l y s a t e w a s sheal~ed 5 t i m e s b y p a s s a g e t h r o u g h a 2 7 - g a u g e n e e d l e a t m a x i m u m t h u m b p r e s s u r e . N e x t 0.61 g CsCI w a s a d d e d t o 1.0 m l l y s a t e . O c c a s i o n a l l y t h e r e s u l t i n g s o l u t i o n w a s i n c u b a t e d a t 60~ for l h . O m i t t i n g t h i s i n c u b a t i o n h a d n o effect. F i n a l l y 0.4 m l o f t h e CsCI/GuC1 l y s a t e w a s c e n t r i f u g e d t o e q u i l i b r i u m a t 44,700 r e v s / m i n

(a)

\ (b)

/

E E ~D

(c)

i-701z

1,7055 1-719

Density [g/cm s)

Fxo. 1. Photoelectric scans of CsC1 b u o y a n t density gradients. A. quadruplicatum D N A isolated from early stationary-phase cells was centrifuged a t 44,700 revs]min for 21 h a t 25~ in a CsC1 density gradient. The scans, made a t 265 n m with the photoelectric scanner optics of the B e c k m a n model E ultracentrifuge, show the algal D N A b o t h alone (a) a n d together (b) with a bacterial marker D N A isolated from a Micrococcus species (p = 1.719 g/cm-3). For the purposes of comparison the 3rd scan (c) shows E. coli D N A along with the Micrococcus marker centrifuged u n d e r the same conditions. Fie. 2. Thermal denaturation profiles a t 260 rim. The melting profiles, made in 0.12 ~ - p h o s p h a t e buffer (pH 6.8), are shown for native DI~As of b o t h A . quadruplicatum ( Q , m ) a n d E. cell ( 0 ) a n d for r e n a t u r e d double-stranded Agrnenellurn D N A ( • isolated from h y d r o x y a p a t i t e after r e n a t u r a t i o n to a Cot of 30. (a) A s t a n d a r d plot of [O.D. 26~ (at T~176 (at 20~ versus temperature. (b) The same information plotted in differential form: [(O.D. a6~ (at T2) -- O.D.26~ (at T1))/(O.Dfl 6~ (at 100~ -- o.~). 26~ (at 20~ versus (T1 + T2)/2, where T1 a n d T2 are two sequential temperatures differing b y I~ Optical densities are uncorrected for the t h e r m a l expension of water.

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346

T.M.

ROBERTS,

L. C. K L O T Z A N D A. R. L O E B L I C H

III

at 25~ The sample was scanned at 265 n m using the ultraviolet scanner optics of the centrifuge, and the peak area was measured using a K and E planimeter. P e a k area was then converted directly into a D N A concentration in ~g D N A / m l b y use of the calibrated absorbance scale of the instrument. A sample calculation proceeds as follows: a p e a k area a t 265 n m of 2.5 cm 2 is measured. This corresponds to an area of 2-5/0'96 cm 2 or 2"6 cm 2 at 260 nm. A n 0.4 ml sample gives a sample column length of 23 em on t h e scanner output, and the instrument is calibrated so t h a t 1 tzg D N A / m l should give a sample absorbance height of 0.06 cm. Hence a 1 tzg D N A / m l solution should give a t o t a l p e a k area of (23 • cm s or 1-38 cm 2, and our sample contains 2.6/1.38 = 1.89 ~g DNA/ml. Since the volume increase on adding CsC! to the original lysate is 1.17, the original lysate m u s t have h a d a D N A concentration of 1.89 • 1-17 = 2.2/zg DNA/ml. Alternatively, the D N A concentration in the sample solution was obtained b y comparing the sample peak area with t h a t o f a D N A s t a n d a r d of known concentration. B o t h methods gave similar results, and b o t h were reproducible to a 15~ average error.

3.

Results

(a) Initial D N A characterization: CsCl buoyant density and Tm T h e o n l y p r e v i o u s c h a r a c t e r i z a t i o n of t h e I)RIA o f Agmenellum was a r e p o r t b y S t a n i e r et al. (1971) of t h e b u o y a n t d e n s i t y of its D N A in n e u t r a l CsCl: p ---- 1.708 g cm -3 ~- 0.001 g c m - a r e l a t i v e t o E. coli a t p = 1.710 g cm - s . F i g u r e 1 shows t h e CsCl b a n d i n g p a t t e r n of t h e D N A o f A. quadruplicatum as m e a s u r e d b y t h e u l t r a v i o l e t s c a n n e r optics a t 265 nm in t h e Spinco m o d e l E ultracentrifuge. T h e p e a k is q u i t e s y m m e t r i c a l a n d sharp, showing l i t t l e base c o m p o s i t i o n h e t e r o g e n e i t y . N o satellites a r e visible e v e n a t h i g h scale m a g n i f i c a t i o n u n d e r c o n d i t i o n s o f s a m p l e o v e r l o a d i n g ( d a t a n o t shown). M e a s u r e m e n t of t h e p e a k d e n s i t y versus a b a c t e r i a l m a r k e r gives p ---- 1"7012 g c m -3, c o r r e s p o n d i n g t o a I ) N A base c o m p o s i t i o n of 48~/o g u a n i n e plus c y t o s i n e (G-t-C) r e l a t i v e t o E. coli D N A c o m p o s i t i o n of 5 0 ~ (G~-C) ( S c h i l d k r a u t et al., 1962). F i g u r e 2(a) shows t h e t h e r m a l d e n a t u r a t i o n profile of t h e A. quadraplicatum I ) N A along w i t h t h a t of a n E. coli D N A s t a n d a r d . T h e m e l t of t h e algal D N A is q u i t e s h a r p w i t h a Tm (the t e m p e r a t u r e a t which haft t h e final h y p e r c h r o m i c shift is r e a c h e d ) o f 89.2~ • 0.5~ r e l a t i v e t o a n E. coli D N A T m of 90-0~ c o r r e s p o n d i n g t o a (G~-C) c o n t e n t o f 4 8 ~ ( M a r m u r & I ) o t y , 1962) in g o o d a g r e e m e n t w i t h t h e b u o y a n t d e n s i t y results. A differential p l o t of t h e m e l t of Agmenellum D N A while showing a s o m e w h a t s h a r p e r t r a n s i t i o n p e a k i n g a degree or so b e l o w t h a t of t h e E. coli D N A , is e x t r e m e l y similar t o t h a t o f t h e b a c t e r i a l I ) N A (Fig. 2(b)). (b) D N A renaturation lcinetics F i g u r e 3(a) shows a t y p i c a l e x p e r i m e n t c o m p a r i n g t h e r e n a t u r a t i o n o f t h e I ) N A o f

Agmenellum w i t h t h a t o f E. coli. These results, o b t a i n e d b y following t h e r e n a t u r a t i o n v i a o p t i c a l h y p e r e h r o m i c i t y a t 260 nm~ are g r a p h e d as a s e c o n d - o r d e r r e a c t i o n ( W e t m u r is plotted versus time (s). See Experimental Procedure for the details of such plots. The value of p used was 0"75 (or, p = 0.81 if corrected for single-strand structure) in reasonably good agreement with the expected values of p for kinetics performed under nucleation rate-limiting conditions with O.D.~ ~ taken as the O.D.2e~ of sheared native DNA. See Roberts et al. (1976) and Ran (1975) for details. The ratio of corrected k2 values obtained from the plot shown is k' A.,.ad,~pz~ca,~m/k ~ E. coZ, = 0"95 in good agreement with the result of the calculation made using the initial slopes of the reciprocal second-order plot.

CHARACTERIZATION I-9

I

OF BLUE-GREEN

I

ALGAL

I

GENOME

347

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Time ( ~ ) x I0 -s

FIG. 3. T h e k i n e t i c s o f D N A r e n a t u r a t i o n as m e a s u r e d b y optical h y p e r c h r o m i c i t y a t 260 n m for D N A f r o m AgmeneUum ( 9 and E. coli (0). (a) A t y p i c a l e x p e r i m e n t g r a p h e d as a reciprocal s e c o n d - o r d e r plot o f [(o.D.o~O -

o.D.~o)/(o.n.~o

-

o.n.~o)]

versus t i m e (s). T h e r e n a t u r a t i o n , c a r r i e d o u t i n 0-12 u - p h o s p h a t e b u f f e r ( p H 6.8), w a s r u n a t 68~ w i t h a n initial D N A c o n c e n t r a t i o n o f 67.0 ~ g ] m l for Agmenellum a n d 69.5 ~ g ] m l for E . eoli. T h e solid lines r e p r e s e n t t h e initial r a t e s of r e a c t i o n . F r o m t h e slopes o f t h e s e lines k 2 v a l u e s were c a l c u l a t e d (after correction to a f r a g m e n t l e n g t h o f 500 nucleotides) as 0-231 1 m o 1 - 1 s -1 for Agmenellum D N A a n d 0.244 1 m o l - z s - z for E . coli DNA. (b) T h e s a m e e x p e r i m e n t a l d a t a r e p l o t t e d a c c o r d i n g to t h e linearizing e q u a t i o n of R a u (.197.5}. Here p/[(o-:o., ~ ~

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348

T . M. R O B E R T S ,

L . C. K L O T Z

AND

A. R. LOEBLICH

III

& Davidson, 1968). For a perfect second-order reaction, such plots of the reciprocal of the fraction of DNA remaining single-stranded Co/G versus time (t) would generate straight lines. The second-order rate constant (k2) would be obtained by dividing the slope of the line by Go, the initial concentration of DNA nucleotides, and the kinetic complexity of the DNA examined (N~) would be given by the equation N x = Nstandard (]r

(1)

Several points must be noted concerning this plot. First, in the case of both DNAs the renaturations are dominated at early times by a first-order component resulting from the formation of intra-strand, non-specific base-pairing (Rau, 1975; Rau & Klotz, unpublished results). This first-order process causes the initial (temperature equilibration being complete at 2 min) curvature in the plot. Second, under the conditions used in this experiment (0.18 •-Na +, 65~ DNA concentration of 50 ~g/ml, and DNA strand length ~___500 bases), the linear portion of the plot, which appears after the process of intra-strand base-pairing has ceased, continues out only to 20 to 30% of reaction. This linear portion comprises what we will call the initial slope of reaction, i.e. we ignore the first-order processes at the very beginning of the reaction. After 20 to 30~ of the DNA has renatured the actual course of reaction begins to drop increasingly far below the line representing the initial slope. This deviation of optical renaturation plots from the course of an ideal second-order reaction has been noted before (Bak et at., 1971) and is described ill detail in the work of Rau (1975) and I~au & Klotz (unpublished results). (An analogous effect is seen in renaturation followed by $1 nuclease digestion (Morrow, 1974; Smith et al., 1975).) Reliable kinetic complexities can be obtained from such curved plots by comparison of/c 2 values derived from initial slopes, providing care is taken to compare the k2 values obtained with those of a control DNA matched as closely as possible in kinetic complexity, Tin, and base composition heterogeneity to the DNA under examination. Table 1 shows the results of a series of renaturations comparing Agmenellum DNA with E. coli DNA at several concentrations and two fragment sizes, and various temperatures.The ratio ofrenaturation rate constants (/c2)obtained from initial slopes for the two DNAs is independent of concentration, temperature and fragment size (within the limits of error of the technique). Averaging all the data on/c 2 values for the temperature range from 64.5~ to 68~ we find k2 values of 0.208 4- 0.0241 tool- 1s- ~for Agmenellum DNA and 0.218 4- 0.030 1 tool- 1 s - 1 for E. coli DNA. Use of equation (1) gives a kinetic complexity of 2.8 • 10~ 4- 0.3 • 109 daltons for Agmenellum, assuming E. coli has a kinetic complexity of 2.7 • 109 daltons (Klotz & Zimm, 1972b). Alternatively the same data can be calculated by comparing the ratios of/~2 values for the two organisms obtained in each experiment (see the right-hand column in Table 1). This method has the advantage of averaging out any small errors in renaturation temperature or data handling from experiment to experiment. Calculated in this manner the ratio of kAgmenellum/bS, 2 /~2 colt is once again 0.95 4- 0.05 yielding the same complexity for Agmenellum DNA. However, the average deviation from the mean drops from roughly 11% to 5%. Rau and Klotz (Rau, 1975; Rau & Klotz, unpublished results) have developed an equation for linearizing the data obtained from optical renaturation of DNA of a single kinetic class. Figure 3(b) shows the data from Figure 3(a) replotted according to this linearizing function (see Experimental Procedure). The renaturation curve is

.9

0 I o

~. o

~. o

,-~ ~

~ ~

~ ~

~ ~

~. o

~. o

~ ~

~. o

~. o

o

~

o

~

o

~

o

~5

~

o

~5

,~

I

o~

o

~o

O0

O0

O0

O0

~0

~0

§

o~

~

~

~

o

~

! o

o

o

o

o

o

~o 0

350

T. M. R O B E R T S ,

L . C. K L O T Z

AND

A. R. LOEBLICH

lI1

linear to 5 0 ~ reaction. This suggests that any repeated DNA present comprises 5 ~ or less of the total. Kinetic complexity calculations from these linearized plots are in close agreement with those obtained from initial slope calculations (the ratio k~ 4. ~,~r,pzic~t,m/k ~ E. co~t = 0"95).

Optical renaturations were seldom followed past the haft-point of reaction. To obtain a more complete perspective, renaturation kinetics of A. q~a~ruplica~um DNA were measured using hydroxyapatite. Figure 4 gives results of hydroxyapatite renaturation plotted both as a Cot plot (Britten & Kohne, 1968) and as a standard reciprocal second-order plot. Also shown are a few E. coli DNA points run to characterize this particular batch of hydroxyapatite and to demonstrate the degree of completion typical of hydroxyapatite renaturation in tl~s laboratory. The algal DNA renatures with second-order kinetics from under 10~/o to 7 0 ~ of reaction as predicted by theory for hydroxyapatite renaturation. The endpoint of renaturation occurs when roughly 90 to 92% of the DNA binds hydroxyapatite. The kinetic complexity of the blue-green's DI~A may be calculated from this experiment, though of course the limited number of points measured, particularly on the E. coli curve, limits the precision of this measurement. Such a calculation gives a kinetic complexity for Agmenellum DNA of 2.3 • 10~ daltons in relatively good agreement with the optical data. When the double-strand-containing DNA was isolated by hydroxyapatite chromatography (after renaturation to 80 to 90~o completion) and then melted in the spectrophotometer, the resulting denaturation profile was extremely similar to that of native DNA. The Tm of the renatured material was only 1 to 1.8~ lower than that of native DNA. This behavior closely approximates that of E. coli DNA treated similarly (Britten & Kotme, 1968; Allen et al., 1975) and indicates very little mismatching in the renatured product (Fig. 2). (c) D N A per cell measurement Preliminary measurements of DNA per cell were made v/a the diaminobenzoic acid technique (Kissane & Robins, 1958; Holm-Hansen et al., 1968) on cells growing at relatively slow growth rates, 15 to 20 hours per doubling (see Table 2). The values of DNA per cell obtained, ranging from 8.5 • 109 to 13 • 109 daltons per cell, are surprising in light of the already measured kinetic complexity. Unfortunately our preliminary work and previous measurements of D/qA per cell for other blue-green algae (e.g. Mann & Carr, 1974) may be criticized on one important point: the method of DNA estimation used does not allow for the control of positively interfering substances. The measurement of DNA-enhanced fluorescence of ethidium bromide (Klotz & Zimm, 1972b), which we commonly use, does allow for control against positively interfering substances but requires clear, colorless lysates, which cannot be obtained from blue-green algal ceils. Thus, the need arose to find a new method of DNA per cell measurement. The method developed to meet the above need utilizes two properties of DNA to distinguish DNA-induced signal from non-DNA signal: DNAs characteristic density and its ultraviolet spectrum. Cell lysates were banded in a mixed GuCl/CsC1 gradient in the model E ultracentrifuge. The number of cells in the lysate is known by counting, and the amount of DNA can be obtained by measuring the peak area on the traces of the ultraviolet light scammr optics. DNA concentration is obtained either directly from the peak area using the calibrated optical density scale built into the

CHARACTERIZATION

OF BLUE-GREEN

ALGAL

GENOME

351

0 I0-

(a)

20 50 4050 60 70 BO SO , O-I

lO0 0"01

, 1.0

I I0

"\'"--~176 100

Col (tool I -t s d)

1

1

I

I

I

I

I 2-0

I 5.0

I 4.0

(b)

5

•

2

i ll".,~ 0

I 0.4

I I 0 . 8 ].0

Col ( mol I-I s-I )

FIG. 4. T h e k i n e t i c s o f r e n a t u r a t i o n as m e a s u r e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y for A . quadruplicatum D N A ( O ) a n d E. coli D N A ( O ) . R e a s s o e i a t i o n s were c a r r i e d o u t in 0.12 r ~ - p h o s p h a t e buffer ( p H 6.8) a t 63~ A t v a r i o u s t i m e s p o r t i o n s o f D N A were a p p l i e d to a h y d r o x y a p a t i t e c o l u m n a t 60~ S i n g l e - s t r a n d e d m a t e r i a l w a s e l u t e d w i t h 0.12 M - p h o s p h a t e buffer ( p H 6.8) a n d d o u b l e - s t r a n d e d m a t e r i a l w i t h 0.60 M - p h o s p h a t e buffer ( p H 6.8). T h e f r a c t i o n of D N A b i n d i n g to h y d r o x y a p a t i t e as d o u b l e s t r a n d s (R) h a s b e e n c o r r e c t e d for z e r o - t i m e b i n d i n g in t h e m a n n e r of D a v i d s o n etal. (1973). T h e d a t a a r e s h o w n b o t h a s a t r a d i t i o n a l Oot p l o t (a) in t h e m a n n e r o f B r i t t e n & K o h n e (1968) a n d a s a reciprocal s e c o n d o r d e r plot o f t h e s a m e d a t a (b). I n t h i s case, t h e reciprocal of t h e d e c i m a l f r a c t i o n o f t h e D N A r e m a i n i n g s i n g l e - s t r a n d e d 1/(1 - - B) is p l o t t e d versus Cot. T h e a r r o w s m a r k t h e G0t~ values.

23

352

T. M. R O B E R T S ,

L . C. K L O T Z

A N D A. R . L O E B L I C H

III

instrument, or indirectly by comparing peak areas of lysates with peak areas of DNA standard solutions. Results are identical with the two methods. T h a t the signal is due to DNA is evidenced by two observations. (1) The supposed DNA peak appears in the same position on the gradient as do the peaks of pure DNA samples while pure RNA or protein samples appear at the cell bottom or meniscus, respectively. This is only a rough indication of purity since the gradient is very steep. (2) More precise evidence of DNA purity is obtained b y following peak area as a function of the scanned wavelength, yielding an absorption spectrum for the sample peak. This reveals a DNA-like spectrum with O.D.2s0 to O.D.2s0 ratios of 1-7 :E 0"1 (see Fig. 5). Similar ratios are I

I

I

I

I

I

1

I

4

5

I

(~50

250

270

290

5 I0

Wavelength of scon (nm)

FIO. 5. The absorbance spectrum of the DNA peak in a GuCI/CsC1 density gradient. A lysate consisting of 3.0 ml of 6 M-GuC1, 0.5 ml of 16.66% Sarcosyl, and 1.0 ml of cells in 1.0 M-Tris (pH 9.0) was sheared 5 times by passage through a 27-gauge needle. A total of 1-0 ml of the lysate was then added to 0.61 g CsC1 and the resulting solution was centrifuged to equilibrium in the model E ultracentrifuge at 44,700 revs/min at 25~ The resulting gradient was t h e n scanned at various wavelengths, and the area under the peak was measured with a K and E planimeter. The graph above shows the "absorbance spectrum" obtained b y graphing peak area (cm 2) v e r s u s the wavelength of the scan. I t was impossible to make scans at 255 n m or at wavelengths shorter t h a n 240 rim.

obtained for very pure control DNA samples showing o.D,2s 0 to o.D.280 ratios of 1.85 to 1.90 and hyperchromic shifts of 35 to 40~/o when measured on spectrophotometers. Using this method of DNA estimation, cellular DNA content was obtained for A. quadruplicatum cells growing at different measured rates of growth (see Table 2). The results are in good agreement with those obtained by the diaminobenzoie acid method, i.e. DNA per cell is 8.6 • 109 =t= 0.9 • 109 daltons under all conditions tested. This value should be a minimum estimate since some cellular DNA m a y not have been freed of protein or membrane components and hence m a y have moved to the meniscus of the gradient.

C H A R A C T E R I Z A T I O N OF B L U E - G R E E N ALGAL GENOME

353

TABLE 2

D N A per cell in Agmenellum quadruplicatum Method of measurement

Cell growth condition

DNA/cell (daltons) 10.5 • 109 4- 2.5 • 109

Diaminobcnzoic acid

Log-phase cultures (doubling time 10 to 20 h)

Ultracentrifuge m e t h o d

Log-phase cultures (doubling time 20 h 4- 1 h)

8-6 • 109 -4- 0-7 • 109

Ultracentrifuge m e t h o d

Log-phase cultures (doubling time 7 h •

8.6 • 109 4- 1.4 • 109

Ultracentrifuge m e t h o d

0.5 h)

P o s t log-phase cultures (doubling time 20 h 4- 1 h)

8.2 • 10 9 4- 0.4 • 109

The errors reported are the average deviation from the mean. All measurements were made at least in duplicate. The cultures described as post log-phase were allowed to grow until the rate of increase of optical density a t 620 n m was at least 80% below the maximal value.

(d) Molecular weight of the chromosomal D N A The results of the preceding two sections indicate that under the conditions studied A. quadruplicatum possesses roughly three copies of its genome. An obvious question arises. Are these three copies found as one huge piece of DNA, as three separate pieces, or in some other arrangement? To answer this question we made use of viscoelastic retardation time measurements. The details of the viscoelastic technique are published elsewhere (Chapman et al., 1969; Klotz & Zimm, 1972a,b, Kavenoff & Zimm, 1973; Lauer & Klotz, 1975). For a general review of the viscoelastic technique see Roberts et al. (1976). Preliminary experiments showed t h a t the detergent/proteinase lysis systems used before (Klotz & Zimm, 1972b; Kavenoff & Zimm, 1973) failed to give reproducible results on lysates of AgmeneUum cells. Hence a new lysis mixture was developed utilizing highly denaturing reagents instead of proteinases to complement detergent action in stripping proteins from the DNA. The lysis mixture chosen was 2.76 M-GuC1, 5.53 M-urea, 1.28% (w]v) Sarcosyl, 0.077 M-EDTA at p H 9-3. Figure 6(a) shows the tracing of a chart recorder output for a typical experiment. Figure 6(b) is a semilogarithmic plot of rotor position versus time. From such plots the principal retardation time v can be obtained (~ equals the negative of the reciprocal of the slope). I n this maimer ~ was measured at various cell concentrations for cells growing under the same growth conditions used in the DNA per cell measurements (Fig. 7). Under all conditions measured, 9 was approximately 217 seconds. Since no concentration dependence of r was detected, ~o, the value of T at zero DNA concentration, is just equal to the average value of 9 for all measurements. T~ m a y be corrected to standard conditions of 50~ and water as the solvent b y the formula (Kavenoff & Zimm, 1973)

~'%.5o --

0.00549T r0 - - ,

3237

(2)

where T is the temperature in absolute degrees K, y is the solvent viscosity, and 0-00549 is the viscosity of water in poise at 323~ For our lysates T = 323~ and

IO.O--(b)

r~

|

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/

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,

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I I 200 500 Time ( s )

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I 500

FzQ. 6. Results of a typical viscoelastic r e t a r d a t i o n time experiment. (a) A tracing of a typical c h a r t recorder o u t p u t for a recoil. The sharp ascending spike represents the motion of t h e rotor as it is propelled b y the eddy-current drive. A t time 0 the power to the magnets was shut off. After coasting briefly due to inertia, t h e rotor recoiled finally reaching its equilibrium position 8(~o). The d a t a from curve (a) were t h e n plotted on semi-logarithmic paper (b) a n d T was determined from t h e negative of the reciprocal of the slope. The value o f f is denoted b y a n arrow.

T

T

0"25

0-50

T

I

T

r~

1.0

1.25

1"5

:~00

200

I00

0

0-75 0.D.620 units

FzQ. 7. A g r a p h of r e t a r d a t i o n time as a function of cell concentration. The O.D.620 of t h e cell solution used for each lysate is graphed on t h e abscissa while the ordinate shows t h e value of r obtained. The error bars represent the average deviation from t h e mean. I n each instance T was measured a t least twice a n d usually 4 or more times. Cells from the following cultures were used: ( D ) log-phase, 7 . 0 h 4 - 0 . 5 h doubling time; ( O ) ]og-phase, 2 0 h 4 - l h doubling time; ( 0 ) postlog-phase, 20 h 4- 1 h doubling time.

CHARACTERIZATION

OF BLUE-GREEN

ALGAL

GENOME

355

= 0"0147 poises. Hence T~ s0 = 81 seconds. Lauer & Klotz (1975) determined the relations between r~ and DNA molecular weight (M) in 2.0 M-Na + to be: T~

=

3"8 X 10-Z4M rS~

or

(3) M = 2.45 • 108 (~~176

Substituting row,50 = 81 seconds yields a value of M = 3'9 • 109 -b 0.6 • 109 daltons for Agmenellum chromosomal DNA. There is some question as to ~ h e t h e r it is permissible to use the equation of Lauer & Klotz (1975) for the rather strange combination of high guanidinium ion and urea concentration used in o u r experiments. The d a t a of Ross & Scruggs (1968) suggest t h a t the hydrodynamic properties of double-stranded D N A molecules do not change as cation concentrations are increased from 0.9 M to 2.0 M or as the monovalent cation is changed from sodium to t e t r a m e t h y l - a m m o n i u m . This suggests t h a t the equation for 2 M-Na + should be valid for 2.8 M-Gu +. However, to be sure t h a t this is indeed the case, a limited number of molecular weight determinations were made in the GuCl/ urea lysis mixture on DNAs of known molecular weight. The results of these measurements are shown in Figure 8. Our experimental points agree rather well with the graphed line determined from equation (3). I u no case is the error greater t h a n 20%. While the ~iscoelastic technique should be somewhat more accurate t h a n this,

I

I

I

Yeast

/ -~

I 0

I i

I 2

Log r~

FIG. 8. Molecular w e i g h t as a f u n c t i o n ofT~ . ~~ o w a s m e a s u r e d in t h e G u C l / u r e a ]ysis m i x t u r e for 3 o r g a n i s m s : p h a g e T2, t h e y e a s t Saccharomyces cerevi~ae a n d E. coll. T h e v a l u e o f v~ o o b t a i n e d for e a c h o r g a n i s m w a s t h e n p l o t t e d u s i n g t h e a l r e a d y d e t e r m i n e d m o l e c u l a r w e i g h t s for t h e D N A s : T2, 1.2 • 108 (Freffelder, 1970); y e a s t , 2.0 • 10 ~ ( L a u e r & K l o t z , 1975); E. coli, 2.7 • 109 ( K l o t z & Z i m m , 1972b). T h e solid line is t h e g r a p h e d f o r m of t h e e q u a t i o n M = 2.45 • 10 a (v~176 o b t a i n e d b y L a u e r & K l o t z (1975) for 2-0 M-Na ~ .

356

T. M. R O B E R T S ,

L. C. K L O T Z A N D A. R. L O E B L I C H

III

the limited number of lysates measured (2 or 3 in each case) was not sufficient to obtain optimum accuracy. Thus we feel justified in the use of equation (3) for our lysis condition.

(e ) Controls of molecular weight measurements DNA should be the only species present capable of producing measurable recoil at the cell concentrations used (less than 4 • l0 T cells/rain). I t is still desirable to demonstrate that DNA is the species responsible for the recoil. DNAase is inactive in the lysis system used. Thus we must resort to indirect controls. (1) The recoil is shearsensitive, as expected for DNA. Passage of the lysatc "through a Pasteur pipette removes all recoil. (2) The recoil present is heat sensitive. The Tm of Agmenellum DNA in the GuC1/urea lysis system is roughly 60~ As expected, heating of the lysate to 70~ removes all recoil. I f lysates are made using 0.195 M-Na + with sodium dodecyl sulfate and proteinase to remove protein (a system in which Agmenellum DNA has a Tm value of roughly 90~ heating the lysate to 70~ has no effect on the recoil, while heating to 100~ removes all recoil. (3) The values obtained for r in low-salt lysates give the same molecular weight as those obtained in high salt. In 0.195 M-Na +, Tw.~50 is related to M b y the equation (Klotz & Zimm, 1972b) M :

2"2 • 10s(~ ~ 50) ~176

(4)

Though lysates in this salt are not as stable as their GuC1/urea counterparts, we find rw.~~o for Agmenellum to be roughly 125 seconds corresponding to a molecular weight of 3.9 • 109. The fact that the Iysate retardation times are affected b y salt concentration in the same fashion as the retardation times of pure DNA, indicates that the recoiling species is negatively charged and is most probably DBIA. The above three experiments strongly suggest that the recoil-causing agent contains DNA. The experiments do not demand that DNA alone composes the recoiling species; however, a DNAase control would be equally ineffective against any argument which proposed that the species responsible for the recoil was made up of DNA pieces held together by some other material. Even ff it were granted that DNA molecules alone were responsible for the recoil, the question would still arise as to whether the recoiling molecules were, indeed, single molecules or groups of molecules aggregated in some fashion resulting in a longer retardation time. The absence of a dependence of ~- on cell concentration in the Iysate mixture argues against intercellular aggregation. However, other data can be called upon to lend support to the case against aggregation. The determination by Klotz & Zimm (1972a,b) of molecular weights for the DNA of the phages T7 and T2, and the chromosomes of the bacteria E. coli and Bacillus subtilis was extremely straightforward in that reliable independently obtained values for each of these molecular weights were either known at the time or have since become available (in the case of B. subtilis (Wake, 1973)). In each case, the value of M calculated from viscoelastic results is in good agreement with the independently obtained value. The lysates obtained by Klotz & Zimm obeyed a specific set of rules. (1) r was independent of the shear stress. (2) ~ was independent of the length of rotor windup (A0) even with windup periods many-fold greater t h a n the value of T. (3) The viscosity (7) of the solution was independent of the length of time the rotor had been turning--once again ~ remained constant even if the rotor turned for m a n y times r. (4) ~ was independent of the temperature of measurement after correction

C H A R A C T E R I Z A T I O N OF B L U E - G R E E I ~ ALGAL GEI~OME

357

as per equation (2). (5) I t was possible to calculate from theory the number of mo|ecules present in a solution by using the recoil intensity of the longest relaxation time (Kavenoff & Zimm, 1973). I f care was taken to extrapolate to zero DNA concentration the calculated number of molecules was always less than or equal to the actual number of molecules which would have been present in the lysate if all molecules were intact (Klotz, unpubhshed observation). Whenever DNA of l~axown size is measured and these five rules are obeyed, the measured size is correct. However, in the few cases where lysates have produced abnormally high r values presumably due to aggregation, at least one of these rules has been broken (Roberts, Lauer & Klotz, unpubhshed observation). The GuC1/urea lysates of A. quadruplicatum cells obey all the above rules, r is independent of shear stress over a range from 0.3 • 10 -2 dyne/cm 2 to 1.7 • 10 -2 dyne/cm 2. Both r and solution viscosity are independent of rotor windup time over values of windup ranging from less t h a n one-sixth T to several times r. Lowering the temperature of the lysate from 50~ to 25~ (once a proper incubation is finished) has no significant effect on T. Finally, the number of molecules calculated to be present i n the lysate is generally some 5 to 10~ of the total number of molecules expected if all cells had delivered up their DNA intact. (For comparison, Klotz's original experimer~ts on B. cell indicated an apparent yield from 5 to 25%.) The Agmenellum lysates display only one anomaly; t h a t is, they do show a slight dependence of r and ~/on windup for the period including the first few recoil measurements. The apparent molecular weight in these first few recoils is only 30% elevated at maximum and soon drops off to the steady-state value of 3.9 • 109 which is maintained for at least 48 hours. A brief period of relatively high shear stress can hasten the disappearance of this disturbance. I t is thought t h a t this slightly abnormal behavior is caused by a small amount of aggregated material present in the lysate which is dispersed by rotor motion. One further experiment was performed to examine the possibihty of aggregation from a different angle. I f some agent (a cellular polysaccharide, perhaps) present in the Agmenellum lysates was causing DNA aggregation in spite of the large quantities of detergent, urea and guanidinium present, and the long incubation at high temperatures, this same agent might be capable of aggregating other DNA samples. To test this possibility, an Agmenellum lysate was made, and after measurements were concluded, the lysate was sheared b y passage through a pipette and then used as the lysis buffer for an E. cell lysis. Readings of the retardation time for the E. cell lysate gave the normal value for 9 of an E. cell lysate. As a contrast to our work in the GuCl/urea buffer system where aggregation apparently was not a problem, it is of interest to mention our results in the high salt/high E D T A lysis system of Kavenoff & Zimm (1973). Here we found that the various rules of well-behaved lysates were broken: solution viscosity and r were a function of windup time and shear stress, and the number of molecules calculated to be present from recoil intensity could exceed the total possible mlmber of molecules in solution. In this buffer system, ~ could be three times or more larger than the expected value, with the actual value of T varying with shear stress and windup. Our clifl~culties in this lysis system led us to re-examine the results of Kavenoff & Zimm (1973). While, as t h e y clearly state, they did not experience all of our difA. cu]ties (for them T was independent of windup time and shear stress within certain hmits), a recalculation of their data reveals an error. In some of their lysates the

358

T. M. ROBERTS, L. C. KLOTZ AND A. R. L O E B L I C H I I I

number of molecules calculated from the intensity of recoil is up to tenfold larger than the total number of molecules that could possibly be present in the lysate. Kavenoff & Zimm (personal communication) have discovered a factor of ten error was present in their calculations of numbers of molecules present in solution.

4. Discussion The most obvious finding arising from this work is the great similarity of the DNA isolated from A. quadruplicatum with that of E. coli. The thermal denaturation profiles, renaturation curves and properties of the renatured DNA for the two organisms are strikingly alike (see Figs 2, 3 and 4). A comparison of DNA per cell measurements and the DNA kinetic complexity suggest t h a t even under conditions of very slow cell growth (20-h generation time) cellular DNA content in Agmenellum exceeds the kinetic complexity by a factor of roughly three. Although E. coli may exhibit DNA per cell values two or three times higher than its DNA kinetic complexity, such high values are seen only when cell doubling times are fast relative to the time needed to replicate a chromosome and the ensuing characteristic interval before cell division, the so-called "(C-pD) time" (Cooper & Helmstetter, 1968). For Agmenellum the (C-pD) time has not yet been determined, and the effect of DNA synthesis on the measured value of DNA per cell is not known. However the (C q-D) time of Anacystis nidulans, a close relative (Stanier et al., 1971), is only three hours (Herdman et al., 1970; Mann & Carr, 1974). Hence, it is possible that A. quadruplicatum does indeed possess two or three copies of its chromosome even at growth rates where a bacterial analogue would be expected to possess but one copy. A similar situation apparently occurs in Anacystis where DNA per cell exceeds the kinetic complexity by two- to eightfold. However in contrast to AgmeneUum, DNA content in A~mcystis increases with decreasing generation times (lY[aml & Carr, 1974). While previous DNA per cell measurements on blue-green algae have been troubled b y the lack of controls for positively interfering substances, our measurements in the ultracentrifuge are hmited by the fact that they give only a lower bound for the cellular DNA content. Clearly further work is required before DNA content and its relation to the cell cycle in Agmenellum is fully understood. The idea that the blue-green algae contain more than one chromosome is not a new one, nor are DNA per cell measurements the only evidence to favor the notion. Relying on observations of blue-green algal nucleoids in the light microscope, Fuhs (1969) suggested t h a t large-celled blue-green algae may contain multiple copies of their genome. In particular he found that cells of a filamentous species, Oscillatoria amoena, contain from one up to eight separate chromosome-like structures depending on their position in the filament. Even older is the observation of Gardner (1906) that a single-celled coccoid species of blue-green, Synechocystis aquatilis, much like AgmeneUum contained three chromosome-like structures as viewed in the light microscope. Also supportive of the multiple genome hypothesis is the fact that quantitative fluorescence microscopy reveals that certahl hair cells in Calothrix braunii, a filamentous blue-green, contain one-fifth or two-fifths the DNA of actively growing vegetative cells (Ueda, 1971). The final point which requires discussion is the interpretation of the molecular weight measurements. The most straightforward interpretation has already been

CHARACTERIZATION

OF

BLUE-GREEN

ALGAL

GENOME

359

given: i.e. the molecular weight of the largest DNA molecules found in A. quadruplicatum cells grown under the conditions specified is 3.9 • 109 q- 0.6 • 109. The problem with this statement is that, using the value of 2.8 • 109 q- 0.3 • 109 daltons for the genome size obtained from renaturation kinetics (the hydroxyapatite value is not used since the bulk of the data was obtained optically), the measured molecular weight falls between the monomer size and that expected for a dimeric molecule. Beyond the simple possibility that breakage has reduced the apparent molecular weight of the DNA, there are four possible explanations for this. (1) Replication intermediates of larger than unit genome size m a y be present (Muller & Klotz, 1974); however, the lack of effect on the measured molecular weight of either growth rate or stage in the culture cycle of the cells argues against this hypothesis. (2) The molecules m a y be circular. In this case, the observed r~ would correspond to a molecular weight of twice the stated value, or 8.0 • 109 (Klotz & Zimm, 1972b). This closely approximates the DNA per cell measurement. We view this explanation as unlikely since the measured values o f t ~ w.5ofor both E. coli and B. subtilis correspond to linear molecules when both genomes are known to be circular (Klotz & Zimm, 19725). Indeed, in any situation where an organism is known to contain large circular molecules, the measured ~~w.S0 is expected to be t h a t of the linear form because the linear form, even if it makes up only 20% of the large molecules, will sufficiently dominate relaxation in the solution to mask the presence of circular molecules. This is a consequence of two facts: first, the viscoelastic techoique gives an average molecular weight biased heavily towards the molecules occupying the largest microvolumes in solution; second, linear molecules occupy a much larger volume in solution t h a n do their circular analogues because they are not constrained to hold both ends in the same place. This means that linear forms must be present in extremely low yields if the corresponding circles are to be seen in r~ measurements. (3) The value of 2.8 • 109 q- 0.3 • 109 daltons obtained for the kinetic complexity might be in error. This could be the result of some systematic error in our measurements, but we feel this is rather unlikely since Herdman & Carr (1974) have obtained values for the kinetic complexities of Anacystis nidulans and Anabaena cylindrica which, like our value, are with 5 % of the kinetic complexity of the E. coli genome. However, it is possible t h a t the value of 2.8 • 109 • 0.3 • 109 daltons is slightly in error due to an incorrect assumption for the kinetic complexity of E. coli DNA. At any rate, we expect errors in the kinetic complexity to be relatively small compared to the next source of error. (4) Although the measurement of M b y the viscoelastic technique is qttite precise (the average deviation of the mean is less than 15%, the result of over 50 measurements) the absolute accuracy of the method is more suspect. In standard lysis systems, viscoelastic measurements are probably accurate to within • In the new and relatively unexplored lysis system used here, this error might be as high as 30%. I t is possible to minimize these last two sources of possible error. This can be done by comparing the ratio of the kinetic complexities of Agmenellum and our control organism E. coli with the ratio of molecular weights obtained b y viscoelastic measurements in the urea/GuC1 buffer used:

(MA.q./M s.~') (Kinetic complexityA.q']Kinetic complexity s'~')

1.26 --

- -

1.05

--

1-20.

This removes any uncertainty in the genome size of E. coli and helps to reduce the

360

T. M. R O B E R T S , L. C. KLOTZ AND A. R. L O E B L I C H I I I

extrapolation error in the relation between ~~ and M. The calculation strongly suggests that the measured ~~ for A g m e n e l l u m DNA corresponds to the monomer molecule. (A value of 2 would have been expected for the calculation if the molecular species being measured were the dimer.) Thus, while the presence of dimer molecules in our lysates cannot, be ruled out, it seems probable that the largest molecules found in A . quadruplicatum lysates are unit genomes. : I n summary, the results show that the blue-green alga A . quadruplicatum contains DNA of base composition heterogeneity and kinetic complexity extremely similar to t h a t of E . coli DNA. However, the data suggest that the alga contains multiple copies (two or three) of its genome apparently present as genome-sized molecules. The authors thank Dr Donald Itau for discussion of DhiA renaturation kine.tics, Professor Paul Dory for his generous support and encot.tragement, Malcolm Smart for expert technical assistance in operating the model E ultracentrifuge, and finally Dr Gall Lauer for her suggestions and thoughtftfl reading of this rr/anuscript. This research was supported by the hiational Institutes of Health (grant GM19519) and National Science Foundation (grants GB34293 and BMS74-2251). One of us (T.M.R.) was a National Science Foundation pre-doctoral fellow.

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