CHROMOSOMA

Chromosoma (Berl.) 69, 275-289 (1978)

Genome Structure of

9 by Springer-Vertag 1978

Tetrahymenapyriformiis

S.N. Borchsenius, N.A. Belozerskaya, N.A. Merkulova, V.G. Wolfson and V.I. Vorob'ev Institute of Cytology of the Academy of Sciences of the USSR, Leningrad 190121, Ave. Maklina 32, USSR

Abstract. Reassociation kinetics of D N A from the macronucleus of the cili-

ate, Tetrahymenapyriformis GL, has been studied. The genome size determined by the kinetic complexity of D N A was found to be 2.0 x 108 base pairs (or 1.2 x 1011 daltons). About 90% of the macronuclear D N A fragments 200-300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7-9% at a rate corresponding to moderate repetitive sequences; 3-4~ of such D N A fragments reassociate at Cot practically equal to zero. To investigate the linear distribution of repetitive sequences, D N A fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. D N A double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the D N A repetitive sequences. Another half of the D N A repetitive sequences (short D N A regions about 200-300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called "zero time binding" or " f o l d b a c k " D N A seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback D N A is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original D N A concentration. Heat denaturation and renaturation are reversible and show both hyper- and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsC1 density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear D N A . It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes. 0009-5915/78/0069/0275/$03.00

276

S.N. Borchsenius et al.

Introduction

The study of the molecular structure of the Tetrahymena genome is of great evolutionary interest, particularly in comparison with relevant data on the genome structure of multicellular organisms. As shown earlier for a few strains of Tetrahymena pyrifomis the haploid genome size of this protozoan is about the same as in Drosophila (Allen and Gibson, 1972). The macronuclear DNA (Ma DNA) of Tetrahymena contains a small number of repetitive sequences (Yao and Gorovsky, 1974). The linear distribution of single copy and repetitive nucleotide sequences has been investigated in DNA of various species. In most of the multicellular animals (Davidson et al., 1975) and plants studied (Zimmermann and Goldberg, 1977), the majority of the DNA unique sequences (1,000-2,000 nucleotides in length) intersperse with short repetitive sequences 200-400 nucleotides in length. Besides, there occur long regions of unique sequences (more than 10,000 nucleotides) and clusters of short repetitive sequences (2,000 nucleotides in length). The relative content of such long repetitive regions in different organisms varies from 25 to 65% (Davidson et al., 1975). A similar distribution of unique and repetitive sequences was discovered in the genome of the unicellular eukaryote, Dictyostelium discoideum (Firtel and Kindle, 1975). Another distribution of unique and repeated DNA sequences was observed in Drosophila and the honey bee. The genomes of these insects contain no short regions of repetitive sequences alternating with single-copy sequences. The average length of the repetitive sequences was 5,600 nucleotides (Manning et al., 1975; Crain et al., 1976). In the ciliate, Stylonychia mytilus, and in other I-Iypotrichida the Ma DNA contains no repetitive sequences although the ploidy level of the Ma is extremely high (up to 4,000). The percent of repetitive sequences in the DNA of the micronuclei of these ciliates if found to be of the same order as that of multicellular organisms (Ammermann et al., 1974; Raikov, 1976). In the present article we describe the genome of Tetrahymena pyriformis GL as studied by DNA reassociation kinetics. It is shown that about 90% of the Tetrahymena Ma DNA consists of single copy nucleotide sequences and about 7-9% of the Ma DNA, repetitive sequences. We have found that the Tetrahymena genome contains a relatively high quantity of fold-back self-complementary sequences. This fraction was isolated and characterized by its buoyant density and its ability to reassociate with nonfractionated Ma DNA.

Materials and Methods 7. DNA Preparation. Axenic cultures of Tetrahymena pyriformis amicronucleate strain GL were grown on a standard medium (Irlina and Merculova, 1975). Macronuclei (Ma) were isolated and D N A was extracted and purified as described earlier (Borchsenius et al., 1977). To obtain 14C-DNA 2-14C-thymidine (52 ~xCi/mM) was added to give a concentration of 2 ~tCi/ml, The specific radioactivity of the initial ~4C-DNA preparation was 1,7 x 106 cpm/~tg.

G e n o m e Structure of Tetrahymena pyriformis

277

To produce the " h i g h molecular weight D N A preparation" (8000 b.p. in length) purified D N A was dissolved in a 0.005 M PIPES buffer (pH 6.7) with 0.2 M NaC1 and gel filtered on a Sepharose 2B column equilibrated with the same buffer. We used only the D N A fraction eluted in the free volume of the column. It was precipitated and kept in 80% ethanol at - 10~ C. The molecular weight of this and some other preparations was determined in a Beckman Model E ultracentrifuge.

2. Annealing of DNA and Fractionation by S1-Nuclease Treatment. After high molecular weight D N A was denatured in water at 100 ~ C, the solution was cooled rapidly in ice and annealed at 60 ~ C in a 0.01 M PIPES buffer (pH 6.7) with 0.18 M NaCI. Incubation with Sl-nuclease (Special Bureau of Biologically Active Substances, Novosibirsk, USSR) was carried out in 0.05 M Na-acetate buffer (pH 4.4), 0.005 M PIPES, 0.3 M NaC1, 0.01 m M ZnCI2 and 5.5 m M fl-mercaptoethanol. Sl-nuclease (specific activity 1.06 x i0 s units per rag) was added (1 unit per 10 gg D N A ) and the solution was incubated at 3 7 ~ for 45 rain. The timited D N A hydrolysis with Sl-nuclease was performed as suggested by Britten et al., 1976. In the experiments on reassociation of ~zSI-DNA with nonradioactive D N A heat denaturation and annealing were performed in 0.01 M PIPES buffer (pH 6.8) with 0.17 M NaC1 (Crain et al., 1976). In the experiments on detection of instantly reassociating (foldback) D N A sequences (Table 1) Sl-nuclease treatment of 14C-DNA preparations was carried out in the conditions described by Vogt (1973): 50 units/ml of Sl-nuclease in 10 - a m M ZnSO,~, 0.05 M NaCI, 0.03 M Na-acetate (pH 4.5 for 1 h at 45 ~ C). Incubation with DNase I (Worthington, USA) was carried out at 37 ~ C in 0.01 M tris-HC1 buffer (pH 7.5), 0.01 M MgC12 and 20 gg/ml DNase I. After incubation with the enzyme the samples were precipitated on nitrocellulose filters (HUFS, Czechoslovakia) and the radioactivity was measured in a stahdard toluene scintillator.

3. Hydroxyapatite Fractionalion ofDNA. Preparative fractionation of partially reassociated 1~C-DNA and isolation o f a foldback D N A fraction were performed chroma~ographicaIly on a hydroxyapatite (HAP, Bio-Rad, USA) column ( G r a h a m et al., 1974). Ressociation kinetics curves were based on the results of D N A fractionation on H A P suspensions in centrifuge tubes (Kupriyanova et al., I976). Fractionation procedures and centrifugation were conducted at 55 ~ C. Phosphate buffer (PB) was prepared from an equimolar mixture of NaHzPO4 and Na2HPO4. Single-stranded D N A was eluted with 0.12 M PB and double-stranded D N A with 0.4 M PB (pH 6.8). Radioactivity of the fractions was measured on a counter Mark II (Nuclear Chicago, USA) in the following mixture: 3 ml of sample (I*C-DNA in 0.12 M PB with or without HAP), 3 ml of scintillator (8 mg/ml PPO and 0.2 mg/ml POPOP in toluene) and 2 ml of Triton X-100. A correction was made for the decrease in counting efficiency caused by H A P crystals. In some experiments (Fig. 6), after H A P fractionation, the optical density of the sample was measured at 260 nm. The fractions were then precipitated in 5% trichroacetic acid in the presence of a carrier, the precipitations were collected on nitrocellulose filters and the radioactivity was measured in a standard toluene scintilIa~or.

4. DNA Melting. Changes in the optical density during heating and cooling of D N A solutions in 0.12 M PB was registered on a spectrophotometer SPh-4A (LOMO, USSR). The temperature was measured directly in water-jacketed cuvettes using a calibrated thermopair put in a glass capillary.

5. Equilibrium CsCl Density Gradient Centr~ugation. The CsCI density gradient centrifugation was carried out in a K-32 (Soviet model) ultracentrifuge at 2 0 ~ for 60 h at 34,000 rpm using a fixed-angle rotor U-65 according to the method of F l a m m et ai. (1966), with a slight modification (Borchsenius et al., 1977). The density of the CsC1 solutions was calculated from the refractive indices measured on a refractometer JRF-23.

6. Radioiodination ofDNA. A solution containing 5 gg denatured D N A in 20 gl of 0.3 M Na-acetate buffer (pH 4.5) with 5 g M T1C12 (Ventron, USA) was mixed with 20 l-tl of Nal~SI solution (1.5 mCi without carrier), preliminarily incubated with 0.2 m M Na2SO 4 for 30 rain at 0 ~ C. The mixture

278

S.N. Borchsenius et al.

in a sealed ampoule was heated to 100~ C for 2 min, cooled and diluted in water to 0.5 ml (Prensky, 1975). 12SI-DNA was separated from low molecular weight substances on a column of Sephadex G-25 in water, treated for 15 min at 65~ purified on hydroxyapatite (adsorption and washout in 0.05 M PB followed by elution in 0.4 M PB) and dialysed against water. The original specific radioactivity of the 12SI-DNA preparation, measured after precipitation with trichloracetic acid in a GC-counter (Intertechnique, France), was 2.6 x l0 s cpm/gg.

Results

Measurements of Genome Size Reassociation kinetics of Tetrahymena macronuclear 14C-DNA fragments 240 nucleotides in length are given in Figure 1 (curve 1). The shape of the curve indicates the absence of distinct repetitive classes of D N A nucleotide sequences. To give a more precise description of the reassociation kinetics of single copy and repetitive sequences the non-repetitive 14C-DNA fraction was separated from repetitive D N A on a H A P column after annealing a t C o t - 4 0 . The reassociation kinetics of this fraction are presented by an ideal S-shaped curve (Fig. 1, Curve 2) with a slope (ratio of the Cot values at terminal and initial renaturation points) equal to 100. It is seen that the shape of this curve is close to that of Escherichia coli D N A under identical reassociation conditions (Fig. 1, curve 4). Therefore, this D N A fraction (74% of total M a D N A ) is represented exclusively by single copy sequences. The C0t~/2 value for this D N A fraction determined graphically is 160+10. This value was corrected for the GC content of Tetrahymena D N A according to Wetmur and Davidson (1968). The calculated value of Cotl/2=100+_ 10 is 33 times the Cotl/2=3 for E. coli D N A . Thus, the kinetic complexity of the D N A fraction containing only single copy sequences is 1.5 x 108 base pairs (bp). It follows from this that the genome size of the whole macronucleus is 2 x 108 bp. Reassociation kinetics of the a4C-DNA fraction which adhered to H A P after annealing to Cot = 40 (26% of the genome) are shown in Figure 1 (curve 3). About 15% of this fraction (or about 4% of the genome) is bound to H A P almost immediately after denaturation and even at a minimum initial concentration of ~4C-DNA. A detailed analysis of this D N A subfraction will be given below. Another 15% of this fraction adhered to H A P after annealing in the range of Cot=0.1-20. The remaining 60-70% of the D N A (16-18% of the genome) renatures at a rate characteristic of non-repetitive D N A nucleotide sequences (Fig. 1). Thus, the whole M a D N A of Tetrahymena can be divided into three classes varying in their reassociation rates: I. The greatest part (about 90% of the genome) is composed of single copy sequences. II. About 5% of the M a D N A is characterized by C0t=0.15-0.4, which is 10-50 times lower than the Cot value for non-repetitive sequences. This sug-

Genome Structure of Tetrahymenapyriform&

IOL~

o

''

279

'2

2O 3O .~

4-0

50 6O 70 8O 90 100 ~

4

I . 10-2

. 10 -1

. 10 0

. 101

,,, 10 2

10 3

Cot

Fig. I. Reassociation kinetics of Tetrahymena Ma DNA measured by binding to hydroxyapatite. The 14C-DNA preparations were mixed with fragments of nonradioactive and nonfractionated Tetrahymena Ma DNA. The fragment length of DNA in all the preparations ranged from 230 to 260 nucleotides. Curve 1-total Ma DNA, curve 2-fiaction of unique sequences (see the text), curve 3-fraction reassociated at Cot=40 (containing all repetitive sequences), curve 4-DNA of

E. eoli

gests that no less than 5% of the Ma D N A is represented by sequences repeated tens of times. As shown by later analysis, however, a still larger portion of the genome (7-9%) falls within the repetitive fraction. III. A D N A fraction with nucleotide sequences which form duplexes without annealing makes up 3-4%. This fraction was isolated and then characterized by Sl-nuclease resistance, buoyant density in CsC1 gradient and thermostability.

Distribution of Repetitive Sequences by DNA Length To study the linear distribution of repetitive nucleotide sequences we carried out reassociation of high molecular weight fragments of D N A followed by S l-nuclease digestion. D N A double-stranded fragments were then fractionated by size. A similar approach was employed earlier by Davidson et al. (1975). Sl-nuclease digestion of single-stranded D N A occurs in two stages. A limited number of breaks induced in a molecule (fast stage of limited hydrolysis) is followed by a slower exonucleolytic hydrolysis. To determine the amount of Sl-nuclease needed for the limited hydrolysis, samples of high molecular weight D N A (40 gg/0.1 ml) after annealing to Co t = 20 were added to various quantities of Sl-nuclease. Increase of the enzyme led to a decrease in the quantity of D N A bound to H A P in 0.12 M PB. The enzyme was used in the ratio (1 unit per 10 gg D N A ) required to complete the fast reaction stage. Under such conditions most single-stranded areas are detached from the adjacent double-stranded regions, but no hydrolysis occurs at sites of unpaired D N A bases (Britten et al., 1976). High molecular weight fragments of unlabelled D N A 8000 nucleotides in length were annealed to C o t = 2 0 , treated with Sl-nuclease and fractionated on a Sepharose 2B column (Fig. 2). It is seen that the high molecular weight

280

S.N. Borchsenius et al.

0.8 0.6 0.4 0.2

qr~

C

10 15 20 25 30 35 Fig. 2. Fractionation of TetrahymenaMa DNA fragments after annealing and Sl-nuclease treatment. Gel filtration on a column of Sepharose 2B (2 x60 cm) in 0.12 M PB. The column was initially calibrated with the aid of markers: original DNA preparation (8,000 bp); nucleosomal DNA (150-200 bp); (c) uridine (from left to right). The fraction volume was 2 ml. The fractions denoted by open circles were mixed and then filtered on Sephadex G-75 (see Fig. 3)

0.4 0.3

Fig. 3. Separation of low molecular weight DNA fragments from nucleotides. Fractions 18-26 (see Fig. 2) were filtered on a column of Sephadex G-75 (2.5 x 70 cm) in 0.12 M PB. The volume of eluted fractions was 4 ml

8 0.2 ,,~ 0.1 0

0

5

10

15

20

fraction makes up about 20% of the original D N A . All the material of this fraction is b o u n d to H A P in 0.12 M PB. The molecular weight of this fraction measured by the sedimentation rate in the analytical centrifuge corresponds to 2200 nucleotides. Hence, during reassociation, Sl-nuclease treatment and fractionation of D N A , the fragment length is reduced more than 3 times. In fact, these experimental results show that the M a D N A of Tetrahymena contains long regions of repetitive sequences (no less than 2200 nucleotides). The low molecular weight D N A fraction was separated f r o m nucleotides and short oligonucleotides by gel-filtration on Sephadex G-75 (Fig. 3). As seen f r o m the figure, the fraction of low molecular weight fragments separates perfectly well f r o m nucleotides. This fraction makes up 24% of the original D N A , However, only 20% of the material of this fraction is b o u n d to H A P in 0.12 M PB. Thus double-stranded low molecular weight D N A fragments constitute 4 - 5 % of the original D N A . As seen by the position of this fraction near the nucleosomal D N A marker (Fig. 2) the length of low molecular weight fragments is 200-300 nucleotides. A large a m o u n t of single-stranded D N A in this fraction is accounted for by the conditions of the Sl-nuclease digestion. In Figure 4 the melting curves of isolated low and high molecular weight fractions of D N A are c o m p a r e d to those of native D N A . One can see that these D N A fractions differ considerably in their melting profiles.

Genome Structure of Tetrahymenapyriformis

281 1.00

Fig. 4. Melting of Tetrahymena Ma DNA preparations in 0.12 M PB. Curve 1-reassociated low molecular weight fragments (300 bp) from fractions 4-8 (Fig. 3), curve 2-reassociated high molecular weight fragments (2,000 bp) from fractions 9-13 (Fig. 2), curve 3-original DNA (8,000 bp). Before melting, the reassociated material was separated from single-stranded DNA fragments on hydroxyapatite. After correction for fragment length, the Tm values for these preparations were 76.5~ 81.3~ and 81.6~ respectively

Fig. 5. Fractionation of reassociated t4C-DNA fragments on a column of Sepharose 2B, Gel filtration was carried out in 0.2 M NaC1 with 0.005 M EDTA-Na, pH 7.5. The fraction volume was 2 ml. Column calibration (arrows) is the same as in Figure 2. After denaturation and annealing to Cot=20, 14C-DNA was treated with Sl-nuclease. Material resistant to Sl-nuclease (24% of the original DNA) was separated from nucleotides and single-stranded fragments using Sephadex G-75 and hydroxyapatite chromatography

0.95 % 0.90 "g 0.85

1

0.8O ,,

A,~

3

70

80 T~

i

O.75 60

O ~

90

100

3 2

d. d 1 0

J 10

15

20

25

T h e melting curves of the high m o l e c u l a r weight f r a c t i o n a n d native D N A differ insignificantly. A t the s a m e time, the melting t e m p e r a t u r e of the low m o l e c u l a r weight f r a c t i o n is 5 . 6 ~ lower t h a n t h a t o f native D N A . T h e h y p e r c h r o m i c effect o f the low m o l e c u l a r weight D N A f r a c t i o n is 27%. These d a t a p e r m i t the c o n c l u s i o n t h a t the low m o l e c u l a r weight D N A b o u n d to H A P consists of i m p e r f e c t l y p a i r e d d o u b l e - s t r a n d e d fragments. The low p r e c i s i o n o f base p a i r i n g c o r r e s p o n d s to t h a t o b s e r v e d d u r i n g r e a s s o c i a t i o n o f m o d e r a t e l y repetitive D N A sequences. It is k n o w n t h a t the r e a s s o c i a t i o n rate o f D N A is p r o p o r t i o n a l to f r a g m e n t length. By i n t r o d u c i n g a c o r r e c t i o n for f r a g m e n t length ( W e t m u r a n d D a v i d s o n , 1968) we o b t a i n C o t = 6 2 . 5 (instead o f 20) for the r e a s s o c i a t i o n of f r a g m e n t s 2,200 nucleotides long. C o n s e q u e n t l y , a c o n s i d e r a b l e p a r t o f this f r a c t i o n contains single-copy D N A sequences. The p r o p o r t i o n o f repetitive sequence D N A in this f r a c t i o n m a y be derived f r o m r e a s s o c i a t i o n kinetics. F o r this p u r p o s e the original 1 4 C - D N A p r e p a r a t i o n was a n n e a l e d to C o t = 20 a n d t r e a t e d with S l - n u c l e a s e . N u c l e o t i d e s a n d s i n g l e - s t r a n d e d D N A were separ a t e d by s u b s e q u e n t gel-filtration on S e p h a d e x G-75 and c h r o m a t o g r a p h y on H A P . The m a t e r i a l b o u n d to H A P was eluted with 0.12 M PB and then gelfiltered on S e p h a r o s e 2B (Fig. 5). The a m o u n t of 1 4 C - D N A in the high a n d low m o l e c u l a r weight fractions was 18.1% and 5.9% o f the initial a 4 C - D N A

282

S.N. Borchsenius et al.

Oi 10 20

o. o.o. Oo i

i

i

t

i

i

30

9u

40 5o ~ 60 s~ 70 80

~

90 100

I

10-2

I

10-~

I

100

I

101

I

102

I

103

Fig. 6. Reassociation kinetics of long repetitive sequences of Tetrahymena Ma DNA measured by binding on hydroxyapatite.-The 14C-DNA preparation (long repetitive sequences) was mixed with non-radioactive and nonfractionated Ma DNA (1:200) and fragmented to 300 nucleotides. After annealing and separation on hydroxyapatite the optical density (open circles) and radioactivity (solid circles) were measured in each sample. Curve 1-reassociation kinetics of total Ma DNA, curve 2 of a fraction containing all repetitive sequences (see also Fig. 1). The circles are experimental results while the curves are given for comparison

respectively. These results are nearly consistent with those for unlabelled D N A (Figs. 2, 3). A high molecular weight D N A fraction was collected and 14C-DNA was precipitated with an excess of unfractionated unlabeled Ma DNA. Then the D N A was fragmented (to 300 nucleotides) and used for reassociation kinetics studies (Fig. 6). It is seen from the reassociation curves that about 12% of this D N A fraction (2.1% of the total DNA) bound to HAP without annealing and is likely to contain foldback self-complementary sequences. No reassociation occurs in the Co t range from zero to 0.5, which indicates the absence of highly repetitive sequences in the high molecular weight D N A fraction. Within the range of Cot=0.5-10 more than 20% of the D N A reassociates. Consequently, moderately repeated long nucleotide sequences (above 2,000 bp) make up about 3 4 % of the Ma DNA. Thus the genome of Tetrahymena contains 7-9% of repetitive sequences, among which 4-5% of the genome is represented by short regions (300 bp) interspersed with unique sequences, and 3-4%, by long nucleotide sequences (more than 2,000 bp).

Palindromic Nature of a Part of the Foldback DNA Fragments of Tetrahymena Ma D N A 250 nucleotides long were applied in 0.12 M PB to a HAP column after denaturing at 100 ~ C. It was found that 5 7% of the total D N A was bound to HAP. Similar fractions are commonly called "foldback D N A " (Wilson and Thomas, 1974; Pearlman et al., 1976). About 80% of the isolated foldback D N A was bound to HAP after repeated denaturation.

Genome Structure of Tetrahymenapyriformis

283

Table 1. Detection of instantly reassociating sequences in DNA of the Tetrahymena macronucleus. The unfractionated preparation and the a4C-DNA fractions (specific radioactivity 600 cpm/gg) in amounts of 5-15 p,g were subjected to an exhaustive treatment with enzymes (see Materials and Methods). Denaturation of the samples was made by heating to I00~ C for 5 min in a 0.015 M NaC1 solution directly before addition of Sl-nuclease. DNase I treatment was used as a control Material

Genome portion

Resistance to enzyme treatment (%) S 1-nuclease

DNase I

Without After Without denaturation denaturation denaturation Foldback DNA fraction

5.9

DNA fraction not reassociating after annealing to Cot=40

74.0

Non fractionated DNA preparation

i00.0

75.0

98.5

Table 2. Relative content of instantly reassociating material in the foldback fraction at different initial DNA concentrations. Samples containing 3.5 gg of ~*C labelled foldback DNA at the indicated concentrations were denatured by heating in a 0.015 M NaCI solution, then cooled rapidly and incubated with Sl-nuclease in equal volumes (0.25 ml). Incubation conditions are described in Materials and Methods

72.5

2.1

2.9

10.5

7.7

2.5

DNA concentration mg/ml

% resistance to Sl-nuclease treatment

0.011 0.027 0.055 0.110

75.9 74.5 72.0 74.0

The d a t a given in Table 1 allowed the p r e l i m i n a r y c o n c l u s i o n that no less t h a n 70% of the f o l d b a c k D N A fraction (3 4 % of the whole M a D N A ) consists of D N A nucleotide sequences reassociating with kinetics characteristic of a first order reaction. Therefore, the c o m p l e m e n t a r y sequences f o r m i n g duplexes d u r i n g reassociation m u s t b e l o n g to one D N A strand, while the original d o u b l e helical structure m u s t consist of inverted sequences or p a l i n d r o m e s . O u r suggestion that the b u l k of the foldback D N A fraction is c o m p o s e d of inverted nucleotide sequences has been s u p p o r t e d by the results of two experiments. I. Several c o n c e n t r a t i o n s of foldback D N A were treated with S l - n u c l e a s e (Table 2). It was f o u n d that the relative a m o u n t of S 1-resistant d o u b l e - s t r a n d e d D N A was i n d e p e n d e n t of the D N A c o n c e n t r a t i o n . II. Figure 7 presents changes in the optical density at 260 n m d u r i n g heat d e n a t u r a t i o n and r e n a t u r a t i o n of the foldback fraction (curve 1). This curve shows reversible hyper- a n d h y p o c h r o m i c effects d u r i n g heating and cooling of the solution. The m a i n melting characteristics (Tin = 81 ~ C ; H = 33%) indicate a high degree of c o m p l e m e n t a r i t y of the f o l d b a c k D N A fraction. A sharp decrease of h y p e r c h r o m i c i t y occurs d u r i n g D N A cooling in the range f r o m 82 ~ to 80 ~ C. The two p o r t i o n s of curve 1 that describe melting a n d r e n a t u r a t i o n ,

284

S.N. Borchsenius et al.

5O

% A,-Ao Ao 40

I I

z

i I

3O 2O

O

,

1

. . . . .

3o

T~ Fig. 7. Optical absorption of Tetrahymena Ma DNA preparations as a function of temperature. DNA solutions in 0.12 M PB were heated at the rate of 0.5~ C per min and cooled at the rate of 3~ C per rain in a spectrophotometer cuvette. Curve l-foldback DNA fraction of a length of 250 nucleotides, curve 2-fraction of inverted (palindromic) sequences obtained after S1-nuclease treatment and deproteinization of the foldback DNA fraction, curve 3 -native DNA, curve 4 - t h e DNA fraction of a length of 250 nucleotides which reassociated in the Cot range from 0.01 to 2O

respectively, are n e a r l y s y m m e t r i c a l (T . . . . t . = 81 ~ C; H = - 26%). C h a n g e s in the optical densities of the D N A s o l u t i o n are r e p r o d u c i b l e after r e p e a t e d h e a t i n g and cooling. S l - n u c l e a s e t r e a t m e n t followed b y d e p r o t e i n i z a t i o n a n d dialysis against 0,12 M PB causes an a l m o s t c o m p l e t e loss of reversibility of the h y p e r c h r o m i c effect (Fig. 7, curve 2). This is p r e s u m a b l y c o n n e c t e d with the fact t h a t S1nuclease digests s i n g l e - s t r a n d e d l o o p s which connect d o u b l e - s t r a n d e d self-comp l e m e n t a r y sequences of the f o l d b a c k D N A fraction. U p o n h e a t d e n a t u r a t i o n D N A duplexes resistant to S l - n u c l e a s e are split into s i n g l e - s t r a n d e d D N A chains. T h e latter are c a p a b l e of s u b s e q u e n t r e a s s o c i a t i o n only with the kinetics of a second o r d e r reaction. T h e melting curves of the original u n f r a c t i o n a t e d D N A and the D N A fraction which was r e a s s o c i a t e d in the range of C o t = 0 . 0 1 - 2 0 (Fig. 7, curves 3 a n d 4) are given in the same figure. As expected these melting curves differ significantly. The D N A fraction which was r e a s s o c i a t e d in the r a n g e of Cot = 0.01-20 m a k e s u p 15% of the M a D N A a n d p r o b a b l y contains no f o l d b a c k s e l f - c o m p l e m e n t a r y sequences (see Fig. 1). This D N A f r a c t i o n differs f r o m native

Gerlome Structure of Tetrahymenapyriformis

285

['4c] [3H] 1.74 4

1.73

b3

1.72 1.71

~.' 2

1.70 1.69

0

1.68 1,67 1.66 ,

3O

40

%,

~ ....

50

.._o.H'"

60

I

,

70

Fig. 8. Equilibrium distribution of inverted (palindromic) :4C-DNA sequences (1) and native Tetrahymena Ma 3H-DNA (2) in a CsC1 density gradient. The specific radioactivity of the 14C-DNA preparation was 600 cpm/#g and that of the 3H-DNA was 5000 cpm/gg

D N A and the foldback fraction by less cooperativity of melting and less hyperchromicity. Melting of the native D N A preparation occurs within a narrow temperature range and shows a large hyperchromic effect ( A T + 6 ~ C; Tin+82 ~ C; H = 4 0 % ) . Subsequent cooling results in a rapid decrease of optical density ( H = - 4 % ) , which constitutes only 10% of the original hyperchromic effect. Repeated heating and cooling induce reversible changes of the optical density equivalent to about 7% of the original hyperchromic effect (Fig. 7, curve 3).

Nucleotide Content and Renaturation of Palindromic Sequences The foldback 14C-DNA fraction isolated after Sl-nuclease treatment was subjected to CsC1 density gradient equilibrium centrifugation. Comparison of the foldback t 4 C - D N A fraction (Fig. 8, curve 1) and the native unfractionated 3 H - D N A (curve 2) shows that the average G + C contents of the two are similar. The buoyant density of the foldback fraction after S1 nuclease treatment indicates a perfect double-stranded structure for this D N A . The G + C content of r D N A is 36% as compared to 28% for total Tetrahymena D N A (Engberg and Pearlmann, 1972). These values correspond to 1.696 g . c m 3 and 1.684 g - c m -3 for the buoyant densities of the D N A s in CsCI gradient. In the density gradient profile of foldback 14C-DNA one can see a distinct shoulder whose buoyant density corresponds to that of r D N A . The material in this gradient region constitutes about a quarter of the total radioactivity of inverted sequences of the foldback fraction and is likely to contain r D N A . The nature of the remaining 75-80% of palindromic D N A of this fraction

286

S.N. Borchsenius et al. O,

2O % ."g 40 o c0

60 80

100

t

I Jill11]

1

10-1

I IIl[ll[

I

100

I r I[[[[]

101 Cot

T

I lilt[It

I

102

t I(1111]

1

1 iii

103

Fig.9. Reassociation kinetics of palindromic nucleotide sequences obtained from the foldback fraction of Tetrahymena Ma DNA. The ~zSI-DNA preparation of palindromic sequences was mixed with nonradioactive and nonfractionated Ma D N A (1 : 3,000) and fragmented to 300 nucleotides. Open circles: annealing in 0,12 M PB and separation on hydroxyapatite. Solid circles: annealing in PIPES buffer and Sl-nuclease treatment of reassociated material. The curve is given for comparison (see Fig. 1)

remains to be investigated. The broader 14C-DNA distribution compared to the native D N A is easily explained by the smaller size of the D N A molecules of this fraction. It was of interest to estimate the repetitive frequency of palindromic sequences in the Tetrahymena Ma D N A and the degree of their homology to the sequences of other classes. For this purpose the foldback D N A fraction after S1 nuclease treatment was labelled in vitro with 125I. The fragmented non-radioactive Ma D N A was added in excess to the 125I-DNA and the reassociation kinetics was then investigated (Fig. 9). The results obtained from the experiment permit the conclusion that about 20% of the palindromic sequences are repeated tens of times in the Tetrahymena genome, while the remaining palindromic sequences are likely to be unique. Discussion

The genome structure of Tetrahymena is characterized by a low content of repetitive sequences. Short repetitive sequences constitute 4-5% of the Ma DNA. Assuming that short 300 bp nucleotide sequences alternate with unique regions of the order of 1000 bp, as occurs in metazoa, it may be concluded that only 15% of the Tetrahymena genome is composed of such interspersed regions. About 80% of the unique sequences do not alternate with repetitive sequences. This assumption is supported by the results of experiments on reassociation of short (250 nucleotides) fragments of non-labelled D N A with 14C-DNA fragments of different lengths (Borchsenius et al., 1978). In other organisms studied, with the exception of Drosophila and the bee, 65-80% of the unique D N A regions intersperse with short repetitive sequences constituting about 50% of the genome (Davidson et al., 1975). Another part

Genome Structure of Tetrahymenapyriformis

287

of the D N A repetitive regions of Tetrahymena (3-4%) consist of sequences more than 2000 bp in length. We conclude that the distribution of repetitive sequences in the Ma D N A of Tetrahymena is difficult to refer to any known type of genome organization. It has been recently demonstrated that Tetrahymena ribosomal D N A (rDNA) has a palindromic structure. The extrachromosomal r D N A molecule containing two ribosomal genes separated by a small spacer region is a giant (12.6 x 1 0 6 dalton) palindrome (Karrer and Gall, 1976; Engberg et al., 1976). If the total Ma D N A contains ~ 1% of sequences complementary to rRNA, the r D N A (taking into consideration pre-rRNA processing) should make up no less than 2% of the Ma D N A (Engberg et al., 1976). Fragmentation of the D N A inevitably induces breaks in the long palindromic structure of r D N A and other palindromic sequences. Therefore, D N A with a fragment length of 250 nucleotides must contain some sequences derived from palindromes which reassociate according to second order kinetics. It was shown that 17S and 25S r R N A genes are repeated 200 fold per haploid genome in the Ma of Tetrahymena (Engberg et al., 1976). Consequently, under our experimental conditions a portion of the r D N A palindromic sequences must be classed with the fraction of repetitive sequences. From this viewpoint it is not inconceivable that our evaluation of the amount of palindromic sequences in the Tetrahymena genome (3-4%) is understated. On the other hand, the optical data on nonfragmented material (Fig. 7, curve 3) enable us to conclude that the total amount of inverted sequences in Ma D N A must not exceed 7%. It appears that most of the inverted sequence fraction is composed of palindromic structures, which do not belong to r D N A and are present only once in the Tetrahymena genome. This was demonstrated in the CsC1 centrifugation experiments and by reassociation of 125I-DNA fragments (Figs. 8, 9). A preliminary assessment of the size of these structures based on reassociation of D N A fragments of various length at a minimum Cot yields a value of 230 bp (Borchsenius et al., 1978). At least four hypotheses exist concerning the functions of these structures. I.'Palindromes provide a mechanism preventing the accumulation of mutations in the genome via spontaneous formation of cruciform structures, excision of incorrectly paired bases and repair of breaks (Thomas et al., 1974). II. Palindromes of small length serve as templates for hairpin formation in pre-mRNA. The hairpins in turn may serve as recogition sites for enzymes participating in processing (Ryskov et al., 1973). III. Single-copy palindromes may be present in the structure of all genes as specific signal points (Kupriyanova et al., 1976). IV. Palindromes localized at the ends of linear D N A molecules are indispensable for replication of 5'-ends of maternal strands (Cavalier and Smith, 1974). None of the above hypotheses has been either rejected or supported. Consequently Tetrahymena, whose D N A is characterized by a high content of inverted sequences along with a relatively low content of ordinary repetitive sequences, my be used as a suitable test subject for studying the origin and function of palindromic structures.

288

S.N. Borchsenius et al.

Acknowledgements. The authors express their gratitude to Dr. I.S. Irlina for conducting ciliate culture, Dr. T.N. Osipova for determining DNA molecular weight in an analytical centrifuge, Dr. V.A. Pospelov, for supplying a DNA preparation from mononucleosomes, N.S. Kupriyanova and V.V. Kulguskin for their assistance in preparing lzsI-DNA.

References Allen, S.L., Gibson, J.: Genome amplification and gene expression in the ciliate macronucleus. Biochem. Genet. 6, 293-313 (1972) Ammermann, D., Steinbrfick, G., Berger, L. von, Hennig, W. : The development of the macronucleus in the ciliated protozoan Stylonichia mytilus. Chromosoma (Berl.) 33, 209-238 (1974) Borchsenius, S.N., Belozerskaya, N.A., Merkulova, N.A., Irlina, I.S., Vorob'ev, V.I.: Possible repeated replication of a fraction of nuclear DNA in Tetrahymena during a single S-Phase. Molek. Biol. (Russ.) 11, 171-180 (1977) Borchsenius, S.N., Belozerskaya, N.A., Merkulova, N.A., Vorob'ev, V.I.: Unique, repetitive and palindromic sequences of Tetrahymena macronuclear DNA. Molek. Biol. (Russ.) 12, 676-688 (1978) Britten, R.J., Graham, D.E., Eden, F.C., Painchaud, D.M., Davidson, E.H.: Evolutionary divergence and length of repetitive sequences in sea urchin DNA. J. molec. Evol. 9, 1 23 (1976) Cavalier-Smith, T.: Palindromic base sequences and replication of eukaryote chromosome ends. Nature (Lond.) 250, 467-470 (1974) Crain, W.R., Davidson, E.H., Britten, R.J.: Contrasting patterns of DNA sequence arrangement in Apis mellifera (Honeybee) and Musca domestica (Housefly). Chromosoma (Bed.) 59, 1 12, (1976a) Crain, W.R., Eden, F.C., Pearson, W.R., Davidson, E.H., Britten, R.J.: Absence of short period interspersion of repetitive and nonrepetitive sequences in the DNA of Drosophila melanogaster. Chromosoma (Bed.) 56, 309-326 (1976) Davidson, E.H., Galau, G.A., Angerer, R.S., Britten, R.J. : Comparative aspects of DNA organization in metazoa. Chromosoma (Berl.) ill, 253-259 (1975) Engberg, J., Anderson, P., Leick, V., Collins, J.: Free ribosomal DNA molecules from Tetrahymena pyriformis GL are Giant Palindromes. J. molec. Biol. 104, 455-470 (1976) Engberg, J., Pearlman, R.E.: The amount of ribosomal RNA genes in Tetrahymena pyriformis in different physiological states. Europ. J. Biochem. 26, 393-400 (1972) Firtel, R.A., Kindle, K. : Structural organization of the genome of the cellular slime mold : interspersion of repetitive and single-copy DNA sequences. Cell 5, 401-411 (1975) Flamm, W.G., Bond, H.E., Burr, H.E.: Density gradient centrifugation of DNA in a fixed angle rotor. Biochim. biophys. Acta (Amst.) 129, 310-319 (1966) Graham, D.E., Neufeld, B.R., Davidson, E.H., Britten, R.J. : Interspersion of repetitive and nonrepetitive DNA sequences in the sea urchin genome. Cell 1, 127-136 (1974) Irlina, I.S., Merkulova, N.A.: The growing in volume and mass of Tetrahymena pyriformis for biochemical purposes and the synchronization of division. Cytology (Russ.) 17, 1208-1215 (1975) Karrer, K.M., Gall, J.G.: The macronuclear ribosomal DNA of Tetrahymena pyriformis is a palindrome. J. molec. Biol. 104, 421-454 (1976) Kupriyanova, N.S., Timofeeva, M.Ja., Baev, A.A.: Detection and characterization of the palindromes in a loach genome. Molek. Biol. (Russ.) 10, 412-422 (1976) Manning, J.E., Schmidt, C.W., Davidson, N.: Interspersion of repetitive and nonrepetitive DNA sequences in the Drosophila melanogaster genome. Cell 4, 141-155 (1975) Perlman, S., Phillips, C., Bishop, J.O. : A study of fold-back DNA. Cell 8, 33-42 (1976) Prensky, W. : The radioiodination of the RNA and DNA to high specific activities. In: Methods in cell biology (D.M. Prescott, ed.), vol. 13, pp. 121-152. New York-London: Academic Press 1976 Raikov, I.B. : Evolution of macronuclear organization. Ann. Rev. Genet. 10, 413-440 (1976) Ryskov, A.P., Saunders, G.F., Farashyan, V.R., Georgiev, G.P. : Double-helical regions in nuclear precursor of m-RNA (pre-m RNA). Biochim. biophys. Acta (Amst.) 312, 152 164 (1973)

Genome Structure of Tetrahymenapyriformis

289

Thomas, C.A., Jr., Pyeritz, R.E., Wilson, D.A., Dancis, B.M., Lee, C.S., Bick, M.D., Huang, H.L., Zimm, B.H.: Cyclodromes and palindromes in chromosomes. Cold Spr. Harb. Syrup. quant. Biol. 38, 353-370 (1974) Vogt, V.H.: Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Europ. J. Biochem. 33, 192-200 (1973) Wetmur, J.G., Davidson, N. : Kinetics of renaturation of DNA. J. molec. Biol. 31, 349-370 (1968) Wilson, D.A., Thomas, C.A., Jr. : Palindromes in chromosomes. J. molec. Biol. 84, 115 138 (1974) Yao, M.-C., Gorovsky, M.A.: Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis. Chromosoma (Berl.) 48, 1 18 (1974) Zimmerman, I.L., Goldberg, R.B. : DNA sequence organization in the genome of Nicotiana tabacure. Chromosoma (Berl.) 59, 227 252 (1976)

Received May 6-August 12, 1978 / Accepted July 10, 1978 by J.G. Gall Ready for press August 15, 1978