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Characterization of the nuclear genome of pearl millet
192
Biochimica et Biophysica Acta, 562 (1979) 192--206 © Elsevier/North-Holland Biomedical Press
BBA 99415
C H A R A C T E R I Z A T I O N OF THE N U C L E A R GENOME OF P E A R L MILLET
CHARLES F. WIMPEE and JAMES R.Y. RAWSON *
Departments of Botany and Biochemistry, University of Georgia, Athens, GA 30602 (U.S.A.) (Received August 21st, 1978)
Key words: Nuclear genome; Angiosperm DNA, (Pearl millet)
Summary The nuclear genome of pearl millet has been characterized with respect to its size, b u o y a n t density in CsC1 equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the b u o y a n t density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high T m values implying a high degree of sequence homogeneity. A b o u t half of the single copy DNA is short (750--1400 nucleotide pairs) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.
Introduction The organization of the nuclear genome in angiosperms has been generally characterized as repeated DNA sequences 300--400 nucleotide pairs in length interspersed with single copy DNA sequences 8 0 0 - - 1 5 0 0 nucleotide pairs long [1--5]. This pattern of DNA organization, short period interspersion, was first described in Xenopus [6] and has been found to be the most c o m m o n pattern of genome organization among eukaryotes. The only unusual aspect of DNA * T o w h o m reprint requests should be sent. Abbreviation: Pipes, piperazine-N,N'-bis(2-ethane
sulfonic acid).
193 organization in angiosperms which has emerged thus far is the high percentage of repeated DNA present {50--80%) when compared with animal genomes. A striking exception to the 'Xenopus pattern' has been found in Drosophila [7,8], honeybee [9] and the water mold Achyla [10]. In the 'Drosophila pattern', single copy DNA sequences extend for as long as 10 000 nucleotide pairs without interruption by repeated sequences. The average repeated sequence length in Drosophila is 5600 nucleotide pairs with a small fraction (0.10) less than 500 nucleotide pairs. This particular report is intended to characterize the nuclear genome of the dilpoid monocot pearl millet, Pennisetum americanum (L.) K. Schum. A considerable amount of cytogenetic information on this plant has been tabulated. It normally contains 14 chromosomes although supernumerary chromosomes have been reported [11,12]. We have characterized the nuclear DNA from millet with respect to its buoyant density in CsC1, melting temperature, reassociation kinetics and sequence organization. Materials and Methods
Biological material. Seeds and pollen from a dwarf strain of pearl millet (Tift 23 DB) were obtained from Dr. Wayne W. Hanna of the U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Coastal Plain Station at Tifton, GA. Isolation of DNA. Seeds (200 g) were imbibed in water with constant aeration for 1 h. They were drained, rinsed, and placed in a grinding buffer (600 ml) consisting of 50 mM NaC1, 10 mM Tris-HC1 (pH 8.0), and 10 mM EDTA. The seeds were homogenized for 1 min with a polytron. The homogenate was adjusted to 1/10 vol. 1-butanol, 6% (w/v) p-aminosalicylic acid, plus 1/10 vol. 10% (w/v) tri-iso-propylnaphthalenesulfonate and shaken on a rotary shaker for 20 min. The slurry was extracted for 20 min with an equal volume of chloroform/isoamylalcohol (24 : 1; v/v) and centrifuged at 8000 rev./min for 20 min to separate the phases. The nucleic acids were precipitated at 4°C by adjusting the solution to 0.4 m NaC104 and adding 2 vols. of ethanol. The nucleic acids were pelleted at 3000 rev./min and suspended in 100 ml 0.15 M NaC1/15 mM sodium citrate and plus 1.0 mM EDTA. RNA was digested with 5 mg of pancreatic RNAase and 5000 units of T1 RNAase (both previously heated at 90°C for 10 min in 0.15 M NaC1) for 1 h at 37°C. The mixture was extracted for 15 min with 2 vol. of a phenol mixture containing creseol (10%, v/v), 8-hydroxyquinoline (0.1%, w/v) and saturated with 0.15 M NaC1/15 mM sodium citrate and centrifuged at 9000 rev./min for 20 min. The aqueous phase was extracted with an equal volume of chloroform/isoamylalcohol and centrifuged at 8000 rev./min for 20 min. The DNA was precipitated two times from the aqueous phase with 1/10 vol. of 3 M sodium acetate and 2 vols. of ethanol, and then with 1/10 vol. of 3 M sodium acetate and 0.54 vol. of isopropanol. The DNA was finally purified by banding in CsC1 equilibrium density gradients. The DNA from the CsC1 gradients was dialyzed extensively against 0.15 M NaC1/15 mM sodium citrate and precipitated with 1/10 vol. of 3 M sodium acetate and 2 vols. of ethanol. The purified DNA was suspended in 0.15 M NaCl/15 mM sodium citrate plus 0.1 mM EDTA, and stored at --20°C. 200 g of
194 seeds yielded 20--25 mg of purified DNA. The double, and single-stranded molecular weights of the DNA averaged 1.0 • 10 ~ and 3.0 • 106, respectively. Analytical CsCl equilibrium density gradients. The b u o y a n t density of the DNA was determined by CsC1 equilibrium density gradient centrifugation [ 13 ], using SP01 viral DNA (p = 1.742 g/cc) as a density marker. The samples were centrifuged at 44 000 rev./min for 20 h at 25°C in a Beckman Model E analytical ultracentrifuge equipped with ultraviolet optics. Photographs were scanned at 500 nm in a Gilford spectrophotometer, and measurements were made from the tracings. The mol percent G + C content was calculated from the b u o y a n t density of the DNA according to Schildkraut et al. [14]. Determination of "cellular DNA content from millet pollen. Pollen grains were suspended in water and counted under the light microscope using a hemacytometer. DNA was extracted from the pollen by one of t w o procedures. (1) The pollen was centrifuged at 3000 rev./min and suspended in 0.935 N HC104. The grains were broken using a French press (1500 lb/inch 2) and particulate matter removed from the homogenate by low speed centrifugation. The supernatant was heated at 73°C for 20 min and centrifuged at 15 000 rev./min for 15 min to remove cell debris. ( 2 ) T h e pollen was suspended in water and broken in the French press. The homogenate was made 10% (w/v) trichloroacetic acid and allowed to stand at 4°C for 30 min. The precipitate was collected b y centrifugation at 10 000 rev./min and washed extensively with an ether/ethanol (v/v, 3 : 1) mixture. The pellet was suspended in 0.935 N HC104. The DNA concentration in the HC10, solution was determined by the diphenylamine procedure of Burton [15], using salmon sperm DNA or Escherichia coli DNA as a standard. The DNA content/haploid nucleus was determined b y assuming three nuclei/pollen grain (Hanna, W., personal communication). Shearing and sizing of DNA. DNA was sheared by sonication in 1.0 M NaC1 at 4°C. Different fragment sizes were obtained by varying the sonication time. Single-stranded fragments lengths were determined by alkaline band sedimentation at 20°C in the Beckman Model E analytical ultracentrifuge [16]. Observed sedimentation coefficients were corrected for solvent density and viscosity and converted to molecular weights using the equation of Studier [16]. Band sedimentation in 1 M NaC1 was used to calculated the observed sedimentation coefficient of double-stranded DNA. The double-stranded fragment length was 0 calculated from the S20,w using Freifelder's equation [17]. DNA reassociation. DNA samples, ranging in concentration from 30 pg/ml to 835 pg/ml in either 0.12 M or 0.48 M sodium phosphate (pH 6.8), were sealed in pasteur pipettes and denatured by boiling for 5 min. Samples in 0.12 M sodium phosphate (pH 6.8) were reassociated at 60°C, while those in 0.48 M sodium phosphate (pH 6.8) were reassociated at 73°C and corrected to the equivalent Cot (M nucleotide-s) according to Britten et al. [18]. After reassociation to the appropriate Cot, the samples were quick frozen in solid CO2/ ethanol. Reassociated DNA was separated from single-stranded DNA by h y d r o x y a p a t i t e chromatography [18--20]. DNA samples reassociated in 0.12 M sodium phosphate (pH 6.8) were thawed and loaded directly onto a h y d r o x y a p a t i t e column. Samples reassociated in 0.48 M sodium phosphate (pH 6.8) were diluted to 0.12 M sodium phosphate (pH 6.8) before loading
195 onto the column. Single-stranded DNA was eluted with 0.12 M sodium phosphate (pH 6.8). Double-stranded DNA was eluted with either 0.48 M sodium phosphate (pH 6.8), or by raising the column temperature to 100°C and eluting the denatured fragments with 0.12 M sodium phosphate (pH 6.8). The recovery from the hydroxyapatite columns was greater than 95%. The relative amounts of single- and double-stranded DNA were determined by absorbance at 260 nm in a Beckman Acta MVI spectrophotometer, assuming 1 absorbance unit (A260) is equal to 50 pg DNA/ml. Absorbance at 320 nm was subtracted from that at 260 nm to correct for light scatter. Thermal denaturation o f DNA. DNA samples ranging in concentration from 15 gg/ml to 50 pg/ml were adjusted to 0.12 M sodium phosphate (pH 6.8) by dialysis and melted in a Beckman Acta MVi double-beam spectrophotometer equipped with a jacketed cuvette holder, automatic sample changer, and a platinum temperature probe. For each melt, a reference cuvette containing 0.12 M sodium phosphate (pH 6.8) was included to ensure a linear base line. The temperature was raised quickly to 60°C, then increased at a rate of 0.3-0.5 degrees/min up to 100°C. Separation of strands was monitored by the increase in absorbance at 260 nm. The absorbance was corrected for thermal expansion of water [13]. The hyperchromicity was calculated at the fraction of denatured absorbance between 60°C and 100°C. The Tm was that temperature which produced 50% increase in hyperchromicity. The mol percent G + C content of native DNA was calculated using the equation of Schildkraut and Lifson [21]. $1 nuclease digestion o f reassociated DNA. DNA with a single-stranded length of 8800 nucleotides was dialyzed against 0.18 M NaC1 plus 0.01 M Pipes (piperazine-N-N'-bis(2-ethanesulfonic acid) buffer, pH 6.8. The DNA samples were sealed in glass vials denatured by boiling for 5 min, and reassociated at 60°C to a Cot sufficient to allow only repeated DNA to reassociate (Cot 10). 0.2 ml of each sample was fractionated on hydroxyapatite to recover those molecules containing duplex structures. The double-stranded fraction was dialyzed against 0.12 M sodium phosphate (pH 6.8) and used for thermal denaturation analysis. The remainder of the sample was adjusted to 25 mM sodium acetate (pH 4.5), 0.5 mM ZnSO4, 25 mM 2-mercaptoethanol, and 40 000 units of S1 nuclease (Miles Laboratories, Inc.)/200 ~g of DNA. The samples were incubated for 1 h at 37°C. The S1 reaction was terminated by adding an equal volume of 0.12 M sodium phosphate (pH 6.8) and cooling the samples to 4°C. If radioactive single-stranded E. coli DNA is mixed with reassociated repetitive DNA and then digested with S1 nuclease, more than 95% of the single-stranded radioactive DNA is digested. S1 nuclease-resistant duplexes were separated from digestion products by hydroxyapatite chromatography. Approximately 25 ug of the Sl-resistant DNA was removed from each sample and dialyzed against 0.12 M sodium phosphate (pH 6.8) for melting analysis. The remainder was concentrated to a small volume (0.5--1.0 ml) with 1-butanol [22], dialyzed extensively against 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA and sized on Agarose columns. Sizing o f Sl-resistant reassociated duplexes. The size distribution of Sl-resistant duplexes was determined by gel filtration on an Agarose A50 (BioRad) column previously calibrated with DNA of known duplex lengths. The gel
196 matrix was poured around 6-mm glassbeads in a 92 cm × 1.5 cm column. Samples were chromatographed in 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA, and the DNA content of the column fractions was determined by absorbance at 260 nm. DNA in the excluded volume (greater than 1500 base pairs) was concentrated with 1-butanol and dialyzed against 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA. Single- and double-stranded molecular weights of the excluded fraction were determined by band sedimentation in the analytical ultracentrifuge as described above. Results
Mol percent G + C o f millet DNA Millet DNA banded in analytical CsC1 equilibrium density gradients as a homogenous c o m p o n e n t with a b u o y a n t density of 1.704 g/cc, corresponding to a mol percent G + C of 44.9%. Increasing the DNA c o n t e n t in the analytical CsC1 gradients did n o t reveal any satellite components. Millet DNA melts in 0.12 M sodium phosphate (pH 6.8) with a Tm of 89.5°C and has a hyperchromicity of 28.5%. The G + C c o n t e n t of the DNA calculated from the Tm is 49.7% or 4.8% greater than that calculated from the b u o y a n t density of millet DNA. The discrepancy between these two values may be accounted for by the presence of 5-methylcytosine, which lowers the b u o y a n t density of DNA [23]. Haploid DNA content o f millet Pollen, the immature male gametophyte of higher plants, is a convenient source for determining the DNA c o n t e n t / N chromosomes. The nuclei in pollen are haploid and contain two or three nuclei/pollen (depending upon species). A single pollen grain from millet contains 0.66 pg of DNA and three nuclei. Therefore, the genomic complexity of millet is 0.22 pg or 2.0- 108 nucleotide pairs/N chromosomes. Reassociation kinetics of millet DNA Fig. l a shows the reassociation kinetics of short (300-nucleotide) fragments of millet DNA. The curve drawn through the points is determined by a nonlinear least squares regression of the data assuming second order kinetics and allowing all the parameters to free float [24]. Analysis of the data in this fashion revealed two kinetic components. The lower curves (dashed lines) represent the predicted reassociation.kinetics of the individual components if t h e y existed alone. The observed rate constant for the slowest reassociating c o m p o n e n t (single copy DNA) is 0.0029 M -1 • s -1. The root-mean-square of this fit was 0.0241. A better fit of the data was obtained by holding constant during the analysis the observed rate constant expected for the single copy DNA in an organism with a genome complexity of 0.22 pg or 2.0 • 108 nucleotide pairs. Table I summarizes the computer analysis of the reassociation kinetics calculated in this fashion. The root-mean-square for this fit is 0.0248, only slightly higher than that for the three-float analysis. There are three distinct kinetic corn-
197
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Fig. 1. R e a s s o c i a t i o n k i n e t i c s of s h o r t vs. long f r a g m e n t s of m i l l e t D N A . (a) R e a s s o c i a t i o n k i n e t i c s o f 3 0 0 n u c l e o t i d e long f r a g m e n t s o f m i l l e t D N A . D N A was s h e a r e d b y s o n i c a t i o n t o a n average s i n g l e - s t r a n d e d l e n g t h o f 3 0 0 n u c l e o t i d e s a n d a d j u s t e d to e i t h e r 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) or 0 . 4 8 M s o d i u m p h o s p h a t e ( p H 6.8) a n d d e n a t u r e d b y boiling for 5 rain. S a m p l e s in 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) w e r e r e a s s o c i a t e d a t 6 0 ° C , while t h o s e in 0 . 4 8 M s o d i u m p h o s p h a t e ( p H 6.8) w e r e r e a s s o c i a t e d at 7 3 ° C a n d c o r r e c t e d t o e q u i v a l e n t Cot ( E C 0 t ) a c c o r d i n g to B r i t t e n e t al. [ 1 8 ] . R e a s s o c i a t e d D N A w a s fract i o n a t e d b y h y d r o x y a p a t i t e ( H A P ) c h r o m a t o g r a p h y a n d the relative a m o u n t s of d o u b l e - a n d singles t r a n d e d D N A was d e t e r m i n e d b y a b s o r b a n c e at 2 6 0 n m . T h e c u r v e d r a w n t h r o u g h t the p o i n t s r e p r e s e n t s a least s q u a r e s fit for t w o c o m p o n e n t s , a s s u m i n g s e c o n d o r d e r k i n e t i c s a n d a l l o w i n g all t h e p a r a m e t e r s t o free f l o a t ( r o o t - m e a n - s q u a r e , 0 . 2 5 6 ) . T h e l o w e r c u r v e s ( d a s h e d lines) r e p r e s e n t t h e p r e d i c t e d r e a s s o c i a t i o n kinetics of t h e p u r e c o m p o n e n t s . (b) R e a s s o c i a t i o n kinetics of 8 6 0 0 - n u c l e o t i d e long f r a g m e n t s of m i l l e t D N A . D N A w i t h a n a v e r a g e s i n g l e - s t r a n d e d l e n g t h of 8 6 0 0 n u c l e o t i d e s w a s a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) , d e n a t u r e d b y boiling for 5 m i n , a n d r e a s s o c i a t e d at 6 0 ° C . R e a s s o c i a t e d D N A was f r a c t i o n a t e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y . Material c o n t a i n i n g d u p l e x s t r u c t u r e s w a s t h e r m a l l y e l u t e d f r o m the h y d r o x y a p a t i t e c o l u m n s . T h e relative a m o u n t s o f d o u b l e - a n d s i n g l e - s t r a n d e d D N A w a s d e t e r m i n e d b y a b s o r b a n c e a t 2 6 0 n m . T h e line t h r o u g h the p o i n t s r e p r e s e n t s a least s q u a r e s fit for t w o c o m p o n e n t s allowing all p a r a m e t e r s to free f l o a t ( r o o t - m e a n - s q u a r e , 0 . 0 2 8 5 ) . T h e l o w e r c u r v e s ( d a s h e d lines) 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 k i n e t i c s of t h e p u r e c o m p o n e n t s .
ponents of DNA in the millet genome. A large fraction (0.226) of the genome reassociates too rapidly to resolve with the techniques used in this investigation (Cot < 0.01). This c o m p o n e n t may consist of one or more classes of highly repeated DNA and/or foldback sequences. A moderately repetitive component, comprising 46.7% of the genome, reassociates with an observed second order rate constant of 1.637 M -~ • s -1. A slow c o m p o n e n t (16.7% of the genome), representing single copy sequences, reassociates with an observed second order rate constant of 0.0055 M -1 • s -~. 14% of the DNA failed to reassociate and may be due to degradation or failure of some sequences to form stable duplexes at the criterion used for these experiments. Knowing the kinetic and chemical complexity of the various repetitive components permits the calculation of the reiteration frequency of these fractions. This is done by dividing the chemical complexity of each c o m p o n e n t by its kinetic complexity. Since the second order rate constant of the fastest kinetic c o m p o n e n t and the a m o u n t of zero time binding have n o t been determined, a reiteration frequency cannot be calculated for that fraction of DNA reassociating at Cot values of less than 0.01. The average reiteration frequency of the middle repetitive fraction is 298 copies.
OF MILLET DNA: 300 NUCLEOTIDES
LONG
Fraction of DNA
0.226 0.467 0.167
Component
Rapidly renaturing Middle repetitive Single copy
by the kinetic complexity.
-
-1.637 0.0055
Observed k (M -1 • s -1 )
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-3.13 - 105 3.33 • 107
Kinetic complexity (nucleotide pairs)
4.52 • 107 9.34 - 10 3.34 - 107
Chemical complexity (nucleotide pairs)
-298 1
Reiteration frequency
rate constant for the single copy component for that expected in an organism with a genome complexity of 0.22 pg (2.0 • 108 nucleotide pairs). The root-means q u a r e f o r t h i s f i t is 0 . 0 2 5 6 . F r a c t i o n o f D N A u n r e a s s o c i a t e d , 0 . 1 4 0 . P u r e k = O b s e r v e d k / f r a c t i o n o f D N A . T h e k i n e t i c c o m p l e x i t y is c a l c u l a t e d r e l a t i v e t o t h e g e n o m e c o m p l e x i t y o f E. coli ( 4 . 2 4 1 0 6 n u c l e o t i d e p a i r s ) a n d t h e s e c o n d o r d e r r a t e c o n s t a n t o f E. coli D N A i n o u r l a b ( 0 . 2 5 9 M - 1 - s -1 ). T h e c h e m i c a l c o m p l e x i t y is t h e f r a c t i o n o f D N A t i m e s t h e t o t a l g e n o m e c o m p l e x i t y ( 0 . 2 2 p g o r 2 . 0 • 1 0 8 n u c l e o t i d e p a i r s ) . T h e r e i t e r a t i o n f r e q u e n c y is t h e c h e m i c a l c o m p l e x i t y d i v i d e d
DNA, 300 nucleotides long, was reassociated. The data were analyzed by a non-linear least squares regression assuming second order kinetics and fixing the observed
KINETIC COMPONENTS
TABLE I
~D Go
199
Interspersion of kinetic components The presence or absence of interspersion of repetitive and single c o p y sequences can be determined by comparing the hydroxyapatite binding of reassociated short and long fragments of DNA [6--9,25,26]. Since hydroxyapatite will bind DNA which contains small regions of double-stranded DNA, the observed renaturation rate of DNA will be equal to that of the least complex class of nucleotide sequences contained in a given DNA fragment. If there is no interspersion of the various kinetic components, then the observed rate constants of the individual components will only vary as a function of the square root of the ratio of the molecular weights of the t w o DNA samples being compared; kl/k2 = (LI/L2) °'5, where k~ and k2 are the rate constants for reassociation of short (L1) and long (L2) fragments, respectively [ 27]. Fig. l b shows the reassociation kinetics of fragments of millet DNA 8600 nucleotides long. The data were analyzed in two different ways. Assuming second order kinetics the computer program was: (1) allowed to find the best fit by allowing all parameters to free float (root-mean-square, 0.0285) and (2) the observed rate constants for the two components were fixed as defined by the expression ks6oo = k3oo (Ls6oo/L3oo) °'5, where the subscripts indicate the length of the fragments being compared and k300 is the observed rate constant for the t w o c o m p o n e n t s given in Table I (root-mean-square, 0.0290). Table II summarizes these two approaches for analyses of the reassociation kinetics of DNA 8600 nucleotides in length. When comparing the reassociation kinetics of DNA 300 or 8600 nucleotides long there is a strikingly different fraction of DNA which appears to have reassociated at Cot 0.01. 22% of the DNA 300 nucleotides long and 39% of the DNA 8600 nucleotides long are retained on hydroxyapatite at this Cot. This indicates that both highly repetitive and/or foldback sequences (i.e. sequences which reassociate prior to Cot 0.01) and middle repetitive sequences are contained on the same DNA fragments 8600 nucleotides long. On the other hand the reassociation of the long middle repetitive sequences does n o t appear to
T A B L E II K I N E T I C COMPONENTS OF M I L L E T DNA: 8600 N U C L E O T I D E S L O N G D N A , 8 6 0 0 n u c l e o t i d e s long, w a s r e a s s o c i a t e d . T h e d a t a w e r e a n a l y z e d b y a n o n - l i n e a r l e a s t s q u a r e s regression assuming s e c o n d o r d e r kinetics. First, all p a r a m e t e r s w e r e a l l o w e d t o free float until the l o w e s t r o o t m e a n ~ q u a r e ( 0 . 0 2 8 5 ) w a s f o u n d . N e x t , the observed rate c o n s t a n t s f o r the t w o c o m p o n e n t s w e r e f i x e d as d e f i n e d b y the e x p r e s s i o n w h e r e the s u b s c r i p t s i n d i c a t e the length o f the fragm e n t s being c o m p a r e d and k 3 0 0 is the observed rate c o n s t a n t for the t w o c o m p o n e n t s observed w h e n the D N A is 3 0 0 n u c l e o t i d e s l o n g given in T a b l e I. T h e r o o t - m e a n ~ q u a r e o f this analysis o f 0 . 0 2 9 0 .
(L8600/L300)O.5h300,
Component
Rapidly renaturing Middle r e p e t i t i v e Single c o p y
Free float
F i x observed rate c o n s t a n t
Fraction of DNA *
Observed k (M -1 • s - 1 )
Fraction o f D N A **
Observed k (M -1 • s - l )
0.397 0.199 0.122
~ 10.13 0.0136
0.386 0.185 0.102
-8.80 0.0295
* Fraction DNA unreassociated, 0.076. ** F r a c t i o n D N A u n x e a s s o c i a t e d , 0 . 0 9 9 .
200
significantly alter the renaturation of the single copy sequences, indicating the lack of interspersion of middle repetitive and single c o p y sequences on fragments 8600 nucleotides long.
Average length of single copy sequences Interspersion of repetitive and single copy sequences will cause a certain amount of single copy DNA to be bound to hydroxyapatite at l o w Cot values. Thus, at a Cot at which only repeated sequences are fully reassociated, the amount of unreassociated single copy DNA existing as single-stranded tails on renatured duplexes will increase as fragment length increases. This will be true to a fragment length which is equal to the average distance between repeated sequences. Therefore, an estimate of single copy sequences length can be made by plotting the amount of DNA bound to hydroxyapatite as a function of fragment length [6,26]. From Fig. la, it can be seen that all of the repetitive sequences are fully reassociated by a Cot of 10. Fig. 2 shows the hydroxyapatite binding of millet DNA sheared to various lengths and reassociated to Cot 10. This particular
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T E M P E R A T U R E (°C)
Fig. 2. T h e f r a c t i o n o f m i l l e t D N A c o n t a i n i n g r e p e a t e d s e q u e n c e s , a s a f u n c t i o n o f f r a g m e n t l e n g t h . D N A w a s s h e a r e d t o v a r i o u s f r a g m e n t l e n g t h s a n d a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e (pI-[ 6 . 8 ) . T h e samples w e r e d e n a t u r e d b y b o i l i n g f o r 5 m i n a n d r e a s s o c i a t e d a t 6 0 ° C t o Cot 1 0 . T h e r e a s s o c i a t e d s a m p l e s w e r e fractionated by hydroxyapatite (HAP) chromatography a n d t h e r e l a t i v e a m o u n t s o f d o u b l e - a n d singlestranded D N A w a s d e t e r m i n e d b y a b s o r b a n c e a t 2 6 0 n m . T h e f r a c t i o n o f l o n g f r a g m e n t s u n a b l e t o f o r m s t a b l e d u p l e x e s w a s c o r r e c t e d f o r in estimating t h e f r a c t i o n o f D N A b o u n d t o h y d r o x y a p a t i t e . T h e lines o n t h e g r a p h a r e a a r e s u l t o f linear regression analysis o f t h e d a t a , a n d r e p r e s e n t t w o p o s s i b i l i t i e s f o r a family of curves which could be drawn through the points.
Fig. 3. T h e r m a l d e n a t u r a t i o n p r o f i l e s o f m i l l e t D N A o f various lengths r e a s s o c i a t e d t o Cot 1 0 , c o m p a r e d t o t h a t o f native D N A . D N A samples with single-stranded lengths o f 4 3 0 , 1 4 0 0 , 3 4 5 0 , a n d 7 2 0 0 nucleotides w e r e a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) , d e n a t u r e d b y b o i l i n g f o r 5 r a i n a n d r e a s s o a t 6 0 ° C t o Cot 1 0 . T h e s a m p l e s w e r e f r a c t i o n a t e d b y h y d r o x y a p a t i t e chromatograph),, and the doubles t ~ ' a n d e d f r a c t i o n s w e r e d i a l y z e d a g a i n s t 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) a n d m e l t e d in t h e s p e c t r o photometer.
201
experiment is n o t as informative as desired, because the DNA b o u n d to hydroxyapatite due to foldback sequences has n o t been subtracted. Therefore, we are limited in the extent of the interpretation of these data. The steep rise in the early par of the curve and the change in slope in the range of fragments 750--1400 nucleotides long suggest the presence of some single c o p y DNA of this length interspersed with more rapidly renaturing DNA. The more gradual increase in slope after 1400 nucleotides indicates the presence of a spectrum of single c o p y sequences up to at least 8600 nucleotides long. Extrapolation of the initial part of the curve to the X-axis [26] gives an average length of the DNA reassociating at a Cot 10 to be in the range of 3000--8000 nucleotide pairs long. This value is dependent upon which fit for the data is used for extrapolation (see legend to Fig. 2).
Characterization of repeated sequences by hyperchromicity Another means of estimating the length of repeated sequences is to study the optical melting behavior of reassociated duplexes of varying length. If singlestranded tails are present on DNA which has been renatured to a Cot sufficient to allow repeated sequences to reassociate, the hyperchromicity of the duplex will be lowered relative to that of native DNA and will be proportional to the fraction of DNA existing as a duplex. This decrease in hyperchromicity can be used to calculate the duplex content of the renatured fragments [26]. Millet DNA of various fragment lengths was adjusted to 0.12 M sodium phosphate (pH 8.6), denatured by boiling for 5 min, and renatured to Cot 10. Single- and double-stranded DNA were separated b y hydroxyapatite chromatography. The double-stranded fractions were dialyzed against 0.12 M sodium phosphate (pH 6.8) and melted. Fig. 3 shows the melting profiles of several reassociated DNA samples and Table III summarizes the calculations from these hyperchromicity studies. The duplex content of the fragments b o u n d to hydroxyapatite multiplied by
T A B L E III H Y P E R C H R O M I C I T Y OF M I L L E T DNA C O N T A I N I N G R E A S S O C I A T E D R E P E T I T I V E D U P L E X REGIONS OF VARIOUS LENGTHS F r a g m e n t l e n g t h w a s m e a s u r e d b y alkaline b a n d s e d i m e n t a t i o n . H y p e r c h r o m c i t i y was c a l c u l a t e d f r o m the formula [A260(100°C) w h e r e t h e a b s o r b a n c e w a s c o r r e c t e d for then~nal e x p a n s i o n . T m (Oc) is the t e m p e r a t u r e w h i c h p r o d u c e d 50% increase in h y p e r c h r o m i c i t y . T h e average d u p l e x c o n t e n t o f b o u n d f r a g m e n t s w a s c a l c u l a t e d relative t o the h y p e r c h r o m i c i t y o f native D N A ( 0 . 2 8 5 ) . A f a c t o r o f 0 . 0 2 6 is i n c l u d e d t o c o r r e c t for single-stranded collapse b e t w e e n 6 0 ° C a n d 1 0 0 ° C [ 1 8 ] . T h e a v e r a g e d u p l e x c o n t e n t o f b o u n d f r a g m e n t s is t h e n e q u a l t o ( H - - 0 . 0 2 6 / ( 0 . 2 8 5 - - 0 . 0 2 6 ) . T h e p r o d u c t o f the f r a c t i o n b o u n d a n d t h e a v e r a g e d u p l e x c o n t e n t is the f r a c t i o n o f b a s e s in r e p e t i t i v e regions. T h e a v e r a g e l e n g t h o f d u p l e x / f r a g m e n t is t h e f r a c t i o n d u p l e x t i m e s t h e f r a g m e n t length.
--A260(60°C)]/A260(lO0°C),
Fragment length (nucleotides)
430
Fraction bound to hydroxyapatite Hyperchromicity Tm (°C) Average duplex content of bound fragments F r a c t i o n o f b a s e s in r e p e t i t i v e regions A v e r a g e l e n g t h o f d u p l e x ( n u c l e o t i d e pairs)/ fragment
0.67 0.207 79.8 0.701 0.470 301
1400 0.73 0.192 85.1 0.641 0.468 897
3450
7200
0.76 0.216 87.8 0.734 0.558 2532
0.80 0.216 88.9 0.734 0.587 5285
Native long -0.285 89.5 ----
202 the fragment length gives an estimate of the length of repeated sequences [26]. The average duplex content increases with fragment length over the entire range of fragment sizes studied, indicating that the majority of repetitive sequences in millet DNA are long. The repetitive sequences must be at least 5000 nucleotide pairs in length. An unexpected observation from these studies is that the thermal stability of the reassociated duplexes increases with fragment length. For example, the renatured 7200-nucleotide fragments melts with a Tm of 88.9°C, or only 0.6°C lower than native DNA. Assuming a 1°C depression in Tm corresponds to 1% mismatch in renatured duplexes [30], the renatured 7200-nucleotide fragments have only 0.6% mismatch. This indicates that the repetitive sequences have undergone little evolutionary divergence. This finding is unlike the results of similar experiments performed on other organisms [4,6,24,28,29] where the thermal stability of reassociated duplexes decreases or remains the same with increasing fragment length.
I00 Native - o - ~ C o t fO DNA -o-o-$1 R~istant Cot I0
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70 80 TEMPERATURE
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F i g . 4. A g a c o s e A 5 0 c o l u m n p r o f i l e o f S1 n u c l e a s e - r e s i s t a n t Cot 10 D N A . Millet D N A w i t h a singles t r a n d e d l e n g t h of. 8 8 0 0 n u c l e o t i d e s w a s a d j u s t e d to 0 . 1 8 M N a C I a n d 0 . 0 1 M P i p e s , d e n a t u r e d b y b o i l i n g a n d r e a s s o c i a t e d a t 6 0 ° C to Cot 10. T h e s a m p l e s w e r e a d j u s t e d to t h e $1 b u f f e r c o n d i t i o n s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s a n d t r e a t e d w i t h $ 1 n u c l e a s e f o r 1 h at 3 7 ° C . T h e d i g e s t i o n p r o d u c t s w e r e r e m o v e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y a n d the size d i s t r i b u t i o n o f S l - r e s i s t a n t d u p l e x e s w a s d e t e r m i n e d b y gel f i l t r a t i o n o n an A g a r o s e A S 0 ( B i o - R a d ) c o l u m n in 10 m M Tris-HC1 ( p H 7.6) a n d 1.0 m M E D T A . T h e gel w a s p o u r e d a r o u n d 6 - r a m glass h e a d s in a 92 c m × 1.5 c m c o l u m n . T h e c o l u m n w a s calibrated with DNA of known duplex lengths. Fig. 5. T h e r m a l d e n a t u r a t i o n p r o f i l e s o f Cot 10 D N A b e f o r e a n d a f t e r $1 n u c l e a s e d i g e s t i o n . D N A w i t h a s i n g l e - s t r a n d e d l e n g t h o f 8 8 0 0 n u c l e o t i d e s w a s a d j u s t e d to 0 , 1 8 M NaC1 a n d 0.01 M P i p e s , d e n a t u r e d b y b o i l i n g a n d r e n a t u r e d at 6 0 ° C to Cot 10. A p o r t i o n o f t h e s a m p l e w a s r e m o v e d a n d f r a c t i o n a t e d by hydroxyapatite chromatography. The fraction containing the double-stranded DNA was dialyzed against 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) a n d m e l t e d in t h e s p e c t r o p h o t o m e t e r ( e ) . T h e r e m a i n d e r of t h e s a m p l e w a s a d j u s t e d t o t h e S1 b u f f e r c o n d i t i o n s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , a n d t r e a t e d w i t h S1 n u c l e a s e f o r 1 h at 3 7 ° C . T h e d i g e s t i o n p r o d u c t s w e r e r e m o v e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y , a n d t h e S l - r e s i s t a n t d u p l e x e s d i a l y z e d a g a i n s t 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) a n d m e l t e d in t h e s p e c t r o p h o t o m e t e r (o). A m e l t o f n a t i v e m i l l e t D N A d o n e s i m u l t a n e o u s l y is i n c l u d e d f o r c o m p a r i s o n .
203
Characterization of repeated sequences by $1 nuclease digestion The repetitive sequence length can be determined directly by treating reassociated repetitive duplexes with the single-stranded specific nuclease, $1 and measuring the size of the nuclease-resistant material [26]. Millet DNA with a single-stranded length of 8800 nucleotides was adjusted to 0.18 M NaC1 and 0.01 M Pipes, denatured by boiling and renatured to Cot 10. A portion of the reassociated DNA was fractionated on hydroxyapatite to determine the fraction containing duplex regions. The remaining samples were treated with $1 nuclease to digest away single-stranded tails. The molecules containing repetitive duplexes were separated from the digestion products by hydroxyapatite chromatography, and the size distribution of the duplex molecules was determined by gel filtration on an Agarose A50 column. Fig. 4 shows the Agarose A50 column profile of Sl-resistant DNA reassociated to Cot 10. DNA molecules greater than 1500 nucleotide pairs are excluded from Agarose A50. In the case of millet DNA, 79% of the Sl-resistant duplexes are excluded from the column. This excluded fraction was sized by neutral band sedimentation in the analytical ultracentrifuge and was found to have a mean double-stranded molecular weight of 9000 nucleotide pairs. The singlestranded length of the Sl-resistant duplexes was found by alkaline band sedimentation to be 4000--5000 nucleotides, which means that approximately 50% of the original 8800-nucleotide fragments consisted of repeated sequences. This value is in good agreement with the fraction of bases in repetitive regions determined by hyperchromicity analysis of reassociated DNA. The fraction of Sl-resistant duplexes in the included volume (0.21) had a broad size distribution, averaging about 400 nucleotide pairs. Fig. 5 shows a thermal denaturation profile of Sl-resistant Cot 10 DNA, compared with native DNA and untreated Cot 10 DNA. Both treated and untreated Cot 10 duplexes had high Tm values, within 0.3°C of native DNA. Again, this indicates the presence of highly conserved repetitive DNA sequences. Treatment of Cot 10 duplexes with $1 nuclease increases their relative hyperchromicity by 4.75%. Discussion The 4.8% discrepancy between the G + C contents calculated from the buoyant density and Tm of millet DNA is not uncommon [23]. In wheat DNA, for example, there is a 4% difference in G + C content calculated from the buoyant density and that determined by chemical means [14]. This discrepancy is probably due to the presence of 5-methylcytosine, which decreases the buoyant density of DNA due to an increased volume effect [23]. The absence of resolvable DNA satellites in CsC1 equilibrium density gradients is typical of monocots, although such satellites are common in dicots [31]. However, when millet DNA is complexed with either Hg(II) or Ag÷ and banded in Cs2SO4 gradients several satellites, representing less than 10% of the total DNA, are observed (Wimpee, C.F., unpublished observations). This is consistent with the resolution of satellites in two other monocots, wheat and barley [32]. Some of these satellites are undoubtedly of chloroplast and mitochondrial origin while other may be highly repetitive DNA sequences. Pearl millet contains a large fraction of repeated DNA which is typical for
.204 higher plants, both monocots and dicots [3]. However, the repeated DNA c o n t e n t of millet (69%) is somewhat lower than that estimated from the Cot curves of related grasses (78% in corn, 76% in barley, 92% in rye, 83% in oats) [3]. Reassociation kinetics monitored by h y d r o x y a p a t i t e chromatography tends to overestimated the actual fraction of repetitive classes, thereby underestimating the single copy fraction. An alternative means of estimating repeated DNA c o n t e n t is by extrapolation of a hydroxyapatite-binding curve such as Fig. 3 to zero fragment length [26]. This m e t h o d generally yields a lower percentage of repeated DNA. For example, the repeated DNA c o n t e n t of wheat and rye obtained from hydroxyapatite-binding curves is 75% and 70%, respectively [2,33], as opposed to 92% and 83% from reassociation kinetics [3]. This estimation, however, requires that the hydroxyapatite-binding curve be corrected for zero time binding [26]. The methods used in this investigation did not allow low enough Cot values to be reached to determine the a m o u n t of zero time binding. Cotton is an exception to the general trend of high repeated DNA c o n t e n t in plants with the genome containing only 35% repetitive sequences [ 1]. When the reassociation of millet DNA 8600 nucleotides long is compared to that of DNA 300 nucleotides long (Fig. l a vs lb), it is obvious that nearly all the middle repetitive DNA is found in the rapidly renaturing component. It can, therefore, be concluded t h a t the middle repetitive DNA sequences plus the rapidly renaturing DNA sequences are linked to one another on DNA fragments which are 8600 nucleotides long. Since the rapid reassociation of the long repetitive sequences does not alter the reassociation of the single copy sequences, the single copy and middle repetitive DNA sequences cannot be interspersed with one another on DNA molecules 8600 nucleotides long. The majority of the middle repetitive DNA sequences do not exist as short repeats. Analysis of the hyperchromicity of long DNA fragments and measurement of the size of the $1 nuclease-resistant duplexes of DNA reassociated to C~t 10 argues for the presence of repetitive sequences approximately 5000 nucleotide pairs in length. Approx. half of the single cipy sequences are between 750 and 1400 nucleotides long. The remainder of the single copy sequences range in size from 1400 to at least 8600 nucleotide pairs. The high thermal stability of the repetitive DNA sequences and the large increase in ,thermal stability of repetitive sequences of increasing lenght is unusual in comparison with reassociated repetitive DNA sequences from other organisms. The increase in thermal stability with fragment length cannot be accounted for solely by the effect of length on melting behavior. Calculations based on the theoretical arguments of Crothers et al. [34] and Hayes et al. [35] can be used to predict the T m of DNA of various lengths: T , -- Tm= B/L, where Tn is the melting temperature of native DNA, L is the fragment length, and B is a constant. Using B = 650 [18] the predicted range of thermal stabilities (87.4--88.9°C) is much narrower than the observed range (79.8--88.9°C). One possible explanation for this observation is that much of the repeated DNA of millet is present in long tandem arrays consisting of shorter elements varying slightly from one another in their nucleotide sequence. The shearing of these tandem arrays would thus result in greater probability of mismatch in the reassociated duplexes than if the entire repeat stretch were left intact. When
205 the longer fragments are reassociated, long duplexes with a minimum of mismatch between the shorter elements are favored by a mechanism similar to that of branch chain migration [36]. The Sl-resistant Cot 10 DNA has a melting profile virtually identical to that of native DNA. This is unusual, but it has also been observed in long renatured repeats from at least t w o plants, pea [37] and tobacco [4] and t w o animals, Xenopus [6] and Spisula [28]. What is unusual about millet DNA is that the melting profile of Sl-resistant Cot 10 DNA is similar to that of total, unfractionated Sl-resistant duplexes. In the other studies, the renatured repeats were fractionated b y gel filtration prior to melting. It was found that the duplexes in the excluded volume (i.e. the long repeats) were those with the high thermal stability, while those in the included volume had progressively lower thermal stability as duplex decreased. This implies an evolutionary and perhaps functional difference between long and short repeated sequences [28,37]. This particular question has not been addressed in millet. On the other hand, since an estimated 79% of the repeated DNA is present in long stretches, the melting profile of total Sl-resistant Cot 10 DNA may simply reflect the thermal stability of the major c o m p o n e n t of that DNA. A number of interesting questions have been raised by these experiments. (1) What fraction of the rapidly renaturing DNA are foldback and/or highly repeated DNA sequences? (2) How are the middle repetitive and the rapidly renaturing components organized with respect to one another on long DNA fragments? To answer these questions requires radioactive DNA for following the reassociation kinetics at low Cot values. At present, we have been unable to obtain radioactive DNA of sufficient specific activity to carry out these experiments. The small genome complexity of millet (0.22 pg DNA/1 N nucleus) is unusual for higher plants. The nuclear DNA content places millet at the lower end of a wide range of genome sizes in angiosperms [38]. The only other extensively characterized higher plants with comparable haploid genome complexities are c o t t o n (0.8 pg/1 N nucleus [1], and mung bean, 0.5 pg/1 N nucleus, Murray, M., personal communication). In comparison with more closely releated species, the small nuclear DNA content of millet is even more striking. The nuclei of rye and wheat, for example, contain more than 20 and 30 times the amount of DNA, respectively, than millet. The variation in the nuclear DNA content of these cereal plants provides another indication that there is no simple correlation between genome complexity and biological complexity. In conclusion, the genome of millet consists of 69% repeated sequences. 80% of the repeated sequences are at least 5000 nucleotide pairs in length. 23% of the DNA reassociation prior to Cot 0.01. Another 47% of the DNA consists of middle repetitive sequences repeated approximately 300 times/genome. The repeated DNA of millet also has a high degree of sequence homogeneity, implying that these sequences are either highly conserved or that they have evolved only recently, 27% of the DNA is single copy sequences. A b o u t half of the single c o p y DNA is short (750-.-1400 nucleotide pairs) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotides pairs.
206
Acknowledgements The authors wish to thank Dr. Wayne, W. Hanna for introducing us to the economically important and biologically interesting plant of pearl millet. We also wish to thank Dr. Hanna for his generous supply of biological materil, his enthusiasm and his generous interest in this project. We also wish to thank Ms. Stephanie Curtis for aid in the use of $1 nuclease and for Mrs. Cindy Boerma for many suggestions regarding experimental technique. 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 25 26 27 28 29 30 31 32 33 34 35 36 37 38
W a l b o t , V. a n d D u r e , L.S. ( 1 9 7 6 ) J. Mol. Biol. 1 0 1 , 5 0 3 - - 5 3 6 S m i t h , D . B . a n d Flavell, R . B . ( 1 9 7 7 ) B i o c h i m . B i o p h y s . A c t a 4 7 4 , 8 2 - - 9 7 Flavell, R . B . , B e n n e t t , M.D., S m i t h , J.B. a n d S m i t h , D . B . ( 1 9 7 4 ) B i o c h e m . G e n e t . 12, 2 5 7 - - 2 6 9 Z i m m e r m a n , J . L . a n d G o l d b e r g , R . B . ( 1 9 7 7 ) C h r o m o s o m a 59, 2 2 7 - - 2 5 2 M u r r a y , M . G . , Preisler, R . S . a n d T h o m p s o n , W . F . ( 1 9 7 7 ) C a r n e g i e Inst. Wash. Y e a r b . 76, 2 4 0 - - 2 4 6 D a v i d s o n , E . H . , H o u g h , B . R . , A m e n s o n , C.S. a n d B r i t t e n , R . J . ( 1 9 7 3 ) J. Mol. Biol. 7 7 , 1 - - 2 3 M a n n i n g , J . E . , S c h m i d , C.W. a n d D a v i d s o n , N. ( 1 9 7 5 ) Cell 4, 1 4 1 - - 1 5 5 C r a i n , W . R . , E d e n , F . , P e a r s o n , W., D a v i d s o n , E . H . a n d B r i t t e n , R . J . ( 1 9 7 6 ) C h r o m o s o m a 56, 3 0 9 - 326 C r a i n , W . R . , D a v i d s o n , E . H . a n d B r i t t e n , R . J . ( 1 9 7 6 ) C h r o m o s o m a 59, 1 - - 1 2 H u d s p e t h , M . E . S . , T i m b e r l a k e , W.E. a n d G o l d b e r g , R . B . ( 1 9 7 7 ) P r o c . N a t l . A c a d . Sci., U.S. 74, 4332--4336 P o w e l l , J . B . a n d B u r t o n , G.W. ( 1 9 6 6 ) C r o p Sci. 6, 1 3 1 - - 1 3 4 Pantula, J.V. (1967) Nature 213, 101--102 M a n d e l , M., S c h i l d k r a u t , C.L. a n d M a r m u r , J. ( 1 9 6 8 ) in M e t h o d s in E n z y m o l o g y ( G r o s s m a n d , L. a n d M o l d a v e , K . , e d s . ) , V o l . X I I , p p . 1 8 4 - - 1 9 5 , A c a d e m i c Press, N e w Y o r k S c h i l d l ~ a u t , C . L . , M a r m t t r , J. a n d D o t y , P. ( 1 9 6 2 ) J . Mol. Biol. 4 , 4 3 0 B u r t o n , K. ( 1 9 6 8 ) in M e t h o d s i n E n z y m o l o g y ( G r o s s m a n d , L. a n d M o l d a v e , K., eds.), Vol. XII, p p . 1 6 3 - - 1 6 6 , A c a d e m i c Press, N e w Y o r k S t u d i e r , F.W. ( 1 9 6 5 ) J. Mol. Biol. 11, 3 7 3 - - 3 9 0 F r e i f e l d e r , D. ( 1 9 7 0 ) J. Mol. Biol. 54, 5 6 7 - - 5 7 7 B r i t t e n , R . J . , G r a h a m , D . E . a n d N u e f e l d , B . R . ( 1 9 7 4 ) in M e t h o d s i n E n z y m o l o g y ( G r o s s m a n d , L. a n d M o l d a v e , K . , e d s . ) , V o l X X I X , p p . 3 6 3 - - 4 1 8 , A c a d e m i c Press, N e w Y o r k Britten, R.J. and Kohne, D.E. (1968) Science 161, 529--540 Rawson, J.R.Y. (1975) Biochim. Biophys. Acta 402, 171--178 S c h i l d k r a u t , C . L . a n d L i f s o n , S. ( 1 9 6 5 ) B i o p o l y m e r s 3, 1 9 5 - - 2 0 8 S t a f f o r d , D.W. a n d B i e b e r , D. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A e t a 3 7 8 , 1 8 - - 2 1 K i r k , J . T . O . ( 1 9 6 7 ) J. Mol. Biol. 2 8 , 1 7 1 - - - 1 7 2 P e a r s o n , W . R . , Wu. J. a n d B o n n e r , J. ( 1 9 7 8 ) B i o c h e m i s t r y 17, 5 1 - - 5 9 B r i t t e n , R . J . a n d S m i t h , J. ( 1 9 7 0 ) C a r n e g i e Inst. Wash. Y e a r b . 6 8 , 3 7 8 - - 3 8 6 G r a h a m , D . E . , N e u f e l d , B . R . , D a v i d s o n , E . H . a n d B r i t t e n , R . J . ( 1 9 7 4 ) Cell 1, 1 2 7 - - 1 3 7 W e t m u r , J.(~. a n d D a v i d s o n , N. ( 1 9 6 8 ) J. Mol. Biol. 31, 3 4 9 - - 3 7 0 G o l d b e r g , R . B . , C r a i n , W . R . , R u d e r m a n , J . V . , M o o r e , G.P., B a r n e t t , T . R . , Higgins, R . C . , G e l f a n d , R . A . , G a l a u , G . A , B r i t t e n , R . J . a n d D a v i d s o n , E . H . ( 1 9 7 5 ) C h r o m o s o m a 51, 2 2 5 - - 2 5 1 A n g e r e r , R . C . , D a v i d s o n , E . H . a n d B r i t t e n , R . J . ( 1 9 7 5 ) Cell 6, 2 9 - - 3 9 B o n n e r , T., B r e n n e r , D . J . , N e u f e l d , B . R . a n d B r i t t e n , R . J . ( 1 9 7 3 ) J. Mol. Biol. 81, 1 2 3 - - 1 3 5 Ingle, J., P e a r s o n , G . G . a n d S i n c l a i r , J. ( 1 9 7 3 ) N a t . N e w Biol. 2 4 2 , 1 9 3 - - 1 9 7 R a n j e k a r , P . K . , P a U o t t a , D. a n d L a f o n t a i n e , J. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 5 , 3 0 - - 4 0 Flavell, R . B . a n d S m i t h , D . B . ( 1 9 7 6 ) H e r e d i t y 37, 2 3 1 - - 2 5 2 C r o t h e r s , D . M . , K a l l e n b a c h , N . R . a n d Z i m m , B . H . ( 1 9 6 5 ) J. Mol. Biol. 11, 8 0 2 - - 8 2 0 H a y e s , F . N . , Lilly, E . H . , R a t l i f f , R . L . , S m i t h , D . A . a n d Williams, D . L . ( 1 9 7 0 ) B i o p o l y m e r s 9, 1 1 0 5 - 1117 L e e , C.S., Davis, R . W . a n d D a v i d s o n , N. ( 1 9 7 0 ) J. Mol. Biol. 4 8 , 1 - - 2 2 T h o m p s o n , W . F . ( 1 9 7 6 ) C a r n e g i e Inst. Wash. Y e a r b . 75, 3 5 6 - - 3 6 2 R e e s , H. ( 1 9 7 2 ) B r o o k h a v e n S y r u p . Biol. 23, 3 9 4 - - 4 1 7
Biochimica et Biophysica Acta, 562 (1979) 192--206 © Elsevier/North-Holland Biomedical Press
BBA 99415
C H A R A C T E R I Z A T I O N OF THE N U C L E A R GENOME OF P E A R L MILLET
CHARLES F. WIMPEE and JAMES R.Y. RAWSON *
Departments of Botany and Biochemistry, University of Georgia, Athens, GA 30602 (U.S.A.) (Received August 21st, 1978)
Key words: Nuclear genome; Angiosperm DNA, (Pearl millet)
Summary The nuclear genome of pearl millet has been characterized with respect to its size, b u o y a n t density in CsC1 equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the b u o y a n t density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high T m values implying a high degree of sequence homogeneity. A b o u t half of the single copy DNA is short (750--1400 nucleotide pairs) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.
Introduction The organization of the nuclear genome in angiosperms has been generally characterized as repeated DNA sequences 300--400 nucleotide pairs in length interspersed with single copy DNA sequences 8 0 0 - - 1 5 0 0 nucleotide pairs long [1--5]. This pattern of DNA organization, short period interspersion, was first described in Xenopus [6] and has been found to be the most c o m m o n pattern of genome organization among eukaryotes. The only unusual aspect of DNA * T o w h o m reprint requests should be sent. Abbreviation: Pipes, piperazine-N,N'-bis(2-ethane
sulfonic acid).
193 organization in angiosperms which has emerged thus far is the high percentage of repeated DNA present {50--80%) when compared with animal genomes. A striking exception to the 'Xenopus pattern' has been found in Drosophila [7,8], honeybee [9] and the water mold Achyla [10]. In the 'Drosophila pattern', single copy DNA sequences extend for as long as 10 000 nucleotide pairs without interruption by repeated sequences. The average repeated sequence length in Drosophila is 5600 nucleotide pairs with a small fraction (0.10) less than 500 nucleotide pairs. This particular report is intended to characterize the nuclear genome of the dilpoid monocot pearl millet, Pennisetum americanum (L.) K. Schum. A considerable amount of cytogenetic information on this plant has been tabulated. It normally contains 14 chromosomes although supernumerary chromosomes have been reported [11,12]. We have characterized the nuclear DNA from millet with respect to its buoyant density in CsC1, melting temperature, reassociation kinetics and sequence organization. Materials and Methods
Biological material. Seeds and pollen from a dwarf strain of pearl millet (Tift 23 DB) were obtained from Dr. Wayne W. Hanna of the U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Coastal Plain Station at Tifton, GA. Isolation of DNA. Seeds (200 g) were imbibed in water with constant aeration for 1 h. They were drained, rinsed, and placed in a grinding buffer (600 ml) consisting of 50 mM NaC1, 10 mM Tris-HC1 (pH 8.0), and 10 mM EDTA. The seeds were homogenized for 1 min with a polytron. The homogenate was adjusted to 1/10 vol. 1-butanol, 6% (w/v) p-aminosalicylic acid, plus 1/10 vol. 10% (w/v) tri-iso-propylnaphthalenesulfonate and shaken on a rotary shaker for 20 min. The slurry was extracted for 20 min with an equal volume of chloroform/isoamylalcohol (24 : 1; v/v) and centrifuged at 8000 rev./min for 20 min to separate the phases. The nucleic acids were precipitated at 4°C by adjusting the solution to 0.4 m NaC104 and adding 2 vols. of ethanol. The nucleic acids were pelleted at 3000 rev./min and suspended in 100 ml 0.15 M NaC1/15 mM sodium citrate and plus 1.0 mM EDTA. RNA was digested with 5 mg of pancreatic RNAase and 5000 units of T1 RNAase (both previously heated at 90°C for 10 min in 0.15 M NaC1) for 1 h at 37°C. The mixture was extracted for 15 min with 2 vol. of a phenol mixture containing creseol (10%, v/v), 8-hydroxyquinoline (0.1%, w/v) and saturated with 0.15 M NaC1/15 mM sodium citrate and centrifuged at 9000 rev./min for 20 min. The aqueous phase was extracted with an equal volume of chloroform/isoamylalcohol and centrifuged at 8000 rev./min for 20 min. The DNA was precipitated two times from the aqueous phase with 1/10 vol. of 3 M sodium acetate and 2 vols. of ethanol, and then with 1/10 vol. of 3 M sodium acetate and 0.54 vol. of isopropanol. The DNA was finally purified by banding in CsC1 equilibrium density gradients. The DNA from the CsC1 gradients was dialyzed extensively against 0.15 M NaC1/15 mM sodium citrate and precipitated with 1/10 vol. of 3 M sodium acetate and 2 vols. of ethanol. The purified DNA was suspended in 0.15 M NaCl/15 mM sodium citrate plus 0.1 mM EDTA, and stored at --20°C. 200 g of
194 seeds yielded 20--25 mg of purified DNA. The double, and single-stranded molecular weights of the DNA averaged 1.0 • 10 ~ and 3.0 • 106, respectively. Analytical CsCl equilibrium density gradients. The b u o y a n t density of the DNA was determined by CsC1 equilibrium density gradient centrifugation [ 13 ], using SP01 viral DNA (p = 1.742 g/cc) as a density marker. The samples were centrifuged at 44 000 rev./min for 20 h at 25°C in a Beckman Model E analytical ultracentrifuge equipped with ultraviolet optics. Photographs were scanned at 500 nm in a Gilford spectrophotometer, and measurements were made from the tracings. The mol percent G + C content was calculated from the b u o y a n t density of the DNA according to Schildkraut et al. [14]. Determination of "cellular DNA content from millet pollen. Pollen grains were suspended in water and counted under the light microscope using a hemacytometer. DNA was extracted from the pollen by one of t w o procedures. (1) The pollen was centrifuged at 3000 rev./min and suspended in 0.935 N HC104. The grains were broken using a French press (1500 lb/inch 2) and particulate matter removed from the homogenate by low speed centrifugation. The supernatant was heated at 73°C for 20 min and centrifuged at 15 000 rev./min for 15 min to remove cell debris. ( 2 ) T h e pollen was suspended in water and broken in the French press. The homogenate was made 10% (w/v) trichloroacetic acid and allowed to stand at 4°C for 30 min. The precipitate was collected b y centrifugation at 10 000 rev./min and washed extensively with an ether/ethanol (v/v, 3 : 1) mixture. The pellet was suspended in 0.935 N HC104. The DNA concentration in the HC10, solution was determined by the diphenylamine procedure of Burton [15], using salmon sperm DNA or Escherichia coli DNA as a standard. The DNA content/haploid nucleus was determined b y assuming three nuclei/pollen grain (Hanna, W., personal communication). Shearing and sizing of DNA. DNA was sheared by sonication in 1.0 M NaC1 at 4°C. Different fragment sizes were obtained by varying the sonication time. Single-stranded fragments lengths were determined by alkaline band sedimentation at 20°C in the Beckman Model E analytical ultracentrifuge [16]. Observed sedimentation coefficients were corrected for solvent density and viscosity and converted to molecular weights using the equation of Studier [16]. Band sedimentation in 1 M NaC1 was used to calculated the observed sedimentation coefficient of double-stranded DNA. The double-stranded fragment length was 0 calculated from the S20,w using Freifelder's equation [17]. DNA reassociation. DNA samples, ranging in concentration from 30 pg/ml to 835 pg/ml in either 0.12 M or 0.48 M sodium phosphate (pH 6.8), were sealed in pasteur pipettes and denatured by boiling for 5 min. Samples in 0.12 M sodium phosphate (pH 6.8) were reassociated at 60°C, while those in 0.48 M sodium phosphate (pH 6.8) were reassociated at 73°C and corrected to the equivalent Cot (M nucleotide-s) according to Britten et al. [18]. After reassociation to the appropriate Cot, the samples were quick frozen in solid CO2/ ethanol. Reassociated DNA was separated from single-stranded DNA by h y d r o x y a p a t i t e chromatography [18--20]. DNA samples reassociated in 0.12 M sodium phosphate (pH 6.8) were thawed and loaded directly onto a h y d r o x y a p a t i t e column. Samples reassociated in 0.48 M sodium phosphate (pH 6.8) were diluted to 0.12 M sodium phosphate (pH 6.8) before loading
195 onto the column. Single-stranded DNA was eluted with 0.12 M sodium phosphate (pH 6.8). Double-stranded DNA was eluted with either 0.48 M sodium phosphate (pH 6.8), or by raising the column temperature to 100°C and eluting the denatured fragments with 0.12 M sodium phosphate (pH 6.8). The recovery from the hydroxyapatite columns was greater than 95%. The relative amounts of single- and double-stranded DNA were determined by absorbance at 260 nm in a Beckman Acta MVI spectrophotometer, assuming 1 absorbance unit (A260) is equal to 50 pg DNA/ml. Absorbance at 320 nm was subtracted from that at 260 nm to correct for light scatter. Thermal denaturation o f DNA. DNA samples ranging in concentration from 15 gg/ml to 50 pg/ml were adjusted to 0.12 M sodium phosphate (pH 6.8) by dialysis and melted in a Beckman Acta MVi double-beam spectrophotometer equipped with a jacketed cuvette holder, automatic sample changer, and a platinum temperature probe. For each melt, a reference cuvette containing 0.12 M sodium phosphate (pH 6.8) was included to ensure a linear base line. The temperature was raised quickly to 60°C, then increased at a rate of 0.3-0.5 degrees/min up to 100°C. Separation of strands was monitored by the increase in absorbance at 260 nm. The absorbance was corrected for thermal expansion of water [13]. The hyperchromicity was calculated at the fraction of denatured absorbance between 60°C and 100°C. The Tm was that temperature which produced 50% increase in hyperchromicity. The mol percent G + C content of native DNA was calculated using the equation of Schildkraut and Lifson [21]. $1 nuclease digestion o f reassociated DNA. DNA with a single-stranded length of 8800 nucleotides was dialyzed against 0.18 M NaC1 plus 0.01 M Pipes (piperazine-N-N'-bis(2-ethanesulfonic acid) buffer, pH 6.8. The DNA samples were sealed in glass vials denatured by boiling for 5 min, and reassociated at 60°C to a Cot sufficient to allow only repeated DNA to reassociate (Cot 10). 0.2 ml of each sample was fractionated on hydroxyapatite to recover those molecules containing duplex structures. The double-stranded fraction was dialyzed against 0.12 M sodium phosphate (pH 6.8) and used for thermal denaturation analysis. The remainder of the sample was adjusted to 25 mM sodium acetate (pH 4.5), 0.5 mM ZnSO4, 25 mM 2-mercaptoethanol, and 40 000 units of S1 nuclease (Miles Laboratories, Inc.)/200 ~g of DNA. The samples were incubated for 1 h at 37°C. The S1 reaction was terminated by adding an equal volume of 0.12 M sodium phosphate (pH 6.8) and cooling the samples to 4°C. If radioactive single-stranded E. coli DNA is mixed with reassociated repetitive DNA and then digested with S1 nuclease, more than 95% of the single-stranded radioactive DNA is digested. S1 nuclease-resistant duplexes were separated from digestion products by hydroxyapatite chromatography. Approximately 25 ug of the Sl-resistant DNA was removed from each sample and dialyzed against 0.12 M sodium phosphate (pH 6.8) for melting analysis. The remainder was concentrated to a small volume (0.5--1.0 ml) with 1-butanol [22], dialyzed extensively against 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA and sized on Agarose columns. Sizing o f Sl-resistant reassociated duplexes. The size distribution of Sl-resistant duplexes was determined by gel filtration on an Agarose A50 (BioRad) column previously calibrated with DNA of known duplex lengths. The gel
196 matrix was poured around 6-mm glassbeads in a 92 cm × 1.5 cm column. Samples were chromatographed in 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA, and the DNA content of the column fractions was determined by absorbance at 260 nm. DNA in the excluded volume (greater than 1500 base pairs) was concentrated with 1-butanol and dialyzed against 10 mM Tris-HC1 (pH 7.6) and 1.0 mM EDTA. Single- and double-stranded molecular weights of the excluded fraction were determined by band sedimentation in the analytical ultracentrifuge as described above. Results
Mol percent G + C o f millet DNA Millet DNA banded in analytical CsC1 equilibrium density gradients as a homogenous c o m p o n e n t with a b u o y a n t density of 1.704 g/cc, corresponding to a mol percent G + C of 44.9%. Increasing the DNA c o n t e n t in the analytical CsC1 gradients did n o t reveal any satellite components. Millet DNA melts in 0.12 M sodium phosphate (pH 6.8) with a Tm of 89.5°C and has a hyperchromicity of 28.5%. The G + C c o n t e n t of the DNA calculated from the Tm is 49.7% or 4.8% greater than that calculated from the b u o y a n t density of millet DNA. The discrepancy between these two values may be accounted for by the presence of 5-methylcytosine, which lowers the b u o y a n t density of DNA [23]. Haploid DNA content o f millet Pollen, the immature male gametophyte of higher plants, is a convenient source for determining the DNA c o n t e n t / N chromosomes. The nuclei in pollen are haploid and contain two or three nuclei/pollen (depending upon species). A single pollen grain from millet contains 0.66 pg of DNA and three nuclei. Therefore, the genomic complexity of millet is 0.22 pg or 2.0- 108 nucleotide pairs/N chromosomes. Reassociation kinetics of millet DNA Fig. l a shows the reassociation kinetics of short (300-nucleotide) fragments of millet DNA. The curve drawn through the points is determined by a nonlinear least squares regression of the data assuming second order kinetics and allowing all the parameters to free float [24]. Analysis of the data in this fashion revealed two kinetic components. The lower curves (dashed lines) represent the predicted reassociation.kinetics of the individual components if t h e y existed alone. The observed rate constant for the slowest reassociating c o m p o n e n t (single copy DNA) is 0.0029 M -1 • s -1. The root-mean-square of this fit was 0.0241. A better fit of the data was obtained by holding constant during the analysis the observed rate constant expected for the single copy DNA in an organism with a genome complexity of 0.22 pg or 2.0 • 108 nucleotide pairs. Table I summarizes the computer analysis of the reassociation kinetics calculated in this fashion. The root-mean-square for this fit is 0.0248, only slightly higher than that for the three-float analysis. There are three distinct kinetic corn-
197
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Fig. 1. R e a s s o c i a t i o n k i n e t i c s of s h o r t vs. long f r a g m e n t s of m i l l e t D N A . (a) R e a s s o c i a t i o n k i n e t i c s o f 3 0 0 n u c l e o t i d e long f r a g m e n t s o f m i l l e t D N A . D N A was s h e a r e d b y s o n i c a t i o n t o a n average s i n g l e - s t r a n d e d l e n g t h o f 3 0 0 n u c l e o t i d e s a n d a d j u s t e d to e i t h e r 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) or 0 . 4 8 M s o d i u m p h o s p h a t e ( p H 6.8) a n d d e n a t u r e d b y boiling for 5 rain. S a m p l e s in 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) w e r e r e a s s o c i a t e d a t 6 0 ° C , while t h o s e in 0 . 4 8 M s o d i u m p h o s p h a t e ( p H 6.8) w e r e r e a s s o c i a t e d at 7 3 ° C a n d c o r r e c t e d t o e q u i v a l e n t Cot ( E C 0 t ) a c c o r d i n g to B r i t t e n e t al. [ 1 8 ] . R e a s s o c i a t e d D N A w a s fract i o n a t e d b y h y d r o x y a p a t i t e ( H A P ) c h r o m a t o g r a p h y a n d the relative a m o u n t s of d o u b l e - a n d singles t r a n d e d D N A was d e t e r m i n e d b y a b s o r b a n c e at 2 6 0 n m . T h e c u r v e d r a w n t h r o u g h t the p o i n t s r e p r e s e n t s a least s q u a r e s fit for t w o c o m p o n e n t s , a s s u m i n g s e c o n d o r d e r k i n e t i c s a n d a l l o w i n g all t h e p a r a m e t e r s t o free f l o a t ( r o o t - m e a n - s q u a r e , 0 . 2 5 6 ) . T h e l o w e r c u r v e s ( d a s h e d lines) r e p r e s e n t t h e p r e d i c t e d r e a s s o c i a t i o n kinetics of t h e p u r e c o m p o n e n t s . (b) R e a s s o c i a t i o n kinetics of 8 6 0 0 - n u c l e o t i d e long f r a g m e n t s of m i l l e t D N A . D N A w i t h a n a v e r a g e s i n g l e - s t r a n d e d l e n g t h of 8 6 0 0 n u c l e o t i d e s w a s a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) , d e n a t u r e d b y boiling for 5 m i n , a n d r e a s s o c i a t e d at 6 0 ° C . R e a s s o c i a t e d D N A was f r a c t i o n a t e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y . Material c o n t a i n i n g d u p l e x s t r u c t u r e s w a s t h e r m a l l y e l u t e d f r o m the h y d r o x y a p a t i t e c o l u m n s . T h e relative a m o u n t s o f d o u b l e - a n d s i n g l e - s t r a n d e d D N A w a s d e t e r m i n e d b y a b s o r b a n c e a t 2 6 0 n m . T h e line t h r o u g h the p o i n t s r e p r e s e n t s a least s q u a r e s fit for t w o c o m p o n e n t s allowing all p a r a m e t e r s to free f l o a t ( r o o t - m e a n - s q u a r e , 0 . 0 2 8 5 ) . T h e l o w e r c u r v e s ( d a s h e d lines) 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 k i n e t i c s of t h e p u r e c o m p o n e n t s .
ponents of DNA in the millet genome. A large fraction (0.226) of the genome reassociates too rapidly to resolve with the techniques used in this investigation (Cot < 0.01). This c o m p o n e n t may consist of one or more classes of highly repeated DNA and/or foldback sequences. A moderately repetitive component, comprising 46.7% of the genome, reassociates with an observed second order rate constant of 1.637 M -~ • s -1. A slow c o m p o n e n t (16.7% of the genome), representing single copy sequences, reassociates with an observed second order rate constant of 0.0055 M -1 • s -~. 14% of the DNA failed to reassociate and may be due to degradation or failure of some sequences to form stable duplexes at the criterion used for these experiments. Knowing the kinetic and chemical complexity of the various repetitive components permits the calculation of the reiteration frequency of these fractions. This is done by dividing the chemical complexity of each c o m p o n e n t by its kinetic complexity. Since the second order rate constant of the fastest kinetic c o m p o n e n t and the a m o u n t of zero time binding have n o t been determined, a reiteration frequency cannot be calculated for that fraction of DNA reassociating at Cot values of less than 0.01. The average reiteration frequency of the middle repetitive fraction is 298 copies.
OF MILLET DNA: 300 NUCLEOTIDES
LONG
Fraction of DNA
0.226 0.467 0.167
Component
Rapidly renaturing Middle repetitive Single copy
by the kinetic complexity.
-
-1.637 0.0055
Observed k (M -1 • s -1 )
-3.506 0.0323
Pure k ( M -1 • s -1 )
-3.13 - 105 3.33 • 107
Kinetic complexity (nucleotide pairs)
4.52 • 107 9.34 - 10 3.34 - 107
Chemical complexity (nucleotide pairs)
-298 1
Reiteration frequency
rate constant for the single copy component for that expected in an organism with a genome complexity of 0.22 pg (2.0 • 108 nucleotide pairs). The root-means q u a r e f o r t h i s f i t is 0 . 0 2 5 6 . F r a c t i o n o f D N A u n r e a s s o c i a t e d , 0 . 1 4 0 . P u r e k = O b s e r v e d k / f r a c t i o n o f D N A . T h e k i n e t i c c o m p l e x i t y is c a l c u l a t e d r e l a t i v e t o t h e g e n o m e c o m p l e x i t y o f E. coli ( 4 . 2 4 1 0 6 n u c l e o t i d e p a i r s ) a n d t h e s e c o n d o r d e r r a t e c o n s t a n t o f E. coli D N A i n o u r l a b ( 0 . 2 5 9 M - 1 - s -1 ). T h e c h e m i c a l c o m p l e x i t y is t h e f r a c t i o n o f D N A t i m e s t h e t o t a l g e n o m e c o m p l e x i t y ( 0 . 2 2 p g o r 2 . 0 • 1 0 8 n u c l e o t i d e p a i r s ) . T h e r e i t e r a t i o n f r e q u e n c y is t h e c h e m i c a l c o m p l e x i t y d i v i d e d
DNA, 300 nucleotides long, was reassociated. The data were analyzed by a non-linear least squares regression assuming second order kinetics and fixing the observed
KINETIC COMPONENTS
TABLE I
~D Go
199
Interspersion of kinetic components The presence or absence of interspersion of repetitive and single c o p y sequences can be determined by comparing the hydroxyapatite binding of reassociated short and long fragments of DNA [6--9,25,26]. Since hydroxyapatite will bind DNA which contains small regions of double-stranded DNA, the observed renaturation rate of DNA will be equal to that of the least complex class of nucleotide sequences contained in a given DNA fragment. If there is no interspersion of the various kinetic components, then the observed rate constants of the individual components will only vary as a function of the square root of the ratio of the molecular weights of the t w o DNA samples being compared; kl/k2 = (LI/L2) °'5, where k~ and k2 are the rate constants for reassociation of short (L1) and long (L2) fragments, respectively [ 27]. Fig. l b shows the reassociation kinetics of fragments of millet DNA 8600 nucleotides long. The data were analyzed in two different ways. Assuming second order kinetics the computer program was: (1) allowed to find the best fit by allowing all parameters to free float (root-mean-square, 0.0285) and (2) the observed rate constants for the two components were fixed as defined by the expression ks6oo = k3oo (Ls6oo/L3oo) °'5, where the subscripts indicate the length of the fragments being compared and k300 is the observed rate constant for the t w o c o m p o n e n t s given in Table I (root-mean-square, 0.0290). Table II summarizes these two approaches for analyses of the reassociation kinetics of DNA 8600 nucleotides in length. When comparing the reassociation kinetics of DNA 300 or 8600 nucleotides long there is a strikingly different fraction of DNA which appears to have reassociated at Cot 0.01. 22% of the DNA 300 nucleotides long and 39% of the DNA 8600 nucleotides long are retained on hydroxyapatite at this Cot. This indicates that both highly repetitive and/or foldback sequences (i.e. sequences which reassociate prior to Cot 0.01) and middle repetitive sequences are contained on the same DNA fragments 8600 nucleotides long. On the other hand the reassociation of the long middle repetitive sequences does n o t appear to
T A B L E II K I N E T I C COMPONENTS OF M I L L E T DNA: 8600 N U C L E O T I D E S L O N G D N A , 8 6 0 0 n u c l e o t i d e s long, w a s r e a s s o c i a t e d . T h e d a t a w e r e a n a l y z e d b y a n o n - l i n e a r l e a s t s q u a r e s regression assuming s e c o n d o r d e r kinetics. First, all p a r a m e t e r s w e r e a l l o w e d t o free float until the l o w e s t r o o t m e a n ~ q u a r e ( 0 . 0 2 8 5 ) w a s f o u n d . N e x t , the observed rate c o n s t a n t s f o r the t w o c o m p o n e n t s w e r e f i x e d as d e f i n e d b y the e x p r e s s i o n w h e r e the s u b s c r i p t s i n d i c a t e the length o f the fragm e n t s being c o m p a r e d and k 3 0 0 is the observed rate c o n s t a n t for the t w o c o m p o n e n t s observed w h e n the D N A is 3 0 0 n u c l e o t i d e s l o n g given in T a b l e I. T h e r o o t - m e a n ~ q u a r e o f this analysis o f 0 . 0 2 9 0 .
(L8600/L300)O.5h300,
Component
Rapidly renaturing Middle r e p e t i t i v e Single c o p y
Free float
F i x observed rate c o n s t a n t
Fraction of DNA *
Observed k (M -1 • s - 1 )
Fraction o f D N A **
Observed k (M -1 • s - l )
0.397 0.199 0.122
~ 10.13 0.0136
0.386 0.185 0.102
-8.80 0.0295
* Fraction DNA unreassociated, 0.076. ** F r a c t i o n D N A u n x e a s s o c i a t e d , 0 . 0 9 9 .
200
significantly alter the renaturation of the single copy sequences, indicating the lack of interspersion of middle repetitive and single c o p y sequences on fragments 8600 nucleotides long.
Average length of single copy sequences Interspersion of repetitive and single copy sequences will cause a certain amount of single copy DNA to be bound to hydroxyapatite at l o w Cot values. Thus, at a Cot at which only repeated sequences are fully reassociated, the amount of unreassociated single copy DNA existing as single-stranded tails on renatured duplexes will increase as fragment length increases. This will be true to a fragment length which is equal to the average distance between repeated sequences. Therefore, an estimate of single copy sequences length can be made by plotting the amount of DNA bound to hydroxyapatite as a function of fragment length [6,26]. From Fig. la, it can be seen that all of the repetitive sequences are fully reassociated by a Cot of 10. Fig. 2 shows the hydroxyapatite binding of millet DNA sheared to various lengths and reassociated to Cot 10. This particular
100 1.0 43O 0.9 :I
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__
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Fig. 2. T h e f r a c t i o n o f m i l l e t D N A c o n t a i n i n g r e p e a t e d s e q u e n c e s , a s a f u n c t i o n o f f r a g m e n t l e n g t h . D N A w a s s h e a r e d t o v a r i o u s f r a g m e n t l e n g t h s a n d a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e (pI-[ 6 . 8 ) . T h e samples w e r e d e n a t u r e d b y b o i l i n g f o r 5 m i n a n d r e a s s o c i a t e d a t 6 0 ° C t o Cot 1 0 . T h e r e a s s o c i a t e d s a m p l e s w e r e fractionated by hydroxyapatite (HAP) chromatography a n d t h e r e l a t i v e a m o u n t s o f d o u b l e - a n d singlestranded D N A w a s d e t e r m i n e d b y a b s o r b a n c e a t 2 6 0 n m . T h e f r a c t i o n o f l o n g f r a g m e n t s u n a b l e t o f o r m s t a b l e d u p l e x e s w a s c o r r e c t e d f o r in estimating t h e f r a c t i o n o f D N A b o u n d t o h y d r o x y a p a t i t e . T h e lines o n t h e g r a p h a r e a a r e s u l t o f linear regression analysis o f t h e d a t a , a n d r e p r e s e n t t w o p o s s i b i l i t i e s f o r a family of curves which could be drawn through the points.
Fig. 3. T h e r m a l d e n a t u r a t i o n p r o f i l e s o f m i l l e t D N A o f various lengths r e a s s o c i a t e d t o Cot 1 0 , c o m p a r e d t o t h a t o f native D N A . D N A samples with single-stranded lengths o f 4 3 0 , 1 4 0 0 , 3 4 5 0 , a n d 7 2 0 0 nucleotides w e r e a d j u s t e d t o 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) , d e n a t u r e d b y b o i l i n g f o r 5 r a i n a n d r e a s s o a t 6 0 ° C t o Cot 1 0 . T h e s a m p l e s w e r e f r a c t i o n a t e d b y h y d r o x y a p a t i t e chromatograph),, and the doubles t ~ ' a n d e d f r a c t i o n s w e r e d i a l y z e d a g a i n s t 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) a n d m e l t e d in t h e s p e c t r o photometer.
201
experiment is n o t as informative as desired, because the DNA b o u n d to hydroxyapatite due to foldback sequences has n o t been subtracted. Therefore, we are limited in the extent of the interpretation of these data. The steep rise in the early par of the curve and the change in slope in the range of fragments 750--1400 nucleotides long suggest the presence of some single c o p y DNA of this length interspersed with more rapidly renaturing DNA. The more gradual increase in slope after 1400 nucleotides indicates the presence of a spectrum of single c o p y sequences up to at least 8600 nucleotides long. Extrapolation of the initial part of the curve to the X-axis [26] gives an average length of the DNA reassociating at a Cot 10 to be in the range of 3000--8000 nucleotide pairs long. This value is dependent upon which fit for the data is used for extrapolation (see legend to Fig. 2).
Characterization of repeated sequences by hyperchromicity Another means of estimating the length of repeated sequences is to study the optical melting behavior of reassociated duplexes of varying length. If singlestranded tails are present on DNA which has been renatured to a Cot sufficient to allow repeated sequences to reassociate, the hyperchromicity of the duplex will be lowered relative to that of native DNA and will be proportional to the fraction of DNA existing as a duplex. This decrease in hyperchromicity can be used to calculate the duplex content of the renatured fragments [26]. Millet DNA of various fragment lengths was adjusted to 0.12 M sodium phosphate (pH 8.6), denatured by boiling for 5 min, and renatured to Cot 10. Single- and double-stranded DNA were separated b y hydroxyapatite chromatography. The double-stranded fractions were dialyzed against 0.12 M sodium phosphate (pH 6.8) and melted. Fig. 3 shows the melting profiles of several reassociated DNA samples and Table III summarizes the calculations from these hyperchromicity studies. The duplex content of the fragments b o u n d to hydroxyapatite multiplied by
T A B L E III H Y P E R C H R O M I C I T Y OF M I L L E T DNA C O N T A I N I N G R E A S S O C I A T E D R E P E T I T I V E D U P L E X REGIONS OF VARIOUS LENGTHS F r a g m e n t l e n g t h w a s m e a s u r e d b y alkaline b a n d s e d i m e n t a t i o n . H y p e r c h r o m c i t i y was c a l c u l a t e d f r o m the formula [A260(100°C) w h e r e t h e a b s o r b a n c e w a s c o r r e c t e d for then~nal e x p a n s i o n . T m (Oc) is the t e m p e r a t u r e w h i c h p r o d u c e d 50% increase in h y p e r c h r o m i c i t y . T h e average d u p l e x c o n t e n t o f b o u n d f r a g m e n t s w a s c a l c u l a t e d relative t o the h y p e r c h r o m i c i t y o f native D N A ( 0 . 2 8 5 ) . A f a c t o r o f 0 . 0 2 6 is i n c l u d e d t o c o r r e c t for single-stranded collapse b e t w e e n 6 0 ° C a n d 1 0 0 ° C [ 1 8 ] . T h e a v e r a g e d u p l e x c o n t e n t o f b o u n d f r a g m e n t s is t h e n e q u a l t o ( H - - 0 . 0 2 6 / ( 0 . 2 8 5 - - 0 . 0 2 6 ) . T h e p r o d u c t o f the f r a c t i o n b o u n d a n d t h e a v e r a g e d u p l e x c o n t e n t is the f r a c t i o n o f b a s e s in r e p e t i t i v e regions. T h e a v e r a g e l e n g t h o f d u p l e x / f r a g m e n t is t h e f r a c t i o n d u p l e x t i m e s t h e f r a g m e n t length.
--A260(60°C)]/A260(lO0°C),
Fragment length (nucleotides)
430
Fraction bound to hydroxyapatite Hyperchromicity Tm (°C) Average duplex content of bound fragments F r a c t i o n o f b a s e s in r e p e t i t i v e regions A v e r a g e l e n g t h o f d u p l e x ( n u c l e o t i d e pairs)/ fragment
0.67 0.207 79.8 0.701 0.470 301
1400 0.73 0.192 85.1 0.641 0.468 897
3450
7200
0.76 0.216 87.8 0.734 0.558 2532
0.80 0.216 88.9 0.734 0.587 5285
Native long -0.285 89.5 ----
202 the fragment length gives an estimate of the length of repeated sequences [26]. The average duplex content increases with fragment length over the entire range of fragment sizes studied, indicating that the majority of repetitive sequences in millet DNA are long. The repetitive sequences must be at least 5000 nucleotide pairs in length. An unexpected observation from these studies is that the thermal stability of the reassociated duplexes increases with fragment length. For example, the renatured 7200-nucleotide fragments melts with a Tm of 88.9°C, or only 0.6°C lower than native DNA. Assuming a 1°C depression in Tm corresponds to 1% mismatch in renatured duplexes [30], the renatured 7200-nucleotide fragments have only 0.6% mismatch. This indicates that the repetitive sequences have undergone little evolutionary divergence. This finding is unlike the results of similar experiments performed on other organisms [4,6,24,28,29] where the thermal stability of reassociated duplexes decreases or remains the same with increasing fragment length.
I00 Native - o - ~ C o t fO DNA -o-o-$1 R~istant Cot I0
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F i g . 4. A g a c o s e A 5 0 c o l u m n p r o f i l e o f S1 n u c l e a s e - r e s i s t a n t Cot 10 D N A . Millet D N A w i t h a singles t r a n d e d l e n g t h of. 8 8 0 0 n u c l e o t i d e s w a s a d j u s t e d to 0 . 1 8 M N a C I a n d 0 . 0 1 M P i p e s , d e n a t u r e d b y b o i l i n g a n d r e a s s o c i a t e d a t 6 0 ° C to Cot 10. T h e s a m p l e s w e r e a d j u s t e d to t h e $1 b u f f e r c o n d i t i o n s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s a n d t r e a t e d w i t h $ 1 n u c l e a s e f o r 1 h at 3 7 ° C . T h e d i g e s t i o n p r o d u c t s w e r e r e m o v e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y a n d the size d i s t r i b u t i o n o f S l - r e s i s t a n t d u p l e x e s w a s d e t e r m i n e d b y gel f i l t r a t i o n o n an A g a r o s e A S 0 ( B i o - R a d ) c o l u m n in 10 m M Tris-HC1 ( p H 7.6) a n d 1.0 m M E D T A . T h e gel w a s p o u r e d a r o u n d 6 - r a m glass h e a d s in a 92 c m × 1.5 c m c o l u m n . T h e c o l u m n w a s calibrated with DNA of known duplex lengths. Fig. 5. T h e r m a l d e n a t u r a t i o n p r o f i l e s o f Cot 10 D N A b e f o r e a n d a f t e r $1 n u c l e a s e d i g e s t i o n . D N A w i t h a s i n g l e - s t r a n d e d l e n g t h o f 8 8 0 0 n u c l e o t i d e s w a s a d j u s t e d to 0 , 1 8 M NaC1 a n d 0.01 M P i p e s , d e n a t u r e d b y b o i l i n g a n d r e n a t u r e d at 6 0 ° C to Cot 10. A p o r t i o n o f t h e s a m p l e w a s r e m o v e d a n d f r a c t i o n a t e d by hydroxyapatite chromatography. The fraction containing the double-stranded DNA was dialyzed against 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6 . 8 ) a n d m e l t e d in t h e s p e c t r o p h o t o m e t e r ( e ) . T h e r e m a i n d e r of t h e s a m p l e w a s a d j u s t e d t o t h e S1 b u f f e r c o n d i t i o n s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , a n d t r e a t e d w i t h S1 n u c l e a s e f o r 1 h at 3 7 ° C . T h e d i g e s t i o n p r o d u c t s w e r e r e m o v e d b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y , a n d t h e S l - r e s i s t a n t d u p l e x e s d i a l y z e d a g a i n s t 0 . 1 2 M s o d i u m p h o s p h a t e ( p H 6.8) a n d m e l t e d in t h e s p e c t r o p h o t o m e t e r (o). A m e l t o f n a t i v e m i l l e t D N A d o n e s i m u l t a n e o u s l y is i n c l u d e d f o r c o m p a r i s o n .
203
Characterization of repeated sequences by $1 nuclease digestion The repetitive sequence length can be determined directly by treating reassociated repetitive duplexes with the single-stranded specific nuclease, $1 and measuring the size of the nuclease-resistant material [26]. Millet DNA with a single-stranded length of 8800 nucleotides was adjusted to 0.18 M NaC1 and 0.01 M Pipes, denatured by boiling and renatured to Cot 10. A portion of the reassociated DNA was fractionated on hydroxyapatite to determine the fraction containing duplex regions. The remaining samples were treated with $1 nuclease to digest away single-stranded tails. The molecules containing repetitive duplexes were separated from the digestion products by hydroxyapatite chromatography, and the size distribution of the duplex molecules was determined by gel filtration on an Agarose A50 column. Fig. 4 shows the Agarose A50 column profile of Sl-resistant DNA reassociated to Cot 10. DNA molecules greater than 1500 nucleotide pairs are excluded from Agarose A50. In the case of millet DNA, 79% of the Sl-resistant duplexes are excluded from the column. This excluded fraction was sized by neutral band sedimentation in the analytical ultracentrifuge and was found to have a mean double-stranded molecular weight of 9000 nucleotide pairs. The singlestranded length of the Sl-resistant duplexes was found by alkaline band sedimentation to be 4000--5000 nucleotides, which means that approximately 50% of the original 8800-nucleotide fragments consisted of repeated sequences. This value is in good agreement with the fraction of bases in repetitive regions determined by hyperchromicity analysis of reassociated DNA. The fraction of Sl-resistant duplexes in the included volume (0.21) had a broad size distribution, averaging about 400 nucleotide pairs. Fig. 5 shows a thermal denaturation profile of Sl-resistant Cot 10 DNA, compared with native DNA and untreated Cot 10 DNA. Both treated and untreated Cot 10 duplexes had high Tm values, within 0.3°C of native DNA. Again, this indicates the presence of highly conserved repetitive DNA sequences. Treatment of Cot 10 duplexes with $1 nuclease increases their relative hyperchromicity by 4.75%. Discussion The 4.8% discrepancy between the G + C contents calculated from the buoyant density and Tm of millet DNA is not uncommon [23]. In wheat DNA, for example, there is a 4% difference in G + C content calculated from the buoyant density and that determined by chemical means [14]. This discrepancy is probably due to the presence of 5-methylcytosine, which decreases the buoyant density of DNA due to an increased volume effect [23]. The absence of resolvable DNA satellites in CsC1 equilibrium density gradients is typical of monocots, although such satellites are common in dicots [31]. However, when millet DNA is complexed with either Hg(II) or Ag÷ and banded in Cs2SO4 gradients several satellites, representing less than 10% of the total DNA, are observed (Wimpee, C.F., unpublished observations). This is consistent with the resolution of satellites in two other monocots, wheat and barley [32]. Some of these satellites are undoubtedly of chloroplast and mitochondrial origin while other may be highly repetitive DNA sequences. Pearl millet contains a large fraction of repeated DNA which is typical for
.204 higher plants, both monocots and dicots [3]. However, the repeated DNA c o n t e n t of millet (69%) is somewhat lower than that estimated from the Cot curves of related grasses (78% in corn, 76% in barley, 92% in rye, 83% in oats) [3]. Reassociation kinetics monitored by h y d r o x y a p a t i t e chromatography tends to overestimated the actual fraction of repetitive classes, thereby underestimating the single copy fraction. An alternative means of estimating repeated DNA c o n t e n t is by extrapolation of a hydroxyapatite-binding curve such as Fig. 3 to zero fragment length [26]. This m e t h o d generally yields a lower percentage of repeated DNA. For example, the repeated DNA c o n t e n t of wheat and rye obtained from hydroxyapatite-binding curves is 75% and 70%, respectively [2,33], as opposed to 92% and 83% from reassociation kinetics [3]. This estimation, however, requires that the hydroxyapatite-binding curve be corrected for zero time binding [26]. The methods used in this investigation did not allow low enough Cot values to be reached to determine the a m o u n t of zero time binding. Cotton is an exception to the general trend of high repeated DNA c o n t e n t in plants with the genome containing only 35% repetitive sequences [ 1]. When the reassociation of millet DNA 8600 nucleotides long is compared to that of DNA 300 nucleotides long (Fig. l a vs lb), it is obvious that nearly all the middle repetitive DNA is found in the rapidly renaturing component. It can, therefore, be concluded t h a t the middle repetitive DNA sequences plus the rapidly renaturing DNA sequences are linked to one another on DNA fragments which are 8600 nucleotides long. Since the rapid reassociation of the long repetitive sequences does not alter the reassociation of the single copy sequences, the single copy and middle repetitive DNA sequences cannot be interspersed with one another on DNA molecules 8600 nucleotides long. The majority of the middle repetitive DNA sequences do not exist as short repeats. Analysis of the hyperchromicity of long DNA fragments and measurement of the size of the $1 nuclease-resistant duplexes of DNA reassociated to C~t 10 argues for the presence of repetitive sequences approximately 5000 nucleotide pairs in length. Approx. half of the single cipy sequences are between 750 and 1400 nucleotides long. The remainder of the single copy sequences range in size from 1400 to at least 8600 nucleotide pairs. The high thermal stability of the repetitive DNA sequences and the large increase in ,thermal stability of repetitive sequences of increasing lenght is unusual in comparison with reassociated repetitive DNA sequences from other organisms. The increase in thermal stability with fragment length cannot be accounted for solely by the effect of length on melting behavior. Calculations based on the theoretical arguments of Crothers et al. [34] and Hayes et al. [35] can be used to predict the T m of DNA of various lengths: T , -- Tm= B/L, where Tn is the melting temperature of native DNA, L is the fragment length, and B is a constant. Using B = 650 [18] the predicted range of thermal stabilities (87.4--88.9°C) is much narrower than the observed range (79.8--88.9°C). One possible explanation for this observation is that much of the repeated DNA of millet is present in long tandem arrays consisting of shorter elements varying slightly from one another in their nucleotide sequence. The shearing of these tandem arrays would thus result in greater probability of mismatch in the reassociated duplexes than if the entire repeat stretch were left intact. When
205 the longer fragments are reassociated, long duplexes with a minimum of mismatch between the shorter elements are favored by a mechanism similar to that of branch chain migration [36]. The Sl-resistant Cot 10 DNA has a melting profile virtually identical to that of native DNA. This is unusual, but it has also been observed in long renatured repeats from at least t w o plants, pea [37] and tobacco [4] and t w o animals, Xenopus [6] and Spisula [28]. What is unusual about millet DNA is that the melting profile of Sl-resistant Cot 10 DNA is similar to that of total, unfractionated Sl-resistant duplexes. In the other studies, the renatured repeats were fractionated b y gel filtration prior to melting. It was found that the duplexes in the excluded volume (i.e. the long repeats) were those with the high thermal stability, while those in the included volume had progressively lower thermal stability as duplex decreased. This implies an evolutionary and perhaps functional difference between long and short repeated sequences [28,37]. This particular question has not been addressed in millet. On the other hand, since an estimated 79% of the repeated DNA is present in long stretches, the melting profile of total Sl-resistant Cot 10 DNA may simply reflect the thermal stability of the major c o m p o n e n t of that DNA. A number of interesting questions have been raised by these experiments. (1) What fraction of the rapidly renaturing DNA are foldback and/or highly repeated DNA sequences? (2) How are the middle repetitive and the rapidly renaturing components organized with respect to one another on long DNA fragments? To answer these questions requires radioactive DNA for following the reassociation kinetics at low Cot values. At present, we have been unable to obtain radioactive DNA of sufficient specific activity to carry out these experiments. The small genome complexity of millet (0.22 pg DNA/1 N nucleus) is unusual for higher plants. The nuclear DNA content places millet at the lower end of a wide range of genome sizes in angiosperms [38]. The only other extensively characterized higher plants with comparable haploid genome complexities are c o t t o n (0.8 pg/1 N nucleus [1], and mung bean, 0.5 pg/1 N nucleus, Murray, M., personal communication). In comparison with more closely releated species, the small nuclear DNA content of millet is even more striking. The nuclei of rye and wheat, for example, contain more than 20 and 30 times the amount of DNA, respectively, than millet. The variation in the nuclear DNA content of these cereal plants provides another indication that there is no simple correlation between genome complexity and biological complexity. In conclusion, the genome of millet consists of 69% repeated sequences. 80% of the repeated sequences are at least 5000 nucleotide pairs in length. 23% of the DNA reassociation prior to Cot 0.01. Another 47% of the DNA consists of middle repetitive sequences repeated approximately 300 times/genome. The repeated DNA of millet also has a high degree of sequence homogeneity, implying that these sequences are either highly conserved or that they have evolved only recently, 27% of the DNA is single copy sequences. A b o u t half of the single c o p y DNA is short (750-.-1400 nucleotide pairs) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotides pairs.
206
Acknowledgements The authors wish to thank Dr. Wayne, W. Hanna for introducing us to the economically important and biologically interesting plant of pearl millet. We also wish to thank Dr. Hanna for his generous supply of biological materil, his enthusiasm and his generous interest in this project. We also wish to thank Ms. Stephanie Curtis for aid in the use of $1 nuclease and for Mrs. Cindy Boerma for many suggestions regarding experimental technique. 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 25 26 27 28 29 30 31 32 33 34 35 36 37 38
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