Biochimica et Biophysica Acta, 563 (1979) 1--16 © Elsevier/North-Holland Biomedical Press

BBA 99452



Departments of Botany and Biochemistry, University of Georgia, Athens, GA 30602 (U.S.A.) (Received September 19th, 1978)

Key words: DNA sequence organization; Repetitive DNA sequence; Genome complexity; (Euglena gracilis)

Summary The sequence organization of nuclear DNA in the single-celled alga Euglena gracilis has been studied by a combination of techniques: (1) the comparison of the reassociation kinetics of DNA fragments 300, 2000 and 8100 nucleotides long; (2) the reassociation of 32P-labeled DNA fragments of various lengths with driver fragments 300 nucleotides long; (3) the hyperchromicity of DNA structures formed by the reassociation of repetitive sequences; (4) and the direct measurement of the size of the duplex regions of reassociated repetitive DNA resistant to S1 nuclease. The single copy DNA sequences are approximately 1500 nucleotide pairs long and are interspersed with repetitive DNA sequences. The repetitive DNA, consisting of both highly repetitive and middle repetitive sequences, consists of one fraction of nucleotide sequences (0.67) with an average size of 4900 nucleotide pairs and a second fraction {0.33) with an average size of 1000 nucleotide pairs. 34% of the DNA consists of foldback sequences which are present on 45% of the DNA 4000 nucleotides long. Introduction

Repetitive and single copy DNA sequences are interspersed in the genomes of a variety of eukaryotes [1--4]. The interspersion of these DNA sequences is characterized by single copy sequences approximately 1000--2000 nucleotide pairs long immediately adjacent to and bracketed by short {200--400 nucleo* To w h o m reprint requests should be directed, Abbreviation: Pipes, piperzine-N,N'obis(2-ethanesulfonic acid),

tide pairs) repetitive sequences. This pattern of organization of DNA was first demonstrated in Xenopus DNA [1] and has come to be referred to as shortterm interspersion or the 'Xenopus pattern' of DNA organization. A different pattern of DNA sequence organization has been demonstrated in several other eukaryotes (Drosophila melanogaster, honeybee and the water mold, Achyla [5--7] and may be depicted as large repetitive sequences (1000--10 000 nucleotide pairs) linked to single copy sequences 10 000 nucleotide pairs or longer. Such sequence organization has been referred to as long-term interspersion or the 'Drosophila pattern' of DNA organization [6]. Studies of DNA sequence organization in eukaryotes have usually been carried out using organisms which are evolutionary well-defined, although distant with respect to one another. Euglena gracilis is a unicellular alga, classified as a protista [8] and is evolutionarily quite isolated from other lower eukaryotes. The chromosomes in Euglena are also unusual in that they are continually condensed [9]. Initial studies in characterizing the genome of Euglena by reassociation kinetics suggested that there were two kinetic components, and that 36% of the genome consisted of single copy DNA [10]. We have further characterized the kinetics of reassociation of nuclear DNA in Euglena and have found the average size of the repeated DNA sequences somewhat larger than those in Xenopus but interspersed with single copy DNA sequences 1500 nucleotide pairs long. Materials and Methods

Cell growth and DNA isolation. E. gracilis vat. Z was grown in a heterotrophic medium [10]. Total cell DNA with double-stranded and single-stranded molecular weights of 9 • 106 or 13 500 nucleotide pairs and 8 . 1 0 s or 2400 nucleotides, respectively, was prepared as previously described [ 10]. DNA with a single-stranded length of 9000 nucleotides was prepared by a gentler procedure. Euglena cells (1 g wet weight/4 ml)were suspended in 0.15 M NaC1, I mM EDTA and 10 mM Tris-HC1, pH 7.5, plus 2.5% (w/v) sodium dodecyl sulfate. Pronase (predigested for 2 h at 37°C in 0.15 M NaC1) was added to the cell slurry to a final concentration of 200 ~g/ml and the mixture incubated at 37°C for 2 h. The cell lysate was deproteinized with an equal volume of a phenol mixture containing cresol (10%, v/v), 8-hydroxyquinoline (0.1%, w/v) and saturated with 0.15 M NaC1, 1 mM EDTA and 10 mM Tris-HC1 (pH 7.5). The emulsion was shaken at room temperature for 30 min and centrifuged at 6000 rev./min for 10 rain. The aqueous phase was further deproteinized with two volumes of chloroform/isoamyl alcohol (24 : 1, v/v) and the nucleic acids precipitated from the aqueous phase with 0.1 vol. 3 M sodium acetate and 2 vols. ethanol. The precipitate was suspended in 0.15 M NaC1, I mM EDTA and 10 mM Tris-HC1 (pH 7.5) (2 ml/g cells) and digested with 25 pg/ml pancreatic RNAase plus 20 units/ml of T1 RNAase for 90 min at 37°C. The mixture was extracted first with phenol then with chloroform, and the DNA was recovered from the aqueous phase by successive precipitations with ethanol and isopropanol. The DNA was further purified in CsC1 equilibrium density gradients [10]. Labeling of DNA with 32p. Euglena cells were grown in a heterotrophic

medium containing one-tenth the normal phosphate [11]. Cells were adapted to this low phosphate medium for ten generations and then inoculated (1 • 104 cells/ml) into 500 ml of the same medium containing 40 ~Ci/ml [32p]. orthophosphate (New England Nuclear) and grown to late log phase. The specific activity of the 32P-labeled DNA was approximately 200 000 cpm/gg. Molecular weight determinations. DNA was sheared to various singlestranded molecular weights by either sonication [10] or by forcing it through small needles (23 or 27 g a u g e ) w i t h a syringe. Molecular weights of DNA preparations were determined by band sedimentation in the Spinco Model E [12]. Sedimentation coefficients of double-stranded and single-stranded DNA were measured in 1.0 M NaC1 and 0.9 M NaC1/0.1 N NaOH, respectively. The observed sedimentation coefficients were corrected for viscosity and density of the solvents [12]. The S o20,w of double-stranded DNA or single-stranded DNA was converted to molecular weight using the formula of Freifelder [13] or Studier [ 12], respectively. Ranges of molecular weights for preparations of double-stranded DNA were determined by electrophoresis on agarose slab gels [14]. Two sets of molecular weight standards were included on each gel: k-DNA digested with EcoRI [15] and ~-DNA digested with HindIII [16]. Renaturation of DNA. The renaturation of DNA was followed by separating single-stranded from double-stranded DNA in a reaction mix on hydroxyapatite columns [10,17,18]. Hydroxyapatite columns were prepared and used as described previously [10]. 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-sec) according to Britten et al. [18]. Double-stranded structures prepared from low molecular weight: DNA (less than 500 nucleotides) were eluted from hydroxyapatite columns with 0.48 M sodium phosphate (pH 6.7}. Duplex structures prepared from larger single-stranded DNA were eluted from hydroxyapatite columns with 0.12 M sodium phosphate (pH 6.8) at 98°C. The total recovery of DNA from hydroxyapatite columns was always greater than 95%. The data were fit to a curve using a non-linear least squares regression and assuming second order kinetics [ 19]. Preparation of radioactive single copy DNA. Single copy DNA was prepared by allowing DNA 500 nucleotides long to reassociate to Cot 100. The singlestranded single copy DNA was separated from the reassociated DNA by hydroxyapatite chromatography, concentrated by alcohol precipitation and reassociated a second time to Cot 100. Hyperpolymers of single copy DNA were prepared by renaturing the DNA to Cot 20 000 [20]. DNA polymerase I was used to incorporate [3H]TTP into the gaps of the single copy hyperpolymers [20]. The DNA polymerase I reaction was carried out at 15°C for 48 h in 0.2 ml containing 20 ~g hyperpolymer single copy DNA, 37.5 pM of dATP, dCTP and dGTP, 7.5 mM MgC12, 60 mM sodium phosphate (pH 6.8}, 1 mM EDTA, 16 pM [3H]TTP (47 Ci/mmol) and nine units of DNA polymerase I (Boehringer Mannheim). The radioactive DNA was purified from the reactants on a Sephadex G-25 column. A small fraction (0.15) of the deoxyribonucleotides is incorporated into DNA fragments containing selfcomplementary or foldback regions. These were removed by incubating the

D N A to very low Cot values and passing it over a hydroxyapatite column [20]. The single-stranded molecular weight o f the 3H-labeled single copy DNA prepared in this fashion was 300 nucleotides long and had a specific activity of 3 • 106 cpm/~g. Melting curves. The thermal stability o f native and reassociated samples of DNA was measured b y either thermal elutions of radioactive DNA from h y d r o x y a p a t i t e or b y following the hyperchromicity of the DNA in a Beckman VIM double-beam spectrophotometer. The absorbance was corrected for thermal expansion of water [21]. The hyperchromicity of the DNA, H, was equal to [A260 (98°C) --A260 (60°C)]/A260 (98°C). $1 nuclease digestion of single-stranded DNA. $1 nuclease was isolated from crude a-amylase p o w d e r from Aspergillus oryzae [22]. The sulfo-Sephadex chromatographic step was eliminated w i t h o u t altering the ratio of digestion of single-stranded to double-stranded DNA. $1 nuclease digestion of singlestranded DNA was carried o u t in 0.15 M NaC1, 10 mM piperazine-N-N'-bis(2ethanesulfonic acid) (Pipes), 25 mM sodium acetate (pH 4.6), 0.1 mM ZnSO4 and 5 mM 2-mercaptoethanol. A ten-fold excess of enzyme was added to assure complete digestion of all single-stranded DNA. The reaction was carried out at 37°C for 45 rain, and the nuclease reaction was stopped by adding cold 0.12 M sodium phosphate (pH 6.8). Using these conditions, more than 95% of singlestranded 3H-labeled pBR313 DNA was digested. $1 nuclease-resistant duplex structures were separated from digestion products b y h y d r o x y a p a t i t e chromatography and dialyzed against 0 . 1 2 M sodium phosphate (pH 6.8). A portion of the DNA was melted and the remainder of the sample was sized on an Agarose A-50 column. Agarose chromatography. The size distribution of reassociated DNA duplexes was determined by chromatography on Agarose A-50 [3]. Agarose A-50 was poured into a column (92 cm × 1.5 cm) containing 6-ram glass beads [23]. The DNA was chromatographed in 0.12 M sodium phosphate (pH 6.8) and the DNA content o f the column fractions determined by absorbance at 260 nm. The column was calibrated using native DNA and KI as exclusion and inclusion markers, respectively. Scintillation counting. Radiactive DNA samples were precipitated with 5% (w/v) trichloroacetic acid, collected on Millipore filters and counted in a Packard Liquid Scintillation counter [24]. Results

I~enaturation kinetics of total cellular DNA A preliminary study of the renaturation kinetics of E. gracilis DNA revealed two kinetic classes of DNA [10]. Fig. 1 shows the renaturation kinetics o f single-stranded DNA 300 nucleotides long spanning a range o f Cot values from 7 • 10 -s to 4 • 104. Cot values of less than 1 • 10 -2 were achieved using low concentrations o f a:P-labeled DNA. This datum was analyzed three different ways. First, the presence o f t w o second-order kinetic c o m p o n e n t s was assumed and the total nuclear DNA content of Euglena taken to be 3 pg [10]. The observed second-order rate constant for the slowest renaturing c o m p o n e n t was fixed at four different ploidy levels (diploid, tetraploid, hexaploid and octaploid). The

0±~ ° .... °%..°

~° 0 °° ooo °













ECo~" (M- sec)

Fig. I. Reassociation kinetics of ]Euglena D N A fragments 3 0 0 nucleotides long. D N A with a singlestranded fragment length of 3 0 0 nucleotides was reassociated in 0.12 M s o d i u m phosphate ( p H 6.8) at 6 0 ° C or in 0.48 M s o d i u m phosphate ( p H 6.8) at 73°C. Data f r o m the reassoeiation of D N A in 0.48 M s o d i u m phosphate ( p H 6.8) was corrected to equivalent Cot (EC0t) as described by Britten et al. [18]. T h e single-stranded and double-stranded products of the reaction were fractionated o n hyd~oxyapatitc. o, reassoclation of total cell D N A t e, reassociation of 3H-labeled single c o p y D N A in the presence of a driver D N A . T h e curve depicting the reassociation kinetics of the total cell D N A w a s determined b y a least squares fit for three components holding the rate constant for the single copy D N A equal to that expected for an organism with a g e n o m e size of 1.5 pg (root m e a n square 0.0267). T h e curve d r a w n through the points showing the reassociation of single copy D N A w a s determined by a least squares fit for a single c o m p o n e n t and allowing all the parameters to free float (root m e a n square 0.032).

non-linear least squares fit of the data analyzed in this fashion with the lowest root mean square (0.0268) had an observed rate constant of 0.00269 M-I • s-1 for the single copy DNA. Next, the same type o f analysis was performed on the data assuming the presence of three kinetic components. The best fit (root mean square 0.0267) had an observed second-order rate constant for the single copy DNA of 0.00081 M-1 • s-1. Finally, the observed second-order rate constant for the single copy DNA was independently measured by monitoring the renaturation of 3H-labeled single copy DNA in the presence of an excess o f non-radioactive total cell DNA (Fig. 1). The best curve fit through these data was determined by allowing all the parameters in the c o m p u t e r program to free float and assuming the presence of a single kinetic component. The r o o t mean square for this fit was 0.0319 and the observed second-order rate constant for the single copy DNA was 0.00111 M -1 • s -1. This value is most compatible with the observed second-order rate constant for the single copy DNA determined by assuming the presence of three kinetic components and t h a t the genome complexity of Euglena was 1.5 pg (1.36 • 109 nucleotide pairs) or t h a t of a diploid organism. Table I summarizes the best non-linear least squares fit of the data in Fig. 1 assuming three kinetic components. 14% of the DNA reassociates at a Cot value of less than 7 • 10 -5, suggesting t h a t this c o m p o n e n t consists of foldback DNA sequences. 34% of the DNA consists of highly repetitive DNA sequences with an observed second-order rate constant of 0.908 M -~ • s -~. A middle repetitive fraction (31% of the DNA) has an observed second-order rate constant of 0.00439 M -1 • s -1. The single copy DNA consists of 12% of the total DNA.

Interspersion of kinetic components The presence of interspersion of repetitive and single copy sequences can be

TABLE I K I N E T I C C O M P O N E N T S OF Euglena DNA D N A 3 0 0 n u c l e o t i d e s l o n g 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 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 fixing t h e o b s e r v e d r a t e c o n s t a n t f o r t h e single c o p y c o m p o n e n t f o r t h a t o f a cell w i t h a g e n o m e c o m p l e x i c i t y of 1.5 pg ( 1 . 3 6 • 109 ( n u c l e o t i d e pairs). T h e r o o t m e a n s q u a r e f o r this fit is 0 . 0 2 6 7 . F o r t h e 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 is 0 . 0 9 1 . Pure k = o b s e r v e d k + fract 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 to t h e g e n o m e c o m p l e x i t y o f K. coli ( 4 . 2 4 ' 106 n u c l e o t i d e pairs) 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 for r e a s s o c i a t i o n o f E. coli D N A in o u r laborat o r y ( 0 . 2 5 9 M -1 • s - I ) . Component

Fraction of DNA

Observed k (M -z • s -1)

Pure k (M -1 • s -1)

Kinetic complexicity (nucleotide

Reiteration frequency

pairs) Foldback Highly repetitive Middle repetitive Single c o p y

0.136 0.339 0.310 0.124

. 0.908 0.00439 0.00081


. 2.674 0.0142 0.00653

. 4 . 1 1 • 105 7.78 • I 0 ~ 1.68 • 108

1120 5 1

determined by comparing the hydroxyapatite binding of reassociated DNA of various lengths [1,4--7]. If there is interspersion of different kinetic components of DNA, the observed renaturation rate of the DNA will be equal to that of the least complex class of nucleotide sequences contained in a given DNA fragment. The observed rate constant of this least complex class of DNA will vary only as a function of the square root of the ratio of the molecular weights of the two DNA samples being compared; kl/k2 = (LI/L2) °'s, where kl and k2 are the rate constants for reassociation of short (L~) and long (L2) fragments, respectively [25]. Fig. 2 shows the reassociation kinetics of Euglena DNA fragments 2000 and 8100 nucleotides long. The curves drawn through the data were calculated by 0



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DNA sequence organization in the alga Euglena gracilis.

Biochimica et Biophysica Acta, 563 (1979) 1--16 © Elsevier/North-Holland Biomedical Press BBA 99452 DNA SEQUENCE ORGANIZATION IN THE ALGAEUGLENA GRA...
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