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Reassociation kinetics of polyploid hepatocyte DNA
17
Biochimica et Biophysica Acta, 474 (1977) 17--29 © Elsevier/North-Holland Biomedical Press
BBA 98784
REASSOCIATION KINETICS OF POLYPLOID HEPATOCYTE DNA
CHARLES P. ORDAHL
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.) (Received June 10th, 1976)
Summary The state of ploidy of 4c hepatocytes is examined by kinetic analysis of DNA reassociation. The nucleotide sequences present in 4c hepatocyte DNA are found to be complementary to, and present in the same relative abundance as, those of the normal diploid genome. Therefore, these experiments indicate that the increased DNA content of 4c hepatocytes is due to the presence of four haploid genomes and not to selective replication of a subfraction of the diploid genome. The concept of ploidy, typically defined in terms of karyotypic and/or DNA content multiples is thus corroborated and extended by these experiments.
Introduction In the mammalian liver the majority of hepatocytes increase their nuclear DNA content during the course of adult life [ 1 ]. The functional significance of hepatic polyploidization is unknown but may represent a unique growth process [2]. H e p a t o c y t e DNA content increases occur in multiples of the 2c or diploid value and chiefly because of this observation these cells are considered to be polyploid. DNA content alone, however, is not necessarily a reliable index of ploidy because some eucaryotic cells are known to increase DNA content without faithful replication of the entire genome. For example, this occurs in the amphibian oocyte as a result of extensive amplification of the cistrons coding for ribosomal I~NA [3]. In Drosophila, during polytenization of the chromosomes of salivary gland cells, the repeated sequence DNA undergoes a reduction, from 20% to 5%, in its relative abundance [4]. In both these instances the alterations in the relative abundance of certain nucleotide sequences in DNA either causes, or occurs concommitant with a significant increase in cellular DNA content. The question of whether polyploid hepatocytes contain multiple complete genomes is therefore not answered by DNA content determinations. A more
18 direct approach to this question may be made by karyotypic analysis since ploidy is defined in terms of karyotic multiples [5]. Distinct hepatocyte karyotypes are difficult to obtain b u t there is limited evidence that tetraploid karyotypes occur in hepatectomized rat liver undergoing hyperplasia [6]. Karyotypic analysis however remains a morphological approach to a problem which, in conceptual terms, concerns the nucleotide sequences in DNA. Thus one expects the karyotypic complement to correspond to the "unit genome" [7] and contain a full complement of the nucleotide sequences in a haploid genome. A definitive answer to this question is not presently available for the case of hepatic polyploidy. The question may therefore be asked: does the increased DNA c o n t e n t of polyploid hepatocytes represent the presence of additional complete genomes or does it represent the amplified sequences of a subfraction of the genome? Although previous work indicates that these cells possess multiple genomes the methods used cannot exclude the possibility that the DNA c o n t e n t increase is a result of amplification of the nucleotide sequences of a limited subfraction of the genome. Thus two extremes may be imagined; one, the most likely, that polyploidization results replication of all the nucleotide sequences in the genome; and, second, that disproportionate replication of a subfraction of the genome is responsible for the DNA content increase of these cells. If the former case is found to be true this would corroborate classical concepts of ploidy and extend these concepts to the level of the nucleotide sequences in DNA. If, on the other hand, it is found that hepatocyte polyploidization results from nucleotide sequence amplification this information would provide a basis for more critical investigation of the role of polyploid hepatocytes in liver function. To answer this question, I have compared the reassociation kinetics of polyploid hepatocyte DNA to that of diploid kidney DNA. The kinetics of DNA reassociation depend upon the relative concentration of the nucleotide sequences [8]. Amplification of nucleotide sequences alters the relative concentration of the nucleotide sequences and is therefore detectable, within limits, by kinetic analysis of DNA reassociation. The results of these experiments indicate that, as expected, polyploid hepatocyte DNA reassociates with the same kinetics as diploid DNA. Thus these experiments support, at the nucleotide sequence level of DNA, the concept that polyploid hepatocytes possess multiple copies of the diploid genome. In addition, calculation of hypothetical reassociation kinetics permit estimation of some of the kinetic effects expected from the presence of amplified sequence DNA. Recently, a similar experimental approach has been used to demonstrate that uniform replication of the genome is the cause of the two hundred thousand fold increase in DNA c o n t e n t of the silk gland cells o f B o m b y x mori [9]. Methods and Materials
Preparation o f DNA Unlabeled DNA was obtained from nuclei of adult (250--500 g) SpragueDawley rats. Labeled DNA was obtained from nuclei of C-6 rat glial t u m o r cells (American Type Culture Collection) grown in roller bottle culture using modi-
19 lied Ham's F-12 medium with 2 mM K ÷ [10] and 3.3 pCi/ml [3H]thymidine (Amersham Searle) to label DNA. Kidney and cultured cell nuclei were prepared by the m e t h o d of Umana and Dounce [11] with the addition of 1% Triton X-100 (Packard Inst. Co.) to the initial homogenization medium. Polyploid hepatocyte nuclei were prepared by the m e t h o d of Chanda and Dounce [12] except that Triton was used, as above, and for the 2.2 M sucrose spin nuclei were suspended in 2.2 M sucrose and pelleted through a 2.2 M sucrose pad at 7000 rev./min for 1 h in a Spinco SW-25 rotor (Spinco Div., Beckman Inst. Inc.). To extract DNA, nuclei were suspended in 0.15 M NaC1/50 mM KC1/50 mM Tris • HC1 (Sigma Chem. Co.), pH 7.6 with 1% sodium dodecyl sulphate (SDS) added to lyse nuclei and denature protein. To remove protein, lysates were extracted three times with phenol saturated in the above buffer, and once with chloroform/octanol {8 : 1). Extracted DNA, precipitated overnight with 2.5 vols. ethanol at --20°C, was redissolved in the same buffer without SDS and treated with 50 ~tg/ml ribonuclease (RNAase B, Worthington Biochem. Corp.) and then 100 pg/ml pronase (Protease, Sigma Biochem. Co.), each for 1 h at 37°C. Enzymes were removed by phenol and chloroform/octanol extraction as above and the DNA precipitated with ethanol. Purified rat DNA had a ratio of optical absorbance at 260 and 280 nm (260/280) of 1.83--1.85. DNA was sheared by passage through the valve of a french pressure cell (American Inst. Co.) at 30 000 lb/inch 2 [13]. Sheared DNA was passed through Sephadex G100 (Pharmacia Fine Chem.) to remove small molecular weight fragments and stored as an ethanol precipitate at --20 ° C. Sheared DNA migrated at between 6 and 7 S in sucrose gradient centrifugation. Reassociation reactions For reassociation, sheared DNA was dissolved in 0.12 M phosphate buffer, pH 6.8 and aliquots fire sealed in glass capillary tubes, heated to 100°C for 10 min to denature DNA and immediately transferred to a 60°C water bath to permit reassociation. For labeled DNA reassociation reactions, aliquots (5--50 pl) contained 1--5 × 104 cpm labeled DNA (specific activity; 1.8 × l 0 s cpm/pg) and 1 × 10 -3. to 5 mg/ml unlabeled DNA. Unlabeled DNA reassociation reaction aliquots (20--100 pl) contained 0.1--0.5 mg/ml DNA. The concentration and time variable (Cot} for reaction samples was determined by the formula: (absorbance at 260 n m ) × (0.5) = 1 Cot/h, as described by Britten and Kohne [8]. Zero-time samples were chilled on ice immediately after denaturation to prevent reassociation before hydroxyapatite assay. These samples are plotted at Cot 1 • 10 -2 in Figs. 2 and 4. The small amount of reassociation in these samples presumably represents that of highly repeated sequences which reassociate during handling and hydroxyapatite assay. Reassociation reaction aliquots were diluted with 0.12M phosphate buffer and passed through hydroxyapatite columns maintained at 60°C. Unreassociated (single-strand) DNA was eluted with 0.12M phosphate buffer and reassociated (double-strand) DNA with 0.5 M phosphate buffer, both buffers maintained at 60 ° C. Tests showed that procedures for binding and elution of DNA were effective. Labeled DNA fractions from the hydroxyapatite assay were quantitated in liquid scintillation cocktail composed of 86.4% toluene, 3.6% Liquifluor (New
20 England Nuclear) with 10% Biosolv (Beckman Instr. Co.). To quantitate unlabeled DNA in hydroxyapatite assay fractions, the absorbance of each fraction was measured at 260 nm and the absorbance of the unreassociated fraction multiplied by 0.85 to account for the observed hypochromic shift of denatured rat DNA at room temperature.
Flow microfluorometry Nuclear preparations to be subjected to flow microfluorometry were suspended in 0.44 M sucrose made 20% with respect to formalin to fix nuclei, sealed in tubes and shipped on ice to Dr. Paul K. Horan, Los Alamos Scientific Laboratory, N e w Mexico. All flow microfluorometry procedures were performed by him according to methods he has published [14]. Percentages of nuclei under peaks in Fig. 1 were estimated by cutting o u t peaks on paper and weighing. A review of flow microfluorometry technology has been published [15]. Results
Isolation and homogeneity o f polyploid hepatocyte nuclei Polyploid hepatocyte nuclei were isolated from the total liver nuclear population by a modified m e t h o d described by Chanda and Dounce [12]. This procedure takes advantage of the more rapid sedimentation of the larger polyploid nuclei through dense aqueous sucrose than the smaller diploid nuclei [16]. The volume of polyploid hepatocyte nuclei is known to be twice that of diploid hepatocyte nuclei [16,17]. Phase contrast microscopy of isolated polyploid nuclear fractions demonstrated that few (less than 5%) diploid size nuclei contaminated these preparations. Diphenylamine determination [18] of DNA c o n t e n t showed that the polyploid fractions contain twice as much DNA per average nucleus as diploid nuclear fractions from either liver or kidney. More critical demonstration of the homogeneity and DNA c o n t e n t of the polyploid nuclear fractions was made by flow microfluorometry (Fig. 1). In this procedure fixed nuclei are stained with acriflavin, a fluorescent dye which is a quantitative stain for DNA [14,15]. The nuclei, in monodisperse suspension, are individually passed through a beam of laser light to excite the dye and post-excitation fluorescence of each nucleus quantitated by a photoelectric cell and recorded by a pulse height analyzer. Staining, and therefore fluorescent intensity, are linear with respect to DNA content and a display such as that in Fig. 1 shows the number of nuclei containing different amounts of DNA. In the total liver nuclear population the majority of nuclei contain polyploid amounts o f DNA (either 2c or 8c amounts) (Fig. 1, left panel). Fewer than 107o of the nuclei present in isolated polyploid hepatocyte nuclear fractions fluoresce at the 2c value (Fig. 1, right panel). These data, therefore, indicate that the DNA obtained from the polyploid nuclear fractions is, in fact, essentially entirely from polyploid hepatocytes. Reassociation kinetics o f labeled DNA driven kinetically by either polyploid DNA or diploid DNA The kinetics with which denatured DNA reassociates is governed by the
21
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F i g . 1. F l o w m l c x o f l u o r o m e t r i c assay o f D N A c o n t e n t and h o m o g e n e i t y o f f r a c t i o n a t e d live~ nuclei• F l u o r e s c e n t i n t e n s i t y (relative D N A c o n t e n t ) is in arbitrary linear i n c r e m e n t s c o r r e s p o n d i n g t o t h e scale o f a p u l s e h e i g h t a n a l y z e r . T h e p r e d o m i n a n t s h o u l d e r o f t h e 2 c p e a k f o r t o t a l liver n u c l e i f l u o r e s c e s b e l o w t h e e x p e c t e d level w h i l e t h e m i n o r s h o u l d e r is at t h e e x p e c t e d level ( a p p r o x i m a t e l y 1 6 . 5 in this c a s e ) . T h i s is due t o an u n d e r s t a i n i n g artifact, t h e o c c u r r e n c e o f w h i c h is l i m i t e d t o 2 c n u c l e i ( H o r a n and Ordahi, u n p u b l i s h e d o b s e r v a t i o n s ) . C o m p a r i s o n s o f f l u o r e s c e n t i n t e n s i t y b e t w e e n t h e t w o paneis are n o t valid d u e t o d i f f e r e n c e s in t h e scale s e t t i n g s o f t h e pulse h e i g h t analyzer. D i p l o i d k i d n e y n u c l e i ( p a n e l B, solid t r a c i n g ) s e r v e d as a f l u o r e s c e n t i n t e n s i t y standard f o r t h e i s o l a t e d p o l y p l o i d h e p a t o c y t e nuclei• F o r o t h e r d e t a i l s see M e t h o d s and Materials. I n f o r m a t i o n o n t h e t h e o r y , a p p l i c a t i o n and a c c u r a c y o f f l o w m i c r o f l u o r o m e t r y is p u b l i s h e d [ 1 5 , 2 4 ].
DNA concentration, under constant reaction conditions [8,19]. The reassociation of small amounts of labeled DNA can be driven kinetically by excess amounts of homologous unlabeled DNA [20]. In a reaction such as this the kinetics with which labeled DNA is observed to reassociate reflects the kinetics of the unlabeled DNA reassociation. Comparisons between two DNA preparations may be made, therefore, by comparing the kinetics with which each drives the reassociation of labeled DNA. In the experiments described in this section labeled rat C-6 tumor cell DNA is allowed to reassociate with excess amounts of unlabeled DNA from either rat diploid kidney nuclei or polyploid hepatocyte nuclei. If polyploid hepatocyte nuclei contain multiple diploid genomes then DNA from these nuclei would be expected to drive the reassociation of labeled DNA with kinetics identical to that of the diploid DNA driven reaction. This is expected because although each nucleotide sequence is replicated two or more times the relative concentration of all sequences remains constant. If, however, polyploid hepatocytes contain a diploid genome plus the amplified sequences of a subfraction of the diploid genome then the relative concentration (copy frequency) of the amplified sequences is increased while that of the unamplified DNA is decreased. In this latter case the DNA from polyploid nuclei would be expected to reassociate with kinetics which differ from that of diploid DNA and hence drive the reassociation of the labeled DNA differently.
22 Fig. 2 shows the kinetics of reassociation of labeled DNA with diploid DNA (open circles) and polyploid hepatocyte DNA (closed circles). Assay of the e x t e n t of reassociation (%3H as double-stranded DNA) is made by hydroxyapatite chromatography at determined, values of Cot (Cot is defined as; moles of nucleotides DNA × seconds × liter-I; and normalizes the time and concentration variables of reassociation reactions [8]). For further details see Methods and Materials. T h e kinetics of reassociation of labeled DNA with either diploid kidney DNA or polyploid hepatocyte DNA is indistinguishable (Fig. 2). This indicates that both DNA preparations contain homologous nucleotide sequences which are present in the same relative abundance and therefore that multiple complete genomes are present in polyploid hepatocytes. It is essential, however, to estimate the e x t e n t to which the reassociation kinetics of the labeled DNA would be altered if driven by DNA from 4c nuclei resulting from nucleotide sequence amplification. It is possible to quantitatively estimate the extent of these alterations given a mathematic approximation of the kinetics of normal diploid DNA reassociation. DNA reassociation is a second order reaction the. kinetics of which are described by the equation [8] : %DNA reassociated at Cot x = 100
[100/(1 + kx)]
where k is the reassociation rate constant (1~Cot at which reassociation is 50% complete). The solid line in Fig. 2 is the plot obtained by taking the sum of the fractional contribution of four such second reaction components. Because eucaryotic genomes contain both nonrepeated and repeated DNA sequences it is necessary to use a m u l t i c o m p o n e n t expression to approximate their reassociation kinetics. The reassociation rate constants and percentage of the total
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Fig. 2. Reassociation of labeled D N A driven kinetically by D N A f r o m either diploid or polyploid nuclei, O p e n circles are for diploid kidney D N A driven reassociation; closed circles are for polyploid hepatocyte D N A driven reassociation. For details of reaction conditions see M e t h o d s and Materials. Solid line is fitted to the data while the dotted lines represent hypothetical reassociation kinetics of D N A f r o m 4c nuclei resulting f r o m nucleotide sequence amplification (see Results and Table I),
23 DNA in each component are given in Table I. The rate constants of the first three components (kl, k2 and k3) correspond with those expected for repeated sequence DNA while that of the fourth component (k4) corresponds to that nonrepeated sequences [8]. This theoretical curve was fitted by trial and error using varying values for component k and size (P). The curve fits both sets of data equally well and individual points scatter apparently randomly about the line to a maximum of approximately +2%. Given this mathematic approximation of the kinetics of reassociation of rat DNA it is possible to compute the expected reassociation kinetics for DNA from hypothetical 4c nuclei resulting from nucleotide sequence amplification. This requires estimation of the copy frequency increase, relative abundance and reassociation rate constant of amplified sequences in the hypothetical 4c TABLE I C O N S T A N T S A N D M E T H O D F O R C A L C U L A T I O N O F C U R V E S IN F I G U R E 2 T h e s o l u t i o n t o t h e solid line in Fig. 2 is t h e s u m o f t h e f r a c t i o n a l c o n t r i b u t i o n o f f o u r s e c o n d o r d e r c o m p o n e n t s . T h e p e r c e n t a g e o f D N A (P*) r e a s s o c i a t e d at a n y given Cot value (x) is c o m p u t e d as:
P*
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W h e r e , P1 ~-P2 + P3 + P4 = 1 0 0 % . T h e values f o r r e a s s o c i a t i o n r a t e c o n s t a n t (k) a n d p e r c e n t a g e of t h e t o t a l D N A in e a c h c o m p o n e n t (P) a r e ~iven in t h e t o p line o f t h e t a b l e d e s i g n a t e d " s o l i d l i n e " . T h o s e c o m p o n e n t s s u b s c r i p t e d 1, 2 a n d 3 c o r r e s p o n d t o r e p e a t e d s e q u e n c e D N A w h i l e t h a t w i t h t h e s u b s c r i p t 4 r e p r e s e n t s n o n r e p e a t e d D N A . Cases A, B a n d C list t h e c o n s t a n t s u s e d t o c a l c u l a t e t h e c o r r e s p o n d i n g l a b e l e d d o t t e d lines in Fig. 2. T h e s e are f o r 1 0 0 - f o l d , 10-fold a n d 3-fold a m p l i f i c a t i o n , r e s p e c t i v e l y , o f s u b f r a c t i o n s o f n o n r e p e a t e d D N A . T h e c o n s t a n t s k ' P ' , k a a n d Pa are d e f i n e d in t h e i e g e n d to Fig. 3. F o r e a c h case (A, B, o r C) line I s h o w s t h e values f o r k a n d P o f t h e h y p o t h e t i c a l ( u n l a b e l e d ) D N A reassociat i o n (self-reassociation o f t h e h y p o t h e t i c a l D N A ) . L i n e I I s h o w s t h e v a l u e s f o r k a n d P for t h e kinetics w i t h w h i c h t h e h y p o t h e t i c a l D N A w o u l d drive t h e r e a s s o c i a t i o n of labeled D N A (in t h e s e cases t h e v a l u e s f o r k are t h e s a m e in lines I a n d I I b u t P in line I I r e f l e c t s t h e p e r c e n t a g e o f l a b e l e d D N A c o m p l e m e n t a r y t o a m p l i f i e d s e q u e n c e s ) . T h e case d e s i g n a t e d R is f o r a m p l i f i c a t i o n o f r e p e a t e d s e q u e n c e D N A (to a n y copy frequency) or greater than 100-fold amplification of nonrepeated sequence DNA. The resulting v a l u e s f o r t h e f o u r t h c o m p o n e n t (k 4 ~P4) are s u c h t h a t t h e r e s u l t i n g c u r v e w o u l d be essentially similar t o t h a t o f " a " in Fig. 3 o v e r Cot values g r e a t e r t h a n 1 0 2 . T h e m a t h e m a t i c a l a p p r o x i m a t i o n o f l a b e l e d r a t D N A r e a s s o c i a t i o n m a d e h e r e (solid line) differs s o m e w h a t f r o m t h a t m a d e b y o t h e r s [ 2 2 ] a n d p r o b a b l y results f r o m d i f f e r e n c e s in t e m p e r a t u r e a n d salt u n d e r w h i c h r e a s s o c i a t i o n was c o n d u c t e d a n d possibly f r o m d i f f e r e n c e s in t h e m e t h o d o f m o l e c u l a r w e i g h t r e d u c t i o n of D N A b y shearing.
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24 nucleus. The relationship between these variables is plotted in Fig. 3 and the method for their computation given in the legend to Fig. 3. Using these estimates the expected reassociation kinetics were computed for three such hypothetical cases, those of doubling nuclear DNA content by; (a) 100-fold amplification of 1.01% of the diploid genome; (b) 10-fold amplification of 11.1% of the diploid genome; and {c) 3-fold amplification of 50% of the diploid genome. To simplify calculation, in each case, only the amplification of nonrepeated sequence DNA was considered. The expected kinetics of reassociation of labeled DNA with the hypothetical DNA arising from Cases a, b, and c are shown as the dotted lines in Fig. 2. Tabular account of the component sizes and reassociation rate constants for each case is given in Table I. It can be seen in Fig. 2 that the predicted effect of amplified sequences on the kinetics of reassociation with labeled DNA is less than one would intuitively expect. The most pronounced effect would result, not from the increased kinetics of amplified sequence reassociation but, from the reduced reassociation kinetics of labeled DNA complementary to unamplified sequences. This is expected because the relative concentration of unamplified sequences would be reduced two-fold (legend, Fig. 3) and hence their kinetics of reassociation is expected to be two times slower. Amplification of repeated sequence DNA {to any copy frequency) which would cause a doubling of nuclear DNA content is expected to result in a reassociation curve essentially similar to Case a over Cot values greater than 102. This would also be true for any cases resulting from greater than 100-fold amplification of less than 1% of
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Fig. 3. Q u a n t i t a t i v e r e l a t i o n s h i p s b e t w e e n n u c l e o t i d e s e q u e n c e s p r e s e n t in h y p o t h e t i c a l 4 c n u c l e i resulting f r o m n u c l e o t i d e s e q u e n c e a m p l i f i c a t i o n . T o d o u b l e n u c l e a r D N A c o n t e n t a given f r a c t i o n o f the diploid g e n o m e (P') m u s t be a m p l i f i e d to a greater or lesser e x t e n t ( c o p y f r e q u e n c y increase; i) d e p e n d i n g u p o n t h e size o f P~. T h e s e t w o q u a n t i t i e s are r e l a t e d b~': i = 1 + ( 1 0 0 / P ' ) . T h e p e r c e n t a g e o f D N A t h a t a m p l i fied s e q u e n c e s w o u l d c o n s t i t u t e (Pa) is c o m p u t e d as: P a = [ ( P ' ) ( i ) | / 2 . The r e a s s o c i a t i o n rate c o n s t a n t o f a m p l i f i e d s e q u e n c e s (/~a) is related t o their r e a s s o c i a t i o n rate c o n s t a n t b e f o r e a m p l i f i c a t i o n ( h ' ) b y : h a = (i/2)(h'). Graphic r e l a t i o n s h i p b e t w e e n P' ( l e f t hand scale)~ i ( h o r i z o n t a l s c a l e ) and Pa (right hand s c a l e ) is s h o w n . D o u b l i n g n u c l e a r D N A c o n t e n t b y s e q u e n c e a m p l i f i c a t i o n r e d u c e s , b~" half, the relative a b u n d a n c e ( P ) and t h e r e f o r e the values for ~ o f t h o s e s e q u e n c e s w h i c h are n o t r e p l i c a t e d ( u n a m p l i f i e d s e q u e n c e s ) .
25 nonrepeated DNA. All of the hypothetical curves fall outside the observed scatter of the points over at least three decades of Cot value although limited portions of the curves approximate the experimental points. The data in Fig. 3 indicate therefore that polyploid hepatocyte nuclei do not result from nucleotide sequence amplification but rather from complete replication of the entire genome. This conclusion was further tested by a second method of following DNA reassociation kinetics.
Reassociation kinetics of polyploid DNA and diploid DNA as followed by optical absorbance. To test for the possibility that sequence amplification escaped detection in the experiment described above, the reassociation kinetics of polyploid hepatocyte DNA and diploid kidney DNA was followed directly by measuring the 260 nm absorbance of hydroxyapatite chromatography fractions (see Methods and Materials). This approach is potentially more sensitive because under these conditions 50%, or more, of polyploid hepatocyte DNA should be observed to reassociate with kinetics which differ from diploid DNA reassociation if polyploidy results from nucleotide sequence amplification. Fig. 4 shows the results of such an experiment and again both polyploid hepatocyte DNA (closed circles} and diploid kidney DNA (open circles) reassociate with indistinguishable kinetics. The scatter of the data (see legend, Fig. 4) does not obscure the critical point that both DNA preparations possess essentially identical amounts of repeated and nonrepeated sequence DNA. As demonstrated below, this would not be expected if nuclear DNA content were doubled by amplification of the nucleotide sequences of a subfraction of the genome. The reassociation kinetics of these DNA preparations compare favorably with those obtained by others for rat DNA using similar methods [21,22].
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Fig. 4. R e a s s o c i a t i o n k i n e t i c s of d i p l o i d D N A a n d p o l y p l o i d D N A m e a s u r e d b y o p t i c a l a b s o r b a n c e . O p e n circles r e p r e s e n t t h e r e a s s o c i a t i o n o f d i p l o i d k i d n e y D N A ; c l o s e d circles r e p r e s e n t t h e r e a s s o c i a t i o n o f p o l y p l o i d h e p a t o c y t e DNA. F o r d e t a i l s o f r e a c t i o n c o n d i t i o n s see Materials a n d M e t h o d s . T h e s c a t t e r o f t h e s e d a t a is p r i m a r i l y d u e to 2 6 0 n m - a b s o r b i n g m a t e r i a l w h i c h c o - e l u t e s w i t h D N A f~om h y d r o x y a p a t i t e c o l u m n s . T h e c o l u m n s c a n n o t b e w a s h e d free o f t h i s m a t e r i a l n o r c a n its relative c o n t r i b u t i o n t o 2 6 0 n m a b s o r b a n c e b e a c c u r a t e l y q u a n t i t a t e d a n d p r e s u m a b l y it results f r o m p r o g r e s s i v e p a r t i a l d i s s o l u t i o n o f t h e h y d r o x y a p a t i t e crystals. The solid line is f i t t e d to the d a t a w h i l e t h e d o t t e d lines r e p r e s e n t h y p o t h e t i c a l r e a s s o c i a t i o n k i n e t i c s of D N A f r o m 4c n u c l e i r e s u l t i n g f r o m n u c l e o t i d e s e q u e n c e a m p l i f i c a t i o n (see R e s u l t s and T a b l e II).
26 More critical comparison of the kinetics of reassociation of both DNA preparations was made as follows. Both sets of data were divided into three kinetic components; the first, comprising 15% of the DNA, has essentially completed reassociation at Cot 1" 10 -1 and was not scored in these experiments; the second comprises approximately 20% of the DNA and reassociates primarily between Cot values 1 • 10 -1 and 1 X 102; and the third, the nonrepeated component, com.prises approximately 65% of the DNA and reassociates primarily after Cot 1 × 102. While it is acknowledged that there is probably more than one kinetic c o m p o n e n t reassociating between Cot 10 -1 and 102, they cannot be resolved by these data. Therefore, the simplest assumption was made for the purpose of estimation and comparison of the kinetics of reassociation of these two DNA preparations. Estimation of the reassociation rate constant (k) for the second and third components was made by the m e t h o d of Bishop [23] by considering each as an independent second order reaction. The similarity between the values obtained for the diploid DNA preparations is the critical element in these estimations rather than the absolute values obtained. The estimates obtained for the reassociation rate constants of the 2nd and 3rd diploid components are, respectively, 0.236 and 0.00075 while those for the polyploid DNA components are 0.253 and 0.00041. These values axe in excellent agreem e n t and strengthen the conclusion that these two DNA preparations are identical in terms of reassociation kinetics. The solid line in Fig. 4 is the curve obtained by taking the average of the diploid and polyploid values for k of each c o m p o n e n t and plotting the sum of the fractional contribution of the two second order reactions proceeding from a 15% level of reassociation at Cot 1 • 10 -1. The values for k and c o m p o n e n t size used to compute this line are tabulated in Table II. Prediction, based on the solid line in Fig. 4, of the expected kinetics of reas-
T A B L E II CONSTANTS AND METHODS FOR CALCULATION
OF CURVES IN FIGURE 4
All d e s i g n a t i o n s a n d c a l c u l a t i o n p r o c e s s e s are as d e s c r i b e d in t h e l e g e n d t o T a b e l I e x c e p t : ( 1 ) S o l u t i o n t o t h e s o l i d line w a s b a s e d o n a t h r e e - c o m p o n e n t c u r v e w i t h e s t i m a t e s f o r k as d e s c r i b e d in R e s u l t s . E s t i m a t i o n o f k I w a s n o t p o s s i b l e b e c a u s e t h e k i n e t i c s o f r e a s s o e i a t i o n o f t h i s c o m p o n e n t w a s n o t o b s e r v e d in t h e e x p e r i m e n t d e p i c t e d in Fig. 4. T h e v a l u e P I r e p r e s e n t s a r e a s o n a b l e e s t i m a t e o f t h e size o f t h e f i r s t c o m p o n e n t ( e s s e n t i a l l y c o m p l e t e r e a s s o c i a t i o n at Cot 10 -1). ( 2 ) H e r e , o b v i o u s l y , c a l c u l a t i o n s f o r c o m p l e m e n t a r y l a b e l e d D N A a r e n o t n e c e s s a r y b e c a u s e e a c h D N A p r e p a r a t i o n is r e a s s o c i a t i n g o n l y w i t h i t s e l f . (3) R e p e a t e d s e q u e n c e a m p l i f i c a t i o n ( t o a n y c o p y f r e q u e n c y ) o r a m p l i f i c a t i o n o f n o n r e p e a t e d D N A m o r e t h a n 1 0 0 - f o l d w o u l d r e s u l t in a p p r o x i m a t e l y 6 7 . 5 % o f t h e 4c D N A w i t h a r e a s s o c i a t i o n r a t e c o n s t a n t ( k ) l a r g e r t h a n 0 . 0 2 5 . T h i s w o u l d r e s u l t in a c u r v e e s s e n t i a l l y s i m i l a r t o " a " in Fig. 4 excel~t t h a t t h e m a j o r t r a n s i t i o n w o u l d be d i s p l a c e d t o l o w e r Cot v a l u e s . Case
k' ;P'
ka;P a
k 1 ;PI
k2 ;P2
k3 ;P3
Solid line
--
--
(--); 1 5 , 0
0 . 2 4 ~ 5 0 ; 20
0.000527; 65.00
A
0.005;
0.02500; 50.5
(--);
7,5
0 . 1 2 2 2 5 ; 10
0.000264; 32.0
B
0.0005; 11.10
0.00250; 55.0
(--);
7,5
0 . 1 2 2 2 5 ; 10
0.000264; 27.5
C
0.0005; 50.00
0.00075; 75.0
(--);
7,5
0.1225;10
0.000264;
1.01
7.5
R (67.5% DNA with k ~ 0.025)
0.000264; 32.5
27 sociation of hypothetical 4c nuclear DNA resulting, as in the previous section, from 100-fold, 10-fold and 3-fold amplification of nonrepeated DNA is shown by the d o t t e d lines in Fig. 4 labeled a, b and c, respectively. The m e t h o d and constants used for these computations are given in Table II. Here a dramatic effect is expected if 4c nuclei result from nucleotide sequence amplification. In each instance the apparent demarcation between repeated and nonrepeated sequence DNA reassociation would be significantly altered. Each of the hypothetical curves lies far outside the experimental points over at least two decades of Cot values. Amplification of either repeated sequence DNA (to any copy frequency) or of nonrepeated sequence DNA to greater than 100-fold would be expected to result in a curve essentially similar to " a " b u t with greater displacement of the major transition to lower Cot values. These data, and the data presented in Fig. 2 demonstrate no differences in the reassociation kinetics of polyploid hepatocyte DNA, as compared to diploid kidney DNA, which could be attributed to the presence of 50%, or more, amplified sequence DNA. The direct conclusion of these experiments is, therefore, that the increased DNA content of polyploid hepatocytes is due to the presence of multiple genomes and n o t to the amplification of the nucleotide sequences of a subfraction of the genome. Discussion
In the experiments reported here, kinetic analysis of DNA reassociation is used to determine whether the DNA content increase of polyploid hepatocytes is attributable to replication of the genome or to amplification of the nucleotide sequences of only a subfraction of the genome. In the latter case, b u t n o t the former, the reassociation kinetics of DNA from polyploid nuclei would be expected to differ from that of diploid DNA owing to alteration in the relative concentration of the nucleotide sequences. In t w o experiments the kinetics of polyploid DNA reassociation is identical to that of diploid DNA, within the resolution of these methods. The straightforward conclusion therefore is that hepatocyte polyploidization is a result of complete replication of the genome. An essential question, however, is whether failure to detect differences is due, in fact, to kinetic identity or to lack of sensitivity of the methods employed. A definitive answer to this question is n o t possible because it is known that these methods cannot detect kinetic differences if these differences reside in relatively small amounts of the total DNA [7]. For example, changes in the relative number of nucleotide sequence copies per nucleus would certainly go undetected unless these changes detectably affect total nuclear DNA content. Thus these experiments are only intended to detect amplification of limited subfractions of the genome which could cause a doubling of nuclear DNA content. The question of sensitivity is therefore addressed only for those hypothetical cases in which DNA content is doubled by the amplification of the nucleotide sequences of a subfraction of the genome. To double nuclear DNA content by amplification small subfractions of the genome must be amplified to a greater extent than larger subfractions. The relationship between fraction size and extent of amplification are shown in Fig. 3 and permit computation of the expected kinetics of reassociation of DNA
28 from hypothetical nuclei which have doubled DNA content as a result of sequence amplification. Computation of the expected kinetics of reassociation made for four hypothetical cases effectively cover all possible mechanisms of doubling nuclear DNA content by either; (1) amplification limited to subfractions of repeated sequence DNA; or (2) amplification limited to subfractions of nonrepeated DNA in which all amplified sequences are replicated the same n u m b e r of times (homogeneous amplification). For these cases the kinetics with which the hypothetical DNA would reassociate with itself (Fig. 4), or drive the reassociation of labeled DNA (Fig. 2) is significantly different from the experimental observations. Thus, the experimental evidence rules out a wide variety of possible mechanisms of doubling nuclear DNA content by nucleotide sequence amplification. Sequence amplification, if it existed in polyploid hepatocytes, would not necessarily be either homogeneous or limited to the repeated or nonrepeated fractions of the genome. These limitations were imposed because they permit reasonable calculations and are representative of a wide variety of possible cases. More complex schemes for doubling nuclear DNA content by sequence amplification would be expected to produce similar effects on the reassociation kinetics and most conceivable schemes would be detectable by these methods. It would, however, be unrealistic n o t to assume that at some level of complexity of sequence amplification the resulting kinetics would be indistinguishable, by these methods, from that of normal diploid DNA. For example, if 80% of the genome were replicated 2-fold, 10% not replicated at all, and the remaining 10% replicated 4-fold, then the kinetics of reassociation of this hypothetical 4c DNA would probably be indistinguishable from that of normal diploid DNA. To define, however, the point at which resolution is lost between this extreme and the hypothetical cases shown in Fig. 3 and 5 would require extremely complex, and perhaps impossible, calculations. The lower limit of resolution of these kinds of experiments cannot therefore be adequately defined. Possibilities of this kind, however, go b e y o n d the limit of the intended goal of these experiments. Clearly the reassociation kinetics of polyploid hepat o c y t e DNA shows no evidence that amplification of specific subfractions of the genome is responsible for the increased DNA content of these cells. In conclusion, kinetic analysis of DNA reassociation indicates that the increased DNA content of polyploid hepatocytes is attributable to the presence of multiple genomes and not to nucleotide sequence amplification. The concept of ploidy, typically defined in terms of karyotypic and/or DNA content multiples, is therefore corroborated, at the nucleotide sequence level of DNA, by these experiments.
Acknowledgments I would like especially to thank Dr. Paul K. Horan of the University of Rochester Department of Pathology, for generously performing the flow microfluorometric measurements and the Los Alamos Scientific Laboratories for the use of their equipment for these measurements. This work was supported by Training Grants (GM-01981-04, GM-01981-05, PE-00543-03) from the National Institutes of Health.
29
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Carriere, R. (1969) Int. Rev. Cytol. 25, 201--277 EPstein, C.J. (1967) Proc. Natl. Acad. Sci. U.S. 57,327--334 Brown° D.D. and David, I. (1968) Science 160, 272--280 Dickson, E., Boyd, J.B. and Laird, C.D. (1971) J. Mol. BioL 61,615--627 Hughes, D.T. (1966) in Chromosomes Today (Darlington, C.D. and Lewis, K.R., eds.), Vol. I, pp. 118--210, Oliver and Boyd, London Glass, E. (1956) Ch~omosoma 7 , 6 5 5 - 6 6 9 Laird, C.D. (1971) Chromosoma 32, 378--406 Britten, R.J. and Kohne, D.E. (1968) Science 161~ 529--540 Gage, L.P. (1974) J. Mol. Biol. 86, 97--108 Lasher, R.S. and Zagon, I.S. (1972) Brain Res. 41,482--488 Umana, R. and Dounce, A.L. (1964) Exp. Cell Res. 35, 277--292 Chanda, S.K. and Dounce, A.L. (1971) Arch. Biochem. Biophys. 145, 211--219 Hahn, W.E. and Laird, C.D. (1971) Science 173,158--161 Horan, P.K., Rom~ro, A., Steinkamp, J.A. and Peterson, D.F. (1974) J. Natl. Canc. Inst. 52, 843~847 Kraemer, P.M., Deaven, L.L., Crissman, H.A. and VanDilla, M.A. (1972) Adv. CeB Mol. Biol. 2, 47-108 Johnston, I.R., Mathias, A.P., Pennington, F. and Ridge, D. (1968) Biochem. J. 109, 127--135 Epstein, C.J. and Gatens, E.A. (1967) Nature 214, 1050--1051 Bu~ton, K. (1956) Biochem. J. 62, 315--323 Wetmur, J.G. and Davidson, N. (1968) J. Mol. Biol. 31,349--370 Graham, D.E., Neufeld, B.R., Davidson, E.H. and Britten, R.J. (1974) Cell 1,127--137 Melli, M., Whitfield, C., Rao, K.V., Richardson, M. and Bishop, J.O. (1971) Nat. New Biol. 231, 8--12 Holmes, D.S. and Bonnet, J. (1974) Biochemistxy 13, 8 4 1 - 6 4 8 Bishop, J.O. (1972) Binchem. J. 126,171--185 Kraemer, P.M., Deaven, L.L., Crissman, H.A., Steinkamp, J.A. and Petersen, D.F. (1973) Cold Sprin~ Harbor Syrup. Quant. Biol. 38, 133~144
Biochimica et Biophysica Acta, 474 (1977) 17--29 © Elsevier/North-Holland Biomedical Press
BBA 98784
REASSOCIATION KINETICS OF POLYPLOID HEPATOCYTE DNA
CHARLES P. ORDAHL
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.) (Received June 10th, 1976)
Summary The state of ploidy of 4c hepatocytes is examined by kinetic analysis of DNA reassociation. The nucleotide sequences present in 4c hepatocyte DNA are found to be complementary to, and present in the same relative abundance as, those of the normal diploid genome. Therefore, these experiments indicate that the increased DNA content of 4c hepatocytes is due to the presence of four haploid genomes and not to selective replication of a subfraction of the diploid genome. The concept of ploidy, typically defined in terms of karyotypic and/or DNA content multiples is thus corroborated and extended by these experiments.
Introduction In the mammalian liver the majority of hepatocytes increase their nuclear DNA content during the course of adult life [ 1 ]. The functional significance of hepatic polyploidization is unknown but may represent a unique growth process [2]. H e p a t o c y t e DNA content increases occur in multiples of the 2c or diploid value and chiefly because of this observation these cells are considered to be polyploid. DNA content alone, however, is not necessarily a reliable index of ploidy because some eucaryotic cells are known to increase DNA content without faithful replication of the entire genome. For example, this occurs in the amphibian oocyte as a result of extensive amplification of the cistrons coding for ribosomal I~NA [3]. In Drosophila, during polytenization of the chromosomes of salivary gland cells, the repeated sequence DNA undergoes a reduction, from 20% to 5%, in its relative abundance [4]. In both these instances the alterations in the relative abundance of certain nucleotide sequences in DNA either causes, or occurs concommitant with a significant increase in cellular DNA content. The question of whether polyploid hepatocytes contain multiple complete genomes is therefore not answered by DNA content determinations. A more
18 direct approach to this question may be made by karyotypic analysis since ploidy is defined in terms of karyotic multiples [5]. Distinct hepatocyte karyotypes are difficult to obtain b u t there is limited evidence that tetraploid karyotypes occur in hepatectomized rat liver undergoing hyperplasia [6]. Karyotypic analysis however remains a morphological approach to a problem which, in conceptual terms, concerns the nucleotide sequences in DNA. Thus one expects the karyotypic complement to correspond to the "unit genome" [7] and contain a full complement of the nucleotide sequences in a haploid genome. A definitive answer to this question is not presently available for the case of hepatic polyploidy. The question may therefore be asked: does the increased DNA c o n t e n t of polyploid hepatocytes represent the presence of additional complete genomes or does it represent the amplified sequences of a subfraction of the genome? Although previous work indicates that these cells possess multiple genomes the methods used cannot exclude the possibility that the DNA c o n t e n t increase is a result of amplification of the nucleotide sequences of a limited subfraction of the genome. Thus two extremes may be imagined; one, the most likely, that polyploidization results replication of all the nucleotide sequences in the genome; and, second, that disproportionate replication of a subfraction of the genome is responsible for the DNA content increase of these cells. If the former case is found to be true this would corroborate classical concepts of ploidy and extend these concepts to the level of the nucleotide sequences in DNA. If, on the other hand, it is found that hepatocyte polyploidization results from nucleotide sequence amplification this information would provide a basis for more critical investigation of the role of polyploid hepatocytes in liver function. To answer this question, I have compared the reassociation kinetics of polyploid hepatocyte DNA to that of diploid kidney DNA. The kinetics of DNA reassociation depend upon the relative concentration of the nucleotide sequences [8]. Amplification of nucleotide sequences alters the relative concentration of the nucleotide sequences and is therefore detectable, within limits, by kinetic analysis of DNA reassociation. The results of these experiments indicate that, as expected, polyploid hepatocyte DNA reassociates with the same kinetics as diploid DNA. Thus these experiments support, at the nucleotide sequence level of DNA, the concept that polyploid hepatocytes possess multiple copies of the diploid genome. In addition, calculation of hypothetical reassociation kinetics permit estimation of some of the kinetic effects expected from the presence of amplified sequence DNA. Recently, a similar experimental approach has been used to demonstrate that uniform replication of the genome is the cause of the two hundred thousand fold increase in DNA c o n t e n t of the silk gland cells o f B o m b y x mori [9]. Methods and Materials
Preparation o f DNA Unlabeled DNA was obtained from nuclei of adult (250--500 g) SpragueDawley rats. Labeled DNA was obtained from nuclei of C-6 rat glial t u m o r cells (American Type Culture Collection) grown in roller bottle culture using modi-
19 lied Ham's F-12 medium with 2 mM K ÷ [10] and 3.3 pCi/ml [3H]thymidine (Amersham Searle) to label DNA. Kidney and cultured cell nuclei were prepared by the m e t h o d of Umana and Dounce [11] with the addition of 1% Triton X-100 (Packard Inst. Co.) to the initial homogenization medium. Polyploid hepatocyte nuclei were prepared by the m e t h o d of Chanda and Dounce [12] except that Triton was used, as above, and for the 2.2 M sucrose spin nuclei were suspended in 2.2 M sucrose and pelleted through a 2.2 M sucrose pad at 7000 rev./min for 1 h in a Spinco SW-25 rotor (Spinco Div., Beckman Inst. Inc.). To extract DNA, nuclei were suspended in 0.15 M NaC1/50 mM KC1/50 mM Tris • HC1 (Sigma Chem. Co.), pH 7.6 with 1% sodium dodecyl sulphate (SDS) added to lyse nuclei and denature protein. To remove protein, lysates were extracted three times with phenol saturated in the above buffer, and once with chloroform/octanol {8 : 1). Extracted DNA, precipitated overnight with 2.5 vols. ethanol at --20°C, was redissolved in the same buffer without SDS and treated with 50 ~tg/ml ribonuclease (RNAase B, Worthington Biochem. Corp.) and then 100 pg/ml pronase (Protease, Sigma Biochem. Co.), each for 1 h at 37°C. Enzymes were removed by phenol and chloroform/octanol extraction as above and the DNA precipitated with ethanol. Purified rat DNA had a ratio of optical absorbance at 260 and 280 nm (260/280) of 1.83--1.85. DNA was sheared by passage through the valve of a french pressure cell (American Inst. Co.) at 30 000 lb/inch 2 [13]. Sheared DNA was passed through Sephadex G100 (Pharmacia Fine Chem.) to remove small molecular weight fragments and stored as an ethanol precipitate at --20 ° C. Sheared DNA migrated at between 6 and 7 S in sucrose gradient centrifugation. Reassociation reactions For reassociation, sheared DNA was dissolved in 0.12 M phosphate buffer, pH 6.8 and aliquots fire sealed in glass capillary tubes, heated to 100°C for 10 min to denature DNA and immediately transferred to a 60°C water bath to permit reassociation. For labeled DNA reassociation reactions, aliquots (5--50 pl) contained 1--5 × 104 cpm labeled DNA (specific activity; 1.8 × l 0 s cpm/pg) and 1 × 10 -3. to 5 mg/ml unlabeled DNA. Unlabeled DNA reassociation reaction aliquots (20--100 pl) contained 0.1--0.5 mg/ml DNA. The concentration and time variable (Cot} for reaction samples was determined by the formula: (absorbance at 260 n m ) × (0.5) = 1 Cot/h, as described by Britten and Kohne [8]. Zero-time samples were chilled on ice immediately after denaturation to prevent reassociation before hydroxyapatite assay. These samples are plotted at Cot 1 • 10 -2 in Figs. 2 and 4. The small amount of reassociation in these samples presumably represents that of highly repeated sequences which reassociate during handling and hydroxyapatite assay. Reassociation reaction aliquots were diluted with 0.12M phosphate buffer and passed through hydroxyapatite columns maintained at 60°C. Unreassociated (single-strand) DNA was eluted with 0.12M phosphate buffer and reassociated (double-strand) DNA with 0.5 M phosphate buffer, both buffers maintained at 60 ° C. Tests showed that procedures for binding and elution of DNA were effective. Labeled DNA fractions from the hydroxyapatite assay were quantitated in liquid scintillation cocktail composed of 86.4% toluene, 3.6% Liquifluor (New
20 England Nuclear) with 10% Biosolv (Beckman Instr. Co.). To quantitate unlabeled DNA in hydroxyapatite assay fractions, the absorbance of each fraction was measured at 260 nm and the absorbance of the unreassociated fraction multiplied by 0.85 to account for the observed hypochromic shift of denatured rat DNA at room temperature.
Flow microfluorometry Nuclear preparations to be subjected to flow microfluorometry were suspended in 0.44 M sucrose made 20% with respect to formalin to fix nuclei, sealed in tubes and shipped on ice to Dr. Paul K. Horan, Los Alamos Scientific Laboratory, N e w Mexico. All flow microfluorometry procedures were performed by him according to methods he has published [14]. Percentages of nuclei under peaks in Fig. 1 were estimated by cutting o u t peaks on paper and weighing. A review of flow microfluorometry technology has been published [15]. Results
Isolation and homogeneity o f polyploid hepatocyte nuclei Polyploid hepatocyte nuclei were isolated from the total liver nuclear population by a modified m e t h o d described by Chanda and Dounce [12]. This procedure takes advantage of the more rapid sedimentation of the larger polyploid nuclei through dense aqueous sucrose than the smaller diploid nuclei [16]. The volume of polyploid hepatocyte nuclei is known to be twice that of diploid hepatocyte nuclei [16,17]. Phase contrast microscopy of isolated polyploid nuclear fractions demonstrated that few (less than 5%) diploid size nuclei contaminated these preparations. Diphenylamine determination [18] of DNA c o n t e n t showed that the polyploid fractions contain twice as much DNA per average nucleus as diploid nuclear fractions from either liver or kidney. More critical demonstration of the homogeneity and DNA c o n t e n t of the polyploid nuclear fractions was made by flow microfluorometry (Fig. 1). In this procedure fixed nuclei are stained with acriflavin, a fluorescent dye which is a quantitative stain for DNA [14,15]. The nuclei, in monodisperse suspension, are individually passed through a beam of laser light to excite the dye and post-excitation fluorescence of each nucleus quantitated by a photoelectric cell and recorded by a pulse height analyzer. Staining, and therefore fluorescent intensity, are linear with respect to DNA content and a display such as that in Fig. 1 shows the number of nuclei containing different amounts of DNA. In the total liver nuclear population the majority of nuclei contain polyploid amounts o f DNA (either 2c or 8c amounts) (Fig. 1, left panel). Fewer than 107o of the nuclei present in isolated polyploid hepatocyte nuclear fractions fluoresce at the 2c value (Fig. 1, right panel). These data, therefore, indicate that the DNA obtained from the polyploid nuclear fractions is, in fact, essentially entirely from polyploid hepatocytes. Reassociation kinetics o f labeled DNA driven kinetically by either polyploid DNA or diploid DNA The kinetics with which denatured DNA reassociates is governed by the
21
Total Liver r~ucle[
/
40
8I 8c 2%
~-,
4c 56 °/.
2c 42 °/o
Potyploid Hepatocyte nuclei;
& diploid kidr~y nuclei ( ~ ) 8c 4c 2c 92.5 °lo r 1 • •.
~ 30
~o
._ ._2(3
~1
6 z ~
I
~o
~
~
~
I
~o
t
~o
l
,o
Fluorescent
~
~o
,~
~'o
~'o
,
~o
intensity
F i g . 1. F l o w m l c x o f l u o r o m e t r i c assay o f D N A c o n t e n t and h o m o g e n e i t y o f f r a c t i o n a t e d live~ nuclei• F l u o r e s c e n t i n t e n s i t y (relative D N A c o n t e n t ) is in arbitrary linear i n c r e m e n t s c o r r e s p o n d i n g t o t h e scale o f a p u l s e h e i g h t a n a l y z e r . T h e p r e d o m i n a n t s h o u l d e r o f t h e 2 c p e a k f o r t o t a l liver n u c l e i f l u o r e s c e s b e l o w t h e e x p e c t e d level w h i l e t h e m i n o r s h o u l d e r is at t h e e x p e c t e d level ( a p p r o x i m a t e l y 1 6 . 5 in this c a s e ) . T h i s is due t o an u n d e r s t a i n i n g artifact, t h e o c c u r r e n c e o f w h i c h is l i m i t e d t o 2 c n u c l e i ( H o r a n and Ordahi, u n p u b l i s h e d o b s e r v a t i o n s ) . C o m p a r i s o n s o f f l u o r e s c e n t i n t e n s i t y b e t w e e n t h e t w o paneis are n o t valid d u e t o d i f f e r e n c e s in t h e scale s e t t i n g s o f t h e pulse h e i g h t analyzer. D i p l o i d k i d n e y n u c l e i ( p a n e l B, solid t r a c i n g ) s e r v e d as a f l u o r e s c e n t i n t e n s i t y standard f o r t h e i s o l a t e d p o l y p l o i d h e p a t o c y t e nuclei• F o r o t h e r d e t a i l s see M e t h o d s and Materials. I n f o r m a t i o n o n t h e t h e o r y , a p p l i c a t i o n and a c c u r a c y o f f l o w m i c r o f l u o r o m e t r y is p u b l i s h e d [ 1 5 , 2 4 ].
DNA concentration, under constant reaction conditions [8,19]. The reassociation of small amounts of labeled DNA can be driven kinetically by excess amounts of homologous unlabeled DNA [20]. In a reaction such as this the kinetics with which labeled DNA is observed to reassociate reflects the kinetics of the unlabeled DNA reassociation. Comparisons between two DNA preparations may be made, therefore, by comparing the kinetics with which each drives the reassociation of labeled DNA. In the experiments described in this section labeled rat C-6 tumor cell DNA is allowed to reassociate with excess amounts of unlabeled DNA from either rat diploid kidney nuclei or polyploid hepatocyte nuclei. If polyploid hepatocyte nuclei contain multiple diploid genomes then DNA from these nuclei would be expected to drive the reassociation of labeled DNA with kinetics identical to that of the diploid DNA driven reaction. This is expected because although each nucleotide sequence is replicated two or more times the relative concentration of all sequences remains constant. If, however, polyploid hepatocytes contain a diploid genome plus the amplified sequences of a subfraction of the diploid genome then the relative concentration (copy frequency) of the amplified sequences is increased while that of the unamplified DNA is decreased. In this latter case the DNA from polyploid nuclei would be expected to reassociate with kinetics which differ from that of diploid DNA and hence drive the reassociation of the labeled DNA differently.
22 Fig. 2 shows the kinetics of reassociation of labeled DNA with diploid DNA (open circles) and polyploid hepatocyte DNA (closed circles). Assay of the e x t e n t of reassociation (%3H as double-stranded DNA) is made by hydroxyapatite chromatography at determined, values of Cot (Cot is defined as; moles of nucleotides DNA × seconds × liter-I; and normalizes the time and concentration variables of reassociation reactions [8]). For further details see Methods and Materials. T h e kinetics of reassociation of labeled DNA with either diploid kidney DNA or polyploid hepatocyte DNA is indistinguishable (Fig. 2). This indicates that both DNA preparations contain homologous nucleotide sequences which are present in the same relative abundance and therefore that multiple complete genomes are present in polyploid hepatocytes. It is essential, however, to estimate the e x t e n t to which the reassociation kinetics of the labeled DNA would be altered if driven by DNA from 4c nuclei resulting from nucleotide sequence amplification. It is possible to quantitatively estimate the extent of these alterations given a mathematic approximation of the kinetics of normal diploid DNA reassociation. DNA reassociation is a second order reaction the. kinetics of which are described by the equation [8] : %DNA reassociated at Cot x = 100
[100/(1 + kx)]
where k is the reassociation rate constant (1~Cot at which reassociation is 50% complete). The solid line in Fig. 2 is the plot obtained by taking the sum of the fractional contribution of four such second reaction components. Because eucaryotic genomes contain both nonrepeated and repeated DNA sequences it is necessary to use a m u l t i c o m p o n e n t expression to approximate their reassociation kinetics. The reassociation rate constants and percentage of the total
( 10-
o '-~.. •
i 10 -t
i 10 °
',~.
:...
, 10 2
1~01
10 3
, 10 4
--
Cot
Fig. 2. Reassociation of labeled D N A driven kinetically by D N A f r o m either diploid or polyploid nuclei, O p e n circles are for diploid kidney D N A driven reassociation; closed circles are for polyploid hepatocyte D N A driven reassociation. For details of reaction conditions see M e t h o d s and Materials. Solid line is fitted to the data while the dotted lines represent hypothetical reassociation kinetics of D N A f r o m 4c nuclei resulting f r o m nucleotide sequence amplification (see Results and Table I),
23 DNA in each component are given in Table I. The rate constants of the first three components (kl, k2 and k3) correspond with those expected for repeated sequence DNA while that of the fourth component (k4) corresponds to that nonrepeated sequences [8]. This theoretical curve was fitted by trial and error using varying values for component k and size (P). The curve fits both sets of data equally well and individual points scatter apparently randomly about the line to a maximum of approximately +2%. Given this mathematic approximation of the kinetics of reassociation of rat DNA it is possible to compute the expected reassociation kinetics for DNA from hypothetical 4c nuclei resulting from nucleotide sequence amplification. This requires estimation of the copy frequency increase, relative abundance and reassociation rate constant of amplified sequences in the hypothetical 4c TABLE I C O N S T A N T S A N D M E T H O D F O R C A L C U L A T I O N O F C U R V E S IN F I G U R E 2 T h e s o l u t i o n t o t h e solid line in Fig. 2 is t h e s u m o f t h e f r a c t i o n a l c o n t r i b u t i o n o f f o u r s e c o n d o r d e r c o m p o n e n t s . T h e p e r c e n t a g e o f D N A (P*) r e a s s o c i a t e d at a n y given Cot value (x) is c o m p u t e d as:
P*
=
1
l+kl
x
+
2 - - - -
l+k2 x
+
3
l+k3 x
+
4 - - - - '
•
l+k4x
W h e r e , P1 ~-P2 + P3 + P4 = 1 0 0 % . T h e values f o r r e a s s o c i a t i o n r a t e c o n s t a n t (k) a n d p e r c e n t a g e of t h e t o t a l D N A in e a c h c o m p o n e n t (P) a r e ~iven in t h e t o p line o f t h e t a b l e d e s i g n a t e d " s o l i d l i n e " . T h o s e c o m p o n e n t s s u b s c r i p t e d 1, 2 a n d 3 c o r r e s p o n d t o r e p e a t e d s e q u e n c e D N A w h i l e t h a t w i t h t h e s u b s c r i p t 4 r e p r e s e n t s n o n r e p e a t e d D N A . Cases A, B a n d C list t h e c o n s t a n t s u s e d t o c a l c u l a t e t h e c o r r e s p o n d i n g l a b e l e d d o t t e d lines in Fig. 2. T h e s e are f o r 1 0 0 - f o l d , 10-fold a n d 3-fold a m p l i f i c a t i o n , r e s p e c t i v e l y , o f s u b f r a c t i o n s o f n o n r e p e a t e d D N A . T h e c o n s t a n t s k ' P ' , k a a n d Pa are d e f i n e d in t h e i e g e n d to Fig. 3. F o r e a c h case (A, B, o r C) line I s h o w s t h e values f o r k a n d P o f t h e h y p o t h e t i c a l ( u n l a b e l e d ) D N A reassociat i o n (self-reassociation o f t h e h y p o t h e t i c a l D N A ) . L i n e I I s h o w s t h e v a l u e s f o r k a n d P for t h e kinetics w i t h w h i c h t h e h y p o t h e t i c a l D N A w o u l d drive t h e r e a s s o c i a t i o n of labeled D N A (in t h e s e cases t h e v a l u e s f o r k are t h e s a m e in lines I a n d I I b u t P in line I I r e f l e c t s t h e p e r c e n t a g e o f l a b e l e d D N A c o m p l e m e n t a r y t o a m p l i f i e d s e q u e n c e s ) . T h e case d e s i g n a t e d R is f o r a m p l i f i c a t i o n o f r e p e a t e d s e q u e n c e D N A (to a n y copy frequency) or greater than 100-fold amplification of nonrepeated sequence DNA. The resulting v a l u e s f o r t h e f o u r t h c o m p o n e n t (k 4 ~P4) are s u c h t h a t t h e r e s u l t i n g c u r v e w o u l d be essentially similar t o t h a t o f " a " in Fig. 3 o v e r Cot values g r e a t e r t h a n 1 0 2 . T h e m a t h e m a t i c a l a p p r o x i m a t i o n o f l a b e l e d r a t D N A r e a s s o c i a t i o n m a d e h e r e (solid line) differs s o m e w h a t f r o m t h a t m a d e b y o t h e r s [ 2 2 ] a n d p r o b a b l y results f r o m d i f f e r e n c e s in t e m p e r a t u r e a n d salt u n d e r w h i c h r e a s s o c i a t i o n was c o n d u c t e d a n d possibly f r o m d i f f e r e n c e s in t h e m e t h o d o f m o l e c u l a r w e i g h t r e d u c t i o n of D N A b y shearing.
Case
k';P'
~a;Pa
kl;P 1
k2;p 2
k3;p 3
~4;p 4
Solid line
--
--
100; 12
8; 1 5 . 0
0.4; 11.0
0.0010; 62.00
0 . 0 0 1 ; 1.01
0.0500; 50.50
50;
4;
0.2;
5.5
0.0005; 30.50
0.0500:
50; 12
4; 1 5 . 0
0.2; 1 1 . 0
0.0005; 60.09
0.0050; 55.50
50;
4;
0.0050; 11.10
50; 12
0.0015; 75.00
50;
0.0015; 50.00
50; 12
I
6
7.5
A II I B
0.001
;
6
7.5 " 0.2;
5.5
0.0005; 25.50
4; 1 5 . 0
0.2, 1 1 . 0
0.0005; 50.09
4;
0.2; 1 1 0 . 0
0.0005;
0.2; 1 1 , 0
0.0005; 12.00
11.1
I1 I C
1.01
6
7.5
6.00
0 , 0 0 1 ; 50.0 II I
R
4; 1 5 . 0
.
.
.
.
0.0005; 31.00
.
.
.
.
0.0005; 62.00
_ II
24 nucleus. The relationship between these variables is plotted in Fig. 3 and the method for their computation given in the legend to Fig. 3. Using these estimates the expected reassociation kinetics were computed for three such hypothetical cases, those of doubling nuclear DNA content by; (a) 100-fold amplification of 1.01% of the diploid genome; (b) 10-fold amplification of 11.1% of the diploid genome; and {c) 3-fold amplification of 50% of the diploid genome. To simplify calculation, in each case, only the amplification of nonrepeated sequence DNA was considered. The expected kinetics of reassociation of labeled DNA with the hypothetical DNA arising from Cases a, b, and c are shown as the dotted lines in Fig. 2. Tabular account of the component sizes and reassociation rate constants for each case is given in Table I. It can be seen in Fig. 2 that the predicted effect of amplified sequences on the kinetics of reassociation with labeled DNA is less than one would intuitively expect. The most pronounced effect would result, not from the increased kinetics of amplified sequence reassociation but, from the reduced reassociation kinetics of labeled DNA complementary to unamplified sequences. This is expected because the relative concentration of unamplified sequences would be reduced two-fold (legend, Fig. 3) and hence their kinetics of reassociation is expected to be two times slower. Amplification of repeated sequence DNA {to any copy frequency) which would cause a doubling of nuclear DNA content is expected to result in a reassociation curve essentially similar to Case a over Cot values greater than 102. This would also be true for any cases resulting from greater than 100-fold amplification of less than 1% of
gO-
-9O
•~ 8 0 0~70-
-85 ~
o 60
- 8 0 ~r
~ ~J o 40-~ ._ "o 30-
•70 ~ E
~ 20-
-60 ~
65 ~
¢~ 13_
-55
~0-,
Copy
~0 ~00 frequency increase _i
Fig. 3. Q u a n t i t a t i v e r e l a t i o n s h i p s b e t w e e n n u c l e o t i d e s e q u e n c e s p r e s e n t in h y p o t h e t i c a l 4 c n u c l e i resulting f r o m n u c l e o t i d e s e q u e n c e a m p l i f i c a t i o n . T o d o u b l e n u c l e a r D N A c o n t e n t a given f r a c t i o n o f the diploid g e n o m e (P') m u s t be a m p l i f i e d to a greater or lesser e x t e n t ( c o p y f r e q u e n c y increase; i) d e p e n d i n g u p o n t h e size o f P~. T h e s e t w o q u a n t i t i e s are r e l a t e d b~': i = 1 + ( 1 0 0 / P ' ) . T h e p e r c e n t a g e o f D N A t h a t a m p l i fied s e q u e n c e s w o u l d c o n s t i t u t e (Pa) is c o m p u t e d as: P a = [ ( P ' ) ( i ) | / 2 . The r e a s s o c i a t i o n rate c o n s t a n t o f a m p l i f i e d s e q u e n c e s (/~a) is related t o their r e a s s o c i a t i o n rate c o n s t a n t b e f o r e a m p l i f i c a t i o n ( h ' ) b y : h a = (i/2)(h'). Graphic r e l a t i o n s h i p b e t w e e n P' ( l e f t hand scale)~ i ( h o r i z o n t a l s c a l e ) and Pa (right hand s c a l e ) is s h o w n . D o u b l i n g n u c l e a r D N A c o n t e n t b y s e q u e n c e a m p l i f i c a t i o n r e d u c e s , b~" half, the relative a b u n d a n c e ( P ) and t h e r e f o r e the values for ~ o f t h o s e s e q u e n c e s w h i c h are n o t r e p l i c a t e d ( u n a m p l i f i e d s e q u e n c e s ) .
25 nonrepeated DNA. All of the hypothetical curves fall outside the observed scatter of the points over at least three decades of Cot value although limited portions of the curves approximate the experimental points. The data in Fig. 3 indicate therefore that polyploid hepatocyte nuclei do not result from nucleotide sequence amplification but rather from complete replication of the entire genome. This conclusion was further tested by a second method of following DNA reassociation kinetics.
Reassociation kinetics of polyploid DNA and diploid DNA as followed by optical absorbance. To test for the possibility that sequence amplification escaped detection in the experiment described above, the reassociation kinetics of polyploid hepatocyte DNA and diploid kidney DNA was followed directly by measuring the 260 nm absorbance of hydroxyapatite chromatography fractions (see Methods and Materials). This approach is potentially more sensitive because under these conditions 50%, or more, of polyploid hepatocyte DNA should be observed to reassociate with kinetics which differ from diploid DNA reassociation if polyploidy results from nucleotide sequence amplification. Fig. 4 shows the results of such an experiment and again both polyploid hepatocyte DNA (closed circles} and diploid kidney DNA (open circles) reassociate with indistinguishable kinetics. The scatter of the data (see legend, Fig. 4) does not obscure the critical point that both DNA preparations possess essentially identical amounts of repeated and nonrepeated sequence DNA. As demonstrated below, this would not be expected if nuclear DNA content were doubled by amplification of the nucleotide sequences of a subfraction of the genome. The reassociation kinetics of these DNA preparations compare favorably with those obtained by others for rat DNA using similar methods [21,22].
............... ::::::::::::::::::::: 20-
o
"'",. "":";':'".C ~'.. %. ~-.o.
•
~
~ ' o. . ~ .
".G 3 0 . o_
~)~oZSO-
~-.....;.....
. . ~ ~., "-. "~.. ~ "... "..
U 60C
~
"""...~
70"
n~ 80-
,;-'
,b°
,b'co t ,;'
~?~
~b"
Fig. 4. R e a s s o c i a t i o n k i n e t i c s of d i p l o i d D N A a n d p o l y p l o i d D N A m e a s u r e d b y o p t i c a l a b s o r b a n c e . O p e n circles r e p r e s e n t t h e r e a s s o c i a t i o n o f d i p l o i d k i d n e y D N A ; c l o s e d circles r e p r e s e n t t h e r e a s s o c i a t i o n o f p o l y p l o i d h e p a t o c y t e DNA. F o r d e t a i l s o f r e a c t i o n c o n d i t i o n s see Materials a n d M e t h o d s . T h e s c a t t e r o f t h e s e d a t a is p r i m a r i l y d u e to 2 6 0 n m - a b s o r b i n g m a t e r i a l w h i c h c o - e l u t e s w i t h D N A f~om h y d r o x y a p a t i t e c o l u m n s . T h e c o l u m n s c a n n o t b e w a s h e d free o f t h i s m a t e r i a l n o r c a n its relative c o n t r i b u t i o n t o 2 6 0 n m a b s o r b a n c e b e a c c u r a t e l y q u a n t i t a t e d a n d p r e s u m a b l y it results f r o m p r o g r e s s i v e p a r t i a l d i s s o l u t i o n o f t h e h y d r o x y a p a t i t e crystals. The solid line is f i t t e d to the d a t a w h i l e t h e d o t t e d lines r e p r e s e n t h y p o t h e t i c a l r e a s s o c i a t i o n k i n e t i c s of D N A f r o m 4c n u c l e i r e s u l t i n g f r o m n u c l e o t i d e s e q u e n c e a m p l i f i c a t i o n (see R e s u l t s and T a b l e II).
26 More critical comparison of the kinetics of reassociation of both DNA preparations was made as follows. Both sets of data were divided into three kinetic components; the first, comprising 15% of the DNA, has essentially completed reassociation at Cot 1" 10 -1 and was not scored in these experiments; the second comprises approximately 20% of the DNA and reassociates primarily between Cot values 1 • 10 -1 and 1 X 102; and the third, the nonrepeated component, com.prises approximately 65% of the DNA and reassociates primarily after Cot 1 × 102. While it is acknowledged that there is probably more than one kinetic c o m p o n e n t reassociating between Cot 10 -1 and 102, they cannot be resolved by these data. Therefore, the simplest assumption was made for the purpose of estimation and comparison of the kinetics of reassociation of these two DNA preparations. Estimation of the reassociation rate constant (k) for the second and third components was made by the m e t h o d of Bishop [23] by considering each as an independent second order reaction. The similarity between the values obtained for the diploid DNA preparations is the critical element in these estimations rather than the absolute values obtained. The estimates obtained for the reassociation rate constants of the 2nd and 3rd diploid components are, respectively, 0.236 and 0.00075 while those for the polyploid DNA components are 0.253 and 0.00041. These values axe in excellent agreem e n t and strengthen the conclusion that these two DNA preparations are identical in terms of reassociation kinetics. The solid line in Fig. 4 is the curve obtained by taking the average of the diploid and polyploid values for k of each c o m p o n e n t and plotting the sum of the fractional contribution of the two second order reactions proceeding from a 15% level of reassociation at Cot 1 • 10 -1. The values for k and c o m p o n e n t size used to compute this line are tabulated in Table II. Prediction, based on the solid line in Fig. 4, of the expected kinetics of reas-
T A B L E II CONSTANTS AND METHODS FOR CALCULATION
OF CURVES IN FIGURE 4
All d e s i g n a t i o n s a n d c a l c u l a t i o n p r o c e s s e s are as d e s c r i b e d in t h e l e g e n d t o T a b e l I e x c e p t : ( 1 ) S o l u t i o n t o t h e s o l i d line w a s b a s e d o n a t h r e e - c o m p o n e n t c u r v e w i t h e s t i m a t e s f o r k as d e s c r i b e d in R e s u l t s . E s t i m a t i o n o f k I w a s n o t p o s s i b l e b e c a u s e t h e k i n e t i c s o f r e a s s o e i a t i o n o f t h i s c o m p o n e n t w a s n o t o b s e r v e d in t h e e x p e r i m e n t d e p i c t e d in Fig. 4. T h e v a l u e P I r e p r e s e n t s a r e a s o n a b l e e s t i m a t e o f t h e size o f t h e f i r s t c o m p o n e n t ( e s s e n t i a l l y c o m p l e t e r e a s s o c i a t i o n at Cot 10 -1). ( 2 ) H e r e , o b v i o u s l y , c a l c u l a t i o n s f o r c o m p l e m e n t a r y l a b e l e d D N A a r e n o t n e c e s s a r y b e c a u s e e a c h D N A p r e p a r a t i o n is r e a s s o c i a t i n g o n l y w i t h i t s e l f . (3) R e p e a t e d s e q u e n c e a m p l i f i c a t i o n ( t o a n y c o p y f r e q u e n c y ) o r a m p l i f i c a t i o n o f n o n r e p e a t e d D N A m o r e t h a n 1 0 0 - f o l d w o u l d r e s u l t in a p p r o x i m a t e l y 6 7 . 5 % o f t h e 4c D N A w i t h a r e a s s o c i a t i o n r a t e c o n s t a n t ( k ) l a r g e r t h a n 0 . 0 2 5 . T h i s w o u l d r e s u l t in a c u r v e e s s e n t i a l l y s i m i l a r t o " a " in Fig. 4 excel~t t h a t t h e m a j o r t r a n s i t i o n w o u l d be d i s p l a c e d t o l o w e r Cot v a l u e s . Case
k' ;P'
ka;P a
k 1 ;PI
k2 ;P2
k3 ;P3
Solid line
--
--
(--); 1 5 , 0
0 . 2 4 ~ 5 0 ; 20
0.000527; 65.00
A
0.005;
0.02500; 50.5
(--);
7,5
0 . 1 2 2 2 5 ; 10
0.000264; 32.0
B
0.0005; 11.10
0.00250; 55.0
(--);
7,5
0 . 1 2 2 2 5 ; 10
0.000264; 27.5
C
0.0005; 50.00
0.00075; 75.0
(--);
7,5
0.1225;10
0.000264;
1.01
7.5
R (67.5% DNA with k ~ 0.025)
0.000264; 32.5
27 sociation of hypothetical 4c nuclear DNA resulting, as in the previous section, from 100-fold, 10-fold and 3-fold amplification of nonrepeated DNA is shown by the d o t t e d lines in Fig. 4 labeled a, b and c, respectively. The m e t h o d and constants used for these computations are given in Table II. Here a dramatic effect is expected if 4c nuclei result from nucleotide sequence amplification. In each instance the apparent demarcation between repeated and nonrepeated sequence DNA reassociation would be significantly altered. Each of the hypothetical curves lies far outside the experimental points over at least two decades of Cot values. Amplification of either repeated sequence DNA (to any copy frequency) or of nonrepeated sequence DNA to greater than 100-fold would be expected to result in a curve essentially similar to " a " b u t with greater displacement of the major transition to lower Cot values. These data, and the data presented in Fig. 2 demonstrate no differences in the reassociation kinetics of polyploid hepatocyte DNA, as compared to diploid kidney DNA, which could be attributed to the presence of 50%, or more, amplified sequence DNA. The direct conclusion of these experiments is, therefore, that the increased DNA content of polyploid hepatocytes is due to the presence of multiple genomes and n o t to the amplification of the nucleotide sequences of a subfraction of the genome. Discussion
In the experiments reported here, kinetic analysis of DNA reassociation is used to determine whether the DNA content increase of polyploid hepatocytes is attributable to replication of the genome or to amplification of the nucleotide sequences of only a subfraction of the genome. In the latter case, b u t n o t the former, the reassociation kinetics of DNA from polyploid nuclei would be expected to differ from that of diploid DNA owing to alteration in the relative concentration of the nucleotide sequences. In t w o experiments the kinetics of polyploid DNA reassociation is identical to that of diploid DNA, within the resolution of these methods. The straightforward conclusion therefore is that hepatocyte polyploidization is a result of complete replication of the genome. An essential question, however, is whether failure to detect differences is due, in fact, to kinetic identity or to lack of sensitivity of the methods employed. A definitive answer to this question is n o t possible because it is known that these methods cannot detect kinetic differences if these differences reside in relatively small amounts of the total DNA [7]. For example, changes in the relative number of nucleotide sequence copies per nucleus would certainly go undetected unless these changes detectably affect total nuclear DNA content. Thus these experiments are only intended to detect amplification of limited subfractions of the genome which could cause a doubling of nuclear DNA content. The question of sensitivity is therefore addressed only for those hypothetical cases in which DNA content is doubled by the amplification of the nucleotide sequences of a subfraction of the genome. To double nuclear DNA content by amplification small subfractions of the genome must be amplified to a greater extent than larger subfractions. The relationship between fraction size and extent of amplification are shown in Fig. 3 and permit computation of the expected kinetics of reassociation of DNA
28 from hypothetical nuclei which have doubled DNA content as a result of sequence amplification. Computation of the expected kinetics of reassociation made for four hypothetical cases effectively cover all possible mechanisms of doubling nuclear DNA content by either; (1) amplification limited to subfractions of repeated sequence DNA; or (2) amplification limited to subfractions of nonrepeated DNA in which all amplified sequences are replicated the same n u m b e r of times (homogeneous amplification). For these cases the kinetics with which the hypothetical DNA would reassociate with itself (Fig. 4), or drive the reassociation of labeled DNA (Fig. 2) is significantly different from the experimental observations. Thus, the experimental evidence rules out a wide variety of possible mechanisms of doubling nuclear DNA content by nucleotide sequence amplification. Sequence amplification, if it existed in polyploid hepatocytes, would not necessarily be either homogeneous or limited to the repeated or nonrepeated fractions of the genome. These limitations were imposed because they permit reasonable calculations and are representative of a wide variety of possible cases. More complex schemes for doubling nuclear DNA content by sequence amplification would be expected to produce similar effects on the reassociation kinetics and most conceivable schemes would be detectable by these methods. It would, however, be unrealistic n o t to assume that at some level of complexity of sequence amplification the resulting kinetics would be indistinguishable, by these methods, from that of normal diploid DNA. For example, if 80% of the genome were replicated 2-fold, 10% not replicated at all, and the remaining 10% replicated 4-fold, then the kinetics of reassociation of this hypothetical 4c DNA would probably be indistinguishable from that of normal diploid DNA. To define, however, the point at which resolution is lost between this extreme and the hypothetical cases shown in Fig. 3 and 5 would require extremely complex, and perhaps impossible, calculations. The lower limit of resolution of these kinds of experiments cannot therefore be adequately defined. Possibilities of this kind, however, go b e y o n d the limit of the intended goal of these experiments. Clearly the reassociation kinetics of polyploid hepat o c y t e DNA shows no evidence that amplification of specific subfractions of the genome is responsible for the increased DNA content of these cells. In conclusion, kinetic analysis of DNA reassociation indicates that the increased DNA content of polyploid hepatocytes is attributable to the presence of multiple genomes and not to nucleotide sequence amplification. The concept of ploidy, typically defined in terms of karyotypic and/or DNA content multiples, is therefore corroborated, at the nucleotide sequence level of DNA, by these experiments.
Acknowledgments I would like especially to thank Dr. Paul K. Horan of the University of Rochester Department of Pathology, for generously performing the flow microfluorometric measurements and the Los Alamos Scientific Laboratories for the use of their equipment for these measurements. This work was supported by Training Grants (GM-01981-04, GM-01981-05, PE-00543-03) from the National Institutes of Health.
29
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