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Biochimica et Biophysica Acta, 402 ( 1 9 7 5 ) 3 6 3 - - 3 7 1 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- P r i n t e d in The N e t h e r l a n d s

BBA 9 8 3 8 6

DNA REPLICATION BY A POSSIBLE CONTINUOUS-DISCONTINUOUS MECHANISM IN HOMOGENATES OF P H Y S A R U M POL YCEPHALUM CONTAINING DEXTRAN

E.N. BREWER

Division of Radiation Biology, Department of Radiology, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.) (Received March 19th, 1975)

Summary Nuclear DNA synthesis in homogenates of Physarum is greatly stimulated by the presence of dextran in the homogenizing medium. In this cell-free system, the DNA precursor is incorporated approximately equally into two classes of DNA intermediates. One of these is similar in size to that observed previously in the intact organism, i.e. its sedimentation rate in alkaline sucrose density gradients increases, presumably by chain elongation, as the organism progresses through the S phase. The other class (approx. 10 S) is similar to 'Okazaki' fragments. Thus, nuclear DNA synthesis in homogenates of Physarum may occur by a continuous-discontinuous mechanism. Substantial DNA-synthetic activity is obtained b y the addition of dextran to dextran-free homogenates. Maximal activity in this system requires the presence of both the nuclear and post-nuclear supernatant fractions. It is possible that a partial separation and recombination of a DNA polymerase and the endogenous template is effected by this procedure. Introduction We have shown previously that the nuclear DNA of Physarum polycephalum (average mol. wt 2.3 • 10 ~ ) is comprised of single-strand subunits (mol. wt 4 • 10 7 ) which may be the fundamental units of DNA replication in this organism [1,2]. These subunits appear to be replicated in vivo* by continuous elongation of progeny subunits, without the appearance of smaller replication intermediates [2]. * Throughout this paper, t h e t e r m in vivo refers t o t h e i n t a c t organism.

364

Lynch et al. [3] have reported a stimulatory effect of dextran (a polyglucan containing predominantly a, 1 -~ 6 glycosidic linkages) on the synthesis of DNA in nuclei isolated from rat liver. These investigators suggested that dextran might act by reducing the leakage from nuclei of components of the replicative system. In the present communication, we report the stimulatory effect of dextran on DNA synthesis in homogenates and isolated nuclei of Physarum. Synthesis is increased several-fold over that reported previously for homogenates of Physarum prepared in the absence of dextran [4]. DNA synthesis in the present cell-free system appears to be replicative, and may occur by a continuousdiscontinuous mechanism. It is suggested that DNA replication in the intact organism occurs by a similar mechanism, but that the small (Okazaki) intermediates formed by the discontinuous mode of synthesis on the 3' -~ 5' template strands are joined too rapidly to allow their observation by the methods we have utilized [1,2]. Methods and Materials

Cultivation of the organism Macroplasmodia of P. polycephalum, strain M3C, were prepared from stock cultures of microplasmodia as described previously [5,6]. Within such plasmodia, synchronous nuclear divisions occur at 8--9-h intervals [7]. DNA synthesis begins immediately after telophase [8], and elongation of progeny subunits continues for at least 4 h [2]. Preparation of homogenates and isolated nuclei o f Physarum Plasmodia were homogenized at 30 min after MIII (third synchronous metaphase following fusion of microplasmodia) in ice-cold homogenizing medium (12.5 ml per plasmodium) using a Potter-Elvehjem homogenizer equipped with a Teflon pestle. Unless otherwise indicated, the homogenizing medium contained 0.028 M magnesium acetate, 0.06 M EGTA (ethyleneglycol-bis-(~aminoethyl ether) hrhr-tetraacetic acid), and dextran as indicated under Results. The pH was adjusted to 7.6 with NaOH. Homogenates were centrifuged for 5 min at 50 × g, and plasmodial debris removed with a pasteur pipette. Nuclei were obtained by centrifugation of the homogenates at 1500 × g (--dextran) of 4300 × g (+dextran) for 10 min. Nuclear DNA synthesis in vitro as determined by the incorporation o f [3 H]dATP into an acid-insolu ble product To 0.5-ml aliquots of homogenates or nuclear preparations {containing approx. 5 pg DNA) were added 0.05 ml of a solution containing 0.22 M ATP, 1.7 mM dGTP, dCTP and dTTP, and 0.23 mM dATP (including 8.3 #M [3 H] dATP), pH 7.2. Following incubation for 2 h at 35°C, duplicate 0.2-ml aliquots were assayed for incorporation of [3 H] dATP into DNA. To each aliquot was added 2.5 ml of 0.25 M perchloric acid, and the resulting precipitates were recovered by centrifugation at 10 000 × g, taken up in 1 ml of 0.4 M NaOH, and re-precipitated with 2.5 ml of 0.5 M perchloric acid. Acid-insoluble material was then collected for liquid scintillation counting on fiber-glass filters (Gelman Type E) as described previously [4].

365

Alkaline sucrose de.zsity gradient centrifugation of DNA strands synthesized in vitro Homogenates containing dextran (12.5%) were prepared and incubated as described above. Following incubation at 35°C for 45 rain, 1.5 vol. of Mohberg medium [9] {0.25 M sucrose, 0.01 M CaC12, 0.1% Triton, 0.1 M Tris, pH 7.2) were added and nuclei centrifuged down at 4300 × g. The nuclear pellet was resuspended in the same volume of Mohberg medium, and 0.5-ml aliquots (or 0.4 ml + 0.1 ml of [l"C]thymidine-prelabeled nuclei isolated by Mohberg's method) were pelleted at 1500 × g, resuspended in 0.2 ml of 0.15 M NaCl/0.015 M sodium citrate containing 0.005 M EDTA and 2% sarkosyl (pH 7.5), and layered over 5--20% sucrose gradients in 0.1 M NaOH. Sedimentation profiles of the labeled DNA were determined as described previously [ 1,4]. DNA polymerase assay utilizing an exogenous template Homogenates of Physarum contain a DNA polymerase activity (assayed using an exogenous template) which appears to be that responsible for nuclear DNA replication (utilizing the endogenous template) in such homogenates (ref: 10 and Brewer, E.N., in preparation). To determine the level of this activity in preparations obtained from Physarum, plasmodia were homogenized in 12.5 ml of homogenizing medium without dextran (in later experiments plasmodial halves were homogenized in this volume of homogenizing medium), and plasmodial debris removed as described above. To 0.5-ml aliquots of such homogenates, or of post~nuclear supernatant fractions prepared from homogenates by centrifugation at 1500 × g for 10 min, was added 0.05 ml of a suspension of salmon sperm DNA in water (10 mg/ml) followed by 0.05 ml of the standard incorporation medium containing [3 H] dATP. Incubation was for 120 min, and DNA synthesis utilizing the exogenous template was determined as described above. Under these conditions (no dextran), little synthesis on the endogenous template is observed. Materials Dextran (mol. wt 70 000, radioimmunoassay grade), [3 H] dATP (10--12 Ci/mmol), ATP {ultrapure) and unlabeled deoxyribonucleoside triphosphates were purchased from Schwarz/Mann, [1 " C ] t h y m i d i n e (50 Ci/mol) from New England Nuclear, and DNA (salmon sperm, type III) and EGTA from Sigma. Sarkosyl NL 97 (sodium dodecyl sarcosinate) was a generous gift of the CIBA / Geigy Corp. Results

DNA replication in homogenates of Physarum We have reported recently the synthesis of DNA in homogenates of Physarum prepared in the absence of dextran. In this cell-free system, DNA synthesis occurred at an initial rate of about 15% of the in vivo rate, precursor incorporation increased linearly for about 7--8 min, and an overall synthesis of about 2% of the total genome could be demonstrated after a 60-rain incubation period [4]. As seen in Fig. 1, DNA synthesis, utilizing the endogenous template, in homogenates of Physarum is stimulated considerably by the inclusion

366

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10

15

I

I

20 25 °/o Oextr0n

Fig. 1. E f f e c t o f dextran on D N A synthe~s in homogenates o f Physarum using endogenous and exogenous t e m p l a t e s . S e c t o r s ( o n e - s i x t h ) o f a single p l e s m o d i u m w e r e h o m o g e n i z e d a t M I I I + 3 0 r a i n (S p h a s e ) in 2.1 m l o f the h o m o g e n i z i n g m e d i u m c o n t a i n i n g v a r i o u s a m o u n t s o f d e x t r a n as i n d i c a t e d , a n d i n c o r p o r a t i o n o f [ 3 H I d A T P d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s . S e c t o r s o f a s e c o n d p l a s m o d i u m w e r e h o m o g e n i z e d a n d i n c u b a t e d in t h e s a m e w a y , e x c e p t t h a t 0 . 5 m g o f s a l m o n s p e r m D N A ( S i g m a T y p e I I I ) was a d d e d to e a c h 0 . 5 m l o f h o m o g e n a t e p r i o r t o i n c u b a t i o n . I n c o r p o r a t i o n d a t a w e r e n o r m a l i z e d f o r v a r i a t i o n s in t h e r e l a t i v e sizes o f p l a s m o d i a l s e c t o r s b y d e t e r m i n i n g t h e p i g m e n t c o n t e n t [ 1 4 ] o f each, T h e d a t a s h o w n are a v e r a g e s of d u p l i c a t e d e t e r m i n a t i o n s w i t h a v e r a g e d e v i a t i o n s r e p r e s e n t e d b y v e r t i c a l bars. o o, --exogenous DNA; • -', + e x o g e n o u s D N A .

of dextran (10--15% w/v) in the homogenizing medium (modified somewhat from that described previously [4] ). At the same time, the D N A polymerase activity of such homogenates (assayed using an exogenous template) appears to be inhibited by dextran. Endogenous activityis similarly inhibited at the higher dextran levels. The results shown in Fig. I suggest either that a D N A polymerase(s) itself is inhibited by dextran, or that it becomes less accessible to the exogenous template owing, perhaps, to its increased affinity for the endogenous template (it seems unlikely that dextran acts by inhibitingthe breakdown of the reaction

TABLE I E F F E C T OF D E X T R A N OF DNA P O L Y M E R A S E A C T I V I T Y H a l f o f a single p l a s m o d i u m w a s h o m o g e n i z e d in the d e x t r m n - f r e e m e d i u m ( 1 2 . 5 m l ) a t M I I I + 30 rain (S p h a s e ) . N u c l e i w e r e r e m o v e d f r o m t h e h o m 0 g e n a t e b y c e n t r i f u g a t i o n at 1 5 0 0 X g for 10 vain. D e x t r a n w a s a d d e d to a n a l i q u o t o f t h e p o s t - n u c l e a r s u p e r n a t a n t to a c o n c e n t r a t i o n o f 12.5%. T o a s e c o n d a l i q u o t (control) was added water to c o m p e n s a t e for the v o l u m e change resulting from the addition of d e x t r a n ( a p p r o x . 10% v o l u m e ) . D N A s y n t h e s i s utilizing t h e e x o g e n o u s t e m p l a t e w a s d e t e r m i n e d as d e s c r i b e d in t h e t e x t . A v e r a g e d e v i a t i o n s o f d u p l i c a t e d e t e r m i n a t i o n s are i n d i c a t e d . Cell-free s y s t e m

Dextran added

c p m + average deviation

Post-nuclear supernatant fraction Post-nuclear supernatant fraction

+

8 1 7 6 +- 2 0 3 4 6 1 8 -+ 31

367 product by nuclease activity since this effect might be expected with both templates). To distinguish between these possibilities, a plasmodium was homogenized during the S phase in the dextran-free medium and nuclei were removed from the homogenate by centrifugation. As in the case of the homogenate, the activity of the DNA polymerase(s) present in the post-nuclear supernatant fraction is inhibited by the addition of dextran to this fraction (Table I). The time course for DNA synthesis in homogenates of Physarum utilizing the endogenous template is shown in Fig. 2. The rate of synthesis is constant (approx. 25% of the in vivo rate) for about 40--45 min, and synthesis continues at a decreasing rate for at least another 30--60 min. Thus, a total of about 15% of the total genome undergoes replication in homogenates of Physarum contalning dextran, during a 120 min incubation period. For maximal activity, DNA synthesis in the cell-free system requires the presence of Mg2÷, EGTA, and all four deoxyribonucleoside triphosphates, in addition to dextran. The extent of synthesis in homogenates prepared from G2 phase plasmodia is less than 5% that of S phase cultures. Homogenates of cultures pre-treated for 30 min in vivo with cycloheximide (10 pg/ml) exhibit only about 60% the level of synthesis observed in homogenates of untreated controls (Table II).

Apparent dissociation and recombination of the endogenous template and a DNA poly merase(s ) Substantial DNA-synthetic activity (compared to homogenates prepared using medium containing dextran) can be obtained by the addition of dextran to dextran-free homogenates. Approximately half of this activity is associated with the nuclear pellet obtained from such homogenates (following addition of dextran). However, maximal activity in this reconstituted system requires the presence of both the nuclear and the post~nuclear supernatant fractions. By contrast, essentially all of the DNA-synthetic activity found in homogenates of Physarum containing dextran is associated with the nuclear fraction of these homogenates (Table III). 12

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F i g . 2. T i m e c o u r s e for e n d o g e n o u s D N A s y n t h e s i s in h o m o g e n a t e s o f Physarum. A s i n g l e p l a s m o d i u m was homogenized a t M I I I + 3 0 r a i n ( S p h a s e ) , a n d a l i q u o t s i n c u b a t e d for v a r i o u s t i m e s as d e s c r i b e d u n d e r M e t h o d s , e x c e p t t h a t t h e m a g n e s i u m a c e t a t e c o n c e n t r a t i o n in this e x p e r i m e n t w a s 0 . 0 2 M, a n d the A T P c o n c e n t r a t i o n o f the a d d e d i n c u b a t i o n m e d i u m w a s 0 . 1 5 M. A v e r a g e d e v i a t i o n s o f d u p l i c a t e a s s a y s are indicated.

368 T A B L E II C H A R A C T E R I S T I C S OF DNA S Y N T H E S I S IN H O M O G E N A T E S OF PHYSARUM P l a s m o d i a w e r e h o m o g e n i z e d d u r i n g e i t h e r t h e S or G 2 p h a s e of t h e division cycle. H o m o g e n i z a t i o n and i n c u b a t i o n c o n d i t i o n s are d e s c r i b e d u n d e r M e t h o d s . All results s h o w n are a v e r a g e s o f at least t w o i n d e p e n d e n t e x p e r i m e n t s . F o r c y c l o h e x i m i d e p r e t x e a t m e n t , o n e - h a l f of a single p l a s m o d i u m w a s t r a n s f e r r e d t o fresh n u t r i e n t m e d i u m i m m e d i a t e l y f o l l o w i n g t e l o p h a s e , a n d t h e o t h e r h a l f t r a n s f e r r e d to t h e s a m e m e d i u m c o n t a i n i n g 10 ~ g / m l c y c l o h e x i m i d e . H o m o g e n a t e s o f b o t h h a l v e s w e r e p r e p a r e d 30 r a i n later. Culture h o m o g e n i z e d

Incubation medium

Relative incorporation

S phase S phase S phase S phase S phase S phase* G 2 phase

Complete - M g 2+ -EGTA -ATP - d G T P , dCTP, dTTP Complete Complete

100 1 3 4 4 60 4

*

Cycloheximide pretreated.

Alkaline sucrose density gradient profiles of DNA labeled in vitro The sedimentation profiles of D N A synthesized in homogenates obtained at different times in S phase are shown in Fig. 3. It can be seen that about half of the incorporated radioactivity is associated with increasingly heavier (i.e. faster-sedimenting) single-stranded D N A as cultures are harvested later in the S period. However, approximately half of the incorporated radioactivity is found in a peak having a sedimentation coefficient of about 10 S, regardless of the time in S phase at which the culture is homogenized. In order to determine whether these radioactivity peaks represent newly synthesized DNA, a homogenate was prepared from a plasmodium at 30 min after metaphase and mixed with the standard incubation medium. After a 45-min incubation period, half of the reaction mixture was treated with DNAase (250 pg/ml) for an additional

TABLE III D N A - S Y N T H E T I C A C T I V I T Y O F V A R I O U S C E L L - F R E E P R E P A R A T I O N S IN T H E P R E S E N C E OF DEXTRAN ADDED BEFORE OR AFTER HOMOGENIZATION P l a s m o d i a w e r e h o m o g e n i z e d at M I I I + 3 0 r a i n in h o m o g e n i z i n g m e d i u m w i t h or w i t h o u t d e x t r a n , a n d i n c o r p o r a t i o n o f [ 3 H ] d A T P d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s . In s o m e cases, d e x t r a n w a s a d d e d s u b s e q u e n t to h o m o g e n i z a t i o n in t h e a b s e n c e o f d e x t r a n . N u c l e i w e r e g e n t l y r e s u s p e n d e d in h o m o g e n i z i n g m e d i u m or t h e p o s t - n u c l e a r s u p e r n a t a n t f r a c t i o n , to w h i c h d e x t r a n h a d b e e n a d d e d to a final c o n c e n t r a t i o n o f 12.5%. A v e r a g e d e v i a t i o n s for d u p l i c a t e d e t e r m i n a t i o n s are i n d i c a t e d . Experiment

Fraction

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Homogenate Nuclei Supernatant Homogenate Homogenate N u c l e i (in h o m o g e n i z i n g m e d i u m ) N u c l e i (in s u p e r n a t a n t f r a c t i o n ) Supernatant only

II

Dextran* + + + + (+) (+) (+) (+)

* Parentheses indicate that dextran was added to dextran-free preparations.

c p m +- a v e r a g e d e v i a t i o n 17063 16141 400 11919 6207 2806 5763 486

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Fig. 3. A l k a l i n e s u c r o s e d e n s i t y - g r a d i e n t p r o f i l e s o f n u c l e a r D N A s y n t h e s i z e d in h o m o g e n a t e s of P h y . saturn. P l a s m o d i a w e r e h o m o g e n i z e d at 1 5 rain (a) or 6 0 rain ( b ) a f t e r m e t a p h a s e III, i n c u b a t e d f o r 4 5 m i n , a n d a l k a l i n e s u c r o s e d e n s i t y - g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f the l a b e l e d D N A d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s . 1 4 C - p r e l a b e l e d ( n o n - r e p l i c a t i n g ) D N A ( m o l . w t 4 • 1 0 7 ) w a s i n c l u d e d as a m o l e c u l a r - w e i g h t m a r k e r [ 1 ] . F r a c t i o n s w e r e c o l l e c t e d i n t o glass t u b e s , and the l a b e l e d D N A w a s precipit a t e d w i t h 2 . 5 m l o f 0 . 2 5 M p e r c h l o r i c acid and c o l l e c t e d f o r s c i n t i l l a t i o n c o u n t i n g b y f i l t r a t i o n . - - , ~H; . . . . . . , 14C. Fig. 4. E f f e c t o f D N A a s e o n the alkaline s u c r o s e d e n s i t y - g r a d i e n t p r o f i l e of D N A l a b e l e d in vitro. A single p l a s m o d i u m w a s h o m o g e n i z e d at M I l l + 3 0 m i n a n d i n c u b a t e d for 4 5 m i n u n d e r t h e standard c o n d i t i o n & Half o f the l a b e l e d h o m o g e n a t e w a s t h e n t r e a t e d w i t h D N A a s e as d e s c r i b e d u n d e r M e t h o d s . D N A o f e a c h f r a c t i o n w a s p r e c i p i t a t e d w i t h acid and c o u n t e d as d e s c r i b e d f o r Fig. 3. - - - , control; .... •., DNAasetreated.

60 min, and the alkaline sucrose density-gradient patterns obtained for each. As seen in Fig. 4, both radioactive peaks disappeared from the gradient (i.e became acid-soluble) .upon treatment with D N A a s e . Similarly, incubation of the complete reaction mixture (without DNAase) for 0 min results in the absence of acid-insoluble .radioactivity in alkaline sucrose-density gradients (data not shown), indicating that neither radioactive peak is due to anomalous binding of the labeled precursor to D N A or to some other macromolecular species. Discussion There are several lines of evidence to suggest that D N A synthesis observed in homogenates and isolated nuclei of Physarum is of a replicative type. First, homogenates prepared from S-phase plasmodia are more than 20-fold as

370 active than are those prepared from G2-phase cultures. Second, homogenates obtained from cultures pre-treated with cycloheximide are less active in DNA synthesis than are those prepared from control cultures (Table II). Studies on the inhibition of DNA replication in vivo suggest that cycloheximide strongly inhibits DNA chain elongation, rather than the initiation of synthesis of DNA subunits, in this organism [ 11]. Third, only those DNA strands which are being elongated in the intact organism are labeled in homogenates as judged by alkaline sucrose gradient sedimentation profiles, i.e. parental (template) strands are not labeled under these conditions (Fig. 3). Finally, synthesis in the cellfree system requires Mg2÷, ATP, and all four deoxyribonucleoside triphosphates (Table II). Under the present conditions, DNA synthesis in homogenates prepared from plasmodia at 30 min after metaphase (early S period) occurs at an initial rate of about 25% of the in vivo rate, incorporation increases linearly for about 45 min, and after a 2 h incubation period, approx. 15% of the total genome has been replicated (Fig. 2). This value is several-fold higher than that reported for other eukaryotic cell-free systems [3,12]. DNA replication in homogenates of Physarum is greatly stimulated by the presence of dextran (10--15%) in the homogenizing medium (Fig. 1). Lynch et al. [3] suggested that dextran (or Ficoll) inhibits nuclear swelling and leakage from nuclei of components of the replicative system. The present data suggest that the mechanism of dextran stimulation may be a more complex one, since addition of dextran to dextran-free homogenates results in substantial recovery of DNA-synthetic activity. It is possible, therefore, that dextran acts by providing an environment which somehow stabilizes the replication complex. This suggestion is also supported by the fact that cell-free synthesis continues for a m u c h longer period of time in the presence (Fig. 2) than in the absence [4] of dextran. In view of the fact that the extent of DNA synthesis is more than 10-fold higher in the presence than in the absence of dextran, it is tempting to suggest that a substance which performs a similar function may play a role in the DNA replication process in the intact organism. At the same time, it is surprising that DNA polymerase activity, per se, is inhibited by dextran (Fig. 1, Table I). This observation suggests the possibility that the stimulation of endogenous DNA synthesis might be even greater than that reported herein if a dextran substitute, one which does n o t inhibit DNA polymerase activity, could be found. A partial separation of the enzyme (post-nuclear supernatant fraction) from the endogenous template (nuclear pellet) may be effected by homogenization in the absence of dextran, and DNA-synthetic activity can be restored by recombining these two fractions in the presence of dextran (Table III). It is possible, however, that the stimulatory effect of the post-nuclear supernatant fraction on nuclear DNA synthesis is due to a stimulatory factor other than the DNA polymerase(s) present therein {Table I). Clarification of this point awaits further study. In homogenates of Physarum, [3 HI dATP is incorporated to approximately the same e x t e n t into two discrete size classes of DNA as evidenced by their sedimentation profiles in alkaline sucrose-density gradients (Fig. 3). One of

371 these D N A species appears to sediment at a rate which depends u p o n the time in the S period at which cultures are homogenized. This DNA peak is also broader at later times in S phase. In these respects, the heavier DNA species appears to be very similar to D N A progeny strands pulse-labeled in vivo [2]. On the other hand, the lighter (10 S) DNA species sediments at the same rate regardless of the time at which cultures are homogenized. The sedimentation coefficient of this D N A is similar to that of 'Okazaki' fragments [ 1 3 ] . Thus, nuclear DNA synthesis in homogenates of Physarum may occur by a continuous-discontinuous mechanism. It is possible that DNA synthesis in the intact organism occurs by the same mechanism, b u t that the joining of DNA intermediates is t o o rapid to be detectable by the methods we have e m p l o y e d [2]. If so, the results seen in Fig. 3 imply that one or more enzyme functions responsible for joining of the intermediate DNA fragments (DNA ligase?) are missing or inactive in the present cell-free system. Evidence suggesting a continuous-discontinuous mechanism for DNA synthesis in mammalian cell-free systems has also been obtained recently [ 15,16 ].

Acknowledgements I thank Mrs Pauline Ting and Ms Jennifer Rosenthal for excellent technical assistance, and Dr Oddvar Nygaard for reviewing the manuscript. This work was supported by grant GB-40299 from the National Science Foundation (U.S.A.) and by contract W-31-109-ENG-78 with the Atomic Energy Commission, R e p o r t COO-78-334. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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