{'fraction_non_alphanumeric': 0.041458394589826524, 'fraction_numerical': 0.03311526021758306, 'mean_word_length': 4.553990610328638, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Macronuclear DNA in Stentor coeruleus: a first approach to its characterization

The macronuclei of Stentor coeruleus were isolated on a discontinuous sucrose gradient and their DNA was purified by conventional methods. The GC content was 32 mole %. The DNA banded as a single peak on analytical ultracentrifugation at 1-691 g/cm 3 . The molecular weight of the DNA was 5 x 10 6 to 4 x 10 7 daltons. Genome size determined by DNA-DNA reassociation kinetics was 6 x 10 10 daltons. The macronuclear genome was mostly simple, about 85 % being made of non-repetitive sequences. Electron micrograph of Stentor macroauclear DNA spread by a modified Kleinschmidt's technique and shadowed with Pt-Pd. Inset: electron micrograph of a partial denatured DNA molecule. A preliminary study showing AT-rich regions.


INTRODUCTION
Stentor coeruleus is a common freshwater protozoan which contains two different types of nuclei: a nodular macronucleus and several micronuclei (Plate 1, fig. 1). In a general way, genetic continuity is maintained in ciliates by the micronucleus through mitosis and conjugation. In heterotrichs, micronuclear function and formation of the macronucleus is not well understood. Conjugation is observed in Blepharisma (Giese, 1973;Miyake & Beyer, 1973). In Spirostomum, conjugation is rare, but macronuclear development with well-organized chromosomes has been described by Rao (1968). Little information is available for Stentor coeruleus. In this species, conjugation is a very rare event (Plate 1, fig. 2) (Tartar, 1961). It sometimes appears in laboratory cultures, but no reproducible method for inducing it has been reported. It is possible that autogamy occurs, but it has not been reported to date. The macronucleus of ciliates has essentially a metabolic function. A few years ago, the macronucleus was considered simply as highly polyploid, i.e. containing numerous copies of the micronuclear genome. More recently a series of important investigations on the genetic apparatus of Stylonychia on one hand Prescott, Murti & Bostock, 1973) and on Tetrahymena pyriformis and Paramecium aurelia on the other hand (Allen & Gibson, 1972;Yao & Gorovsky, 1974) have been reported. This ultrastructural and biochemical information led to a new concept of the genetic apparatus in ciliate protozoa. Studies on hypotrichs show that important differences exist in the molecular weight, buoyant density and thermal denaturation profiles for the micro-and macronuclear DNA. T. pyriformis and P. aurelia are two ciliates with a much simpler pattern of 278 B. PELVAT AND G. DE HALLER macronuclear development and they do not show these molecular differences. The origin, organization and formation of the macronucleus may thus be different in the various groups of ciliates. To our knowledge, molecular characteristics of heterotrich ciliate DNA have not been studied, except for some GC characteristics in Blepharisma (McCarthy, 1965) and Stentor (Mandel & Muzyka, 1971). In contrast, timing of DNA and RNA synthesis has been extensively studied in the macronucleus of Stentor and Blepharisma during the cellular cycle and regeneration (Giese, 1973;de Terra, 1974;Gavurin & Hirshfield, 1974;Pelvat, Hyvert & de Haller, 1973). Thus characterization of the macronuclear DNA becomes necessary to understand at a molecular level the role that the macronucleus plays in morphogenetic processes. In this paper, we report some of the characteristics of the genome in S. coeruleus, principally the GC content, molecular-weight estimation and renaturation kinetics. We compare our results with those obtained with other free-living ciliates. Our results suggest that the genome of S. coeruleus is more similar to the genetic material of T. pyriformis and P. aurelia than to that of Stylonychia and other hypotrichs studied to date.

MATERIAL AND METHODS
(i) Culture of Stentors The strain of S. coeruleus used in this study was isolated from a pond near Geneva. Clones were grown in plastic dishes (11 capacity) in modified Carter's medium containing 0-5 mM-KCl, 2 mia-NaCl, 0-2 mM-MgCl 2 , 0-5 mM-CaCl 2 and buffered at pH 7 with 0-8 mM-NaH 2 PO 4 . Good cultures (250 animals/ml) could be obtained when feeding the ciliates every day with Chlorogonium elongatum (Cambridge strain 12/2c) axenically grown in artificial medium (de Haller & Rouiller, 1961) at 18 °C with a light:dark cycle of 12:12 h. The flagellates were centrifuged and washed with Carter's medium prior to feeding the ciliates. Stentor with normal daylight illumination, fresh medium and a constant food supply have a generation time of 18-20 h.
(ii) Ultrastructural studies Starved animals were fixed in 3 % glutaraldehyde in 1/15 M phosphate buffer (PB) (Sorensen) pH 7 for 20 min, then a rinse solution of 1/15 M PB was added slowly and the cells were subsequently rinsed for three 20 min periods in rinse solution. The cells were postfixed in 2 % OsO 4 in 1/15 M PB, pH 7-5 and then dehydrated in ethanol and embedded in Spurr or Epon-Araldite mixture. Thin sections were cut on a Reichert 0mU2 ultramicrotome, stained with uranyl acetate and lead citrate, and examined in a Hitachi 7 A electron microscope.
(iii) Isolation of nuclei One to 2 1 of ciliate culture starved for 2 days, was concentrated in a small continuous flow centrifuge and the nuclei isolated as described by Prescott et cd. (1971) with the following modifications: concentrated cells were lysed at 0 °C in 0-01 M Tris-HCl containing 0-5% (vol./vol.) Triton X-100 and 0-004% (wt/vol.) spermidine phosphate trihydrate (pH 6-9). Triton X-100 is known to have an inhibitory effect on DNAse. Stentors were left in this solution for a few minutes and the cells were broken by rapid mixing with a pipette. The nuclei were concentrated by centrifugation (5 min, 1000 g), resuspended in the same medium and 5 ml of this nuclei suspension was layered on a discontinuous sucrose gradient (15 ml 60 %, 15 ml 40 %, 5 ml 20 %) made in a 50 ml round-bottom plastic centrifuge tube. The gradient was run for 10 min at 300 g (600 rev/min) in a MSE super minor centrifuge in a cold room at 2 °C. All the sucrose solutions contained 0-1 % (vol./vol.) of Triton X-100 and 0-01 % (wt/vol.) of spermidine. Macronuclei formed a grey pellet free of contaminants. Isolated macronuclei were either used immediately for preparation of DNA, or quickly frozen at -30 °C.
(iv) Extraction and purification of DNA Routinely, the DNA was extracted from macronuclear preparations or whole cells by Kirby's conventional phenol method (1968) with some modifications. Macronuclei or packed cells were lysed in 2 ml of saline-EDTA (0-1 M-NaCl, 0-1 M -E D T A , pH 8 adjusted with NaOH) containing 1 % SDS, and gently shaken at room temperature for 15 min in a stoppered glass tube with an equal volume of phenol saturated with saline-EDTA (pH 8). The emulsion was centrifuged 10 min at 15000#. The aqueous layer containing the nucleic acids was removed, deproteinized once more by the same treatment and centrifuged. The aqueous phase was then treated with Sevag's solvent (chloroform/isoamyl alcohol 24:1, v/v) to complete deproteinization. The crude DNA was precipitated with 2 vols. of cold ethanol and spooled onto a glass rod. DNA was then dissolved in 0-1 x SSC (1 x SSC = 0-15 M-NaCl, 0-015 M sodium citrate, pH 7) or in Tris-EDTA buffer at pH 7 (10 mM-NaCl, 15 HIM Tris, 1 mM EDTA) and treated, first with 100/jg/ml of RNAse (DNAse-free, Miles) and 1000 units/ml RNAse Tl for 1 h at 37 °C and then with 500 /^g/ml pronase (nuclease-free, Calbiochem) for 1 h at 37 °C. Subsequently, the solution was treated (usually 3 x) with Sevag's solvent until no interphase layer was present. DNA was precipitated with cold ethanol, redissolved in 0-1 x SSC and dialysed against 0-1 x SSC if DNA was used immediately.
When little material was available and undegraded DNA was desired, another method was developed: isolated macronuclei lysed in saline-EDTA at pH 8, containing 1 % SDS, was treated with RNAse (150/ig/ml) for 1 h at 37 °C and subsequently for 2 h at 37 °C with pronase (1 mg/ml). The solution was shaken 2 x with Sevag's solvent and then mixed with solid CsCl to a density of 1-6 g/cm 3 and centrifuged for 30 h at 35000 rev/min in a fixed-angle rotor at 20 °C. DNA was recovered from the bottom of the centrifuge tube. The purity of all DNA samples was estimated by UV absorption spectra with a Beckman 24 spectrophotometer.
(v) Determination of thermal transition {thermal melting) of the DNA The general procedure was first described by Mandel & Marmur (1968) and Marmur & Doty (1962). Thermal melting being dependent on the ionic strength of. 280 B. PELVAT AND G. DE HALLER the solvent, all measurements were made in 0-1 xSSC and the GC content calculated from the T m (temp, for half denaturation) with the following equation, for this SSC dilution: GC = (T m -53-9) 2-44 . Thermal denaturation was followed using a digital Beckman 24 spectrophotometer equipped with a Haake thermostat and coupled to an external recorder for optical density and temperature increase. The cuvettes contained 0-7 ml of DNA dissolved in 0-1 SSC at a concentration of 1 OD/ml. The rate of temperature increase was 1 °C/min during melting.

(vi) Analytical CsCl gradient ultracentrifugation
The method of Mandel, Schildkraut & Marmur (1968) was followed for CsCl gradient centrifugation. The DNA samples were dissolved in 0-1 x SSC and adjusted to a density of 1-710 g/cm 3 with solid CsCl and centrifuged 20 h at 40 000 rev/min in a Spinco model E analytical ultracentrifuge equipped with a photoelectric scanning recording system. M. lysodeikticus DNA (density 1-731) was added as a reference marker for the determination of the buoyant density of the Stentor DNA.
(vii) Sucrose-gradient centrifugation Purified DNA, dissolved in 0-1 M Tris, 001 M EDTA, 0-1 M-NaCl, pH 7, was layered on the top of a 5-20 % (w/v) sucrose density gradient. The tubes were chilled to 10 °C and centrifuged at 25000 rpm in a MSE HS 25 (SW rotor) for 16 h. DNA sedimentation was studied with two markers of known S value and molecular weight: lambda DNA (33 S) and 16 + 23 S ribosomal RNA. At the end of the run, fractions were collected from the bottom of the tube and their optical density read in a spectrophotometer at 260 nm. Determination of the molecular weight of DNA was carried out using the relationship developed by Burgi & Hershey (1963). For 3 H counting the DNA was precipated with an equal volume of 10% TCA, collected on Whatman GF/F glass filters, washed with cold 5 % TCA, cold 95 % ethanol and dried. Radioactivity was measured by liquid scintillation counting and cpm inserted on an absorption profile graphic.

(viii) Chrornatographic determination of the molecular weight on DEAE-celhdose
The method of DEAE-cellulose chromatography coupled with centrifugation (Davila, Charles & Ledoux, 1965;Mingot & Davila, 1974) allows the estimation of the molecular-weight heterogeneity of DNA preparations and was used as a complementary test to sucrose gradient centrifugation. In summary, DNA samples dissolved in 0-01 M phosphate buffer, pH 7, were fixed at pH 7 to the cellulose. The sample and eluants were then placed in the upper part of a pierced tube, and passage through the cellulose facilited by mild centrifugation (100g for 5 min). Each eluate was collected from the lower compartment before loading with the next eluant. This technique allows selective elution of DNA bound to the matrix by modifying the type of buffer and its ionic strength: phosphate buffer with increasing NaCl molarity, followed by NH 4 0H/NaCl and NaOH (for details see Mingot & Davila, 1974 (ix) Preparation of DNA for electron microscopy DNA samples were prepared for electron microscopy by a modified Kleinschmidt method (Davis, Simon & Davidson, 1971;Caro, Sechaud, Boy de la Tour, personal communication, 1975). The spreading solution contained 10 /A of DNA dissolved in Tris/EDTA buffer, 30 /i\ H 2 O, 10/el Tris 1 M -E D T A 0-1 M, 40 /A formamide and 10 [A cytochrome c (0-1%). The hypophase was twice-distilled water. The DNA film was picked up on the surface of the grid, stained with uranyl-acetate, rotary-shadowed with Pt-Pd and observed with a Hitachi 7 A electron microscope. Since partial denaturation studies may provide information on the structure of DNA molecules, we examined Stentor DNA according to the technique used by Murti, Prescott & Pene (1972): DNA dissolved in 0-1 SSC containing 10 % formaldehyde was melted in capillary tubes (15 min at 50 °C) before spreading. DNA filaments were enlarged from electron micrographs with a projector and their contour length determined with an electronic map measurer. Either lambda DNA or simply shadowed latex balls were used as standard. For DNA of normal base composition, molecular weight can be related to length by the following approximation: 1 micron DNA = 1-91 x 10 6 daltons.
The DNA was concentrated, passed through a Chelex-100 column to remove heavy metals and precipitated with 2 vols. of cold ethanol. Precipitated DNA was then dissolved in a minimum volume of 0-12 M PB. DNA was denatured in sealed glass ampoules by heating 15 min at 100 °C at different concentrations. For high DNA concentration and long incubation times, sealed capillaries were used. Heat denatured DNA was immediately incubated at 60 °C in a water bath for different times corresponding to the desired Cot. After reassociation, DNA was cooled, diluted to 2 ml with cold 0-12 M phosphate buffer and immediately applied to a hydroxyapatite column that had been equilibrated with 0-12 M PB at 60 °C. The column consisted of about 1 ml hydroxyapatite (HAP) (packed volume) supported by a disc of porous glass and maintained at 60 °C in a water-jacketed glass tube.
Non-binding single-stranded DNA was eluted with 012 M PB and doublestranded DNA was recovered with 0-4 M PB elution. The ratio, single-stranded/ double stranded allows one to determine, for a given Cot, the percentage of reassociation for a particular DNA. The percentage of reassociation was calculated from OD measurements on the eluates recovered from 0-12 and 0-4 PB elution. E. coli DNA reassociation was followed according to the same system and used as standard. In some experiments, a thermal elution was used for studying stability of hybrids. Reannealed DNA was bound to HAP and the column temperature was B . P E L V A T AND G. D E H A L L E R raised while eluting a fixed concentration of PB (0-12 M PB). As the DNA becomes thermally denatured, it elutes owing to the reduced affinity of the single-stranded for HAP.

ULTRASTRTJCTURE OF THE MACRONUCLEUS
The vegetative macronucleus of S. coeruleus looks like that of other common freshwater ciliates such as Paramecium and Tetrahymena (Plate 1, fig. 3) and it is limited by an envelope consisting of two distinct unit membranes. In the nucleoplasm, small granular dense chromatin bodies, randomly scattered, ranging in size from 0-15 to 0-35 /ira, are very abundant and more or less aggregated. They correspond to what is called 'small bodies' in Paramecium (Sonneborn, 1953;Jurand, Beale & Young, 1962).
Less-numerous larger bodies (0-61 /tm) are thought to be nucleoli. Cytochemical studies have not been made but these masses are probably similar to those found in Paramecium (Ehret & Powers, 1955), and for heterotrichs in Blepharisma (Giese, 1973). S. coeruleus does not typically contain the large nucleoli found in another heterotrich, Climacostomum virens (Peck et al. 1975).
During division, the macronucleus of S. coeruleus condenses to a compact body, but never shows reorganization bands as hypotrichs do. Ultrastructural changes during morphogenetic processes have not been systematically studied in Stentor. Paulin (1973) reports that microtubules appear during the period of macronucleus condensation during regeneration.

DNA CHARACTERIZATION: RESULTS
(i) Determination of GC content Two comparative methods were used: thermal denaturation and analytical isopycnic CsCl centrifugation (Mandel & Marmur, 1968). The hyperchromic shift of Stentor macronuclear DNA due to thermal denaturation in 0-1 SSC was about 35 % and T m (temperature for half denaturation) between 67 and 68 °C (Text- fig. 1). The GC content, calculated following Marmur & Doty (1962) is 31-33 %. Thermal denaturation curves do not reveal heterogeneity: Stentor macronuclear DNA melts as a single component. Isopycnic centrifugation in CsCl with M. lysodeikticus DNA as reference marker (d = 1-731 g/cm 3 ) gives, according to Schildkraut et al.'s relation (1962), a GC content of about 32 %, for a buoyant density of 1-691. This value is in good agreement with that determined by the T m . Text- fig. 2 shows the result of a UV scanner record for Stentor DNA. Stentor DNA shows no detectable satellite band.
(ii) Molecular-weight estimation Three methods were used to estimate the molecular weight and heterogeneity of Stentor macronuclear DNA: DEAE-cellulose chromatography, sucrose-gradient centrifugation and direct measures of electron micrographs. DEAE-cellulose chromatography (Text- fig. 3) and sucrose gradient centrifugation (Text- fig. 4)  somal RNA were used as standards for sucrose-gradient centrifugation. Based upon these standards, the major part of Stentor DNA was estimated to be between 25 and 40S, according to the relation of Burgi & Hershey (1963), and corresponds to a molecular weight of 5 x 10 6 to 4 x 10 7 daltons. Direct measurements on electron micrographs shows molecules of variable length. We unsuccessfully tried to discriminate classes of molecules based upon length, as Ammermann et al. (1974) did for Stylonychia. However, length variation may be an artifact since shearing probably occurs for long molecules. The length of the majority of the molecules and particu" Iarly of the largest ones we found is nevertheless interesting. Out of 121 molecules which were measured, 52 were 15-25 /tm long and correspond to a molecular weight of 3-4 x 10 7 daltons. These molecules probably represent much more than 50 % of the total DNA. Very small (less than 3 fim) and very long molecules (up to 40 /im) are rarer and the largest are difficult to measure.
In Climacostomum, another heterotrich related to Stentor, we have found DNA molecules of the same order of length. Ordinate: optical density. Abscissa: fractions of increasing molecular weight. Fractions 0-4: M.W. 2 X 10 3 to 2 X 10 6 ; fraction 5: 2 x 10 6 ; fraction 6: 6 x 10 6 ; fraction 7: 1, 7 x 10 7 ; fraction 8: 5 x 10' and over. (iii) Renahcration studies The Stentor macronuclear genome was investigated by methods of Britten & Kohne (1966. If the salt concentration, temperature and DNA fragment size (sonicated DNA) are kept constant, the reassociation percentage will depend on the Cot (concentration x incubation time). The reassociation rate will depend on the genomic complexity. Text- fig. 5 shows the reassociation profile oi Stentor DNA in the form of a Cot curve. Zero-time hybridization was found to be less than 2 %. This DNA which reassociates more rapidly than can be measured corresponds to DNA of unknown function, or perhaps to technical artifact. About 10-15% reanneals rapidly, indicating that the major component of this fraction is made of repeated nucleotide sequences. The remainder of the DNA (about 85 %) reassociates with a second-order reaction curve, a kinetic indicating that each sequence is present only once per genome (unique sequences). However, determination of these percentages from renaturation curves may tend to overestimate the number of unique sequences (Hough & Davidson, 1972). No heterogeneity was observed in the more slowly renaturing populations. The Cot value for unique sequences allows one to relate approximately the Cot to the haploid genome size (kinetic complexity, see Wetmur & Davidson, 1968;Britten, 1971). The size of the haploid macronuclear genome in Stentor was calculated with E. coli DNA as reference (Cot \ = 4, genome size = 2-7 x 10 9 daltons) (Cairns, 1963). By using an equation which relates Cot \ to genome size (Britten, 1971) we found about 6-1 x 10 10 daltons per haploid genome for Stentor DNA (Cot \ = 110), which corresponds to a complexity about 20 times greater than for E. coli genome.
Thermal elution was used for studying the stability of hybrids. The melting of some hybrids occurs about 5 °C lower than the native DNA and most other hybrids. As possible explanation to account for the different thermal stability after renaturation, Allen & Gibson (1972) believe that not all copies present in the macronucleus are identical, or more simply, one may hypothesize that a certain degree of mismatching occurs in base pairing during reassociation. DNA base composition has become an important taxonomic criterion (Schildkraut et al. 1962). Generally, the percentage of GC in ciliate DNA is lower than in E. coli DNA (50 % GC): 25-33 % for T. pyriformis strains (Allen & Gibson, 1972), 27-30 % in P. aurelia (Allen & Gibson, 1972Sonneborn, 1975) andin heterotrichs, 3 7 % in Blepharisma (McCarthy, 1965) and 31-33% in S. coeruleus (Mandel & Muzyka, 1971; and this work). The analysis of nucleic acids in free-swimming protozoa is complicated by the fact that very few of them can be cultivated axenically. Additionally, Allen & Gibson (1971) reported that differences in base ratios can be found between bacterized and axenic forms. Cummings (1972) showed that, in P. aurelia, the GC content measured from purified macronuclei is slightly lower (21-26%) than from whole cells (29-30%). In our case, it was interesting to see if the Stentor we used, from local origin, had the same composition as those studied by Mandel & Muzyka (1971), and to compare our results, obtained on isolated macronuclei, with their work on whole cells. Our results are generally in good agreement with theirs, except that we found a 1-2 % lower GC content.
The DNA from many eukaryotes exhibits, in CsCl, one or more minor bands, called satellites. In ciliates, satellite DNA has been found in some strains of P. aurelia (Soldo & Godoy, 1972) and in T. pyriformis strain GL (Sueoka, 1961). Gall (1974) and Engberg et al. (1974) have shown that macronuclear satellite DNA of T. pyriformis GL corresponds to ribosomal cistrons. In another strain, T. pyriformis WH-6, no detectable satellites were found by Yao & Gorovsky (1974) and no marked differences between micro-and macronuclear DNA base composition was observed. In contrast, the DNA of the macronucleus of Stylonychia is quite homogeneous in CsCl gradients when compared to the DNA of the micronucleus (Prescott et al. 1973;Ammermann et al. 1974). S. coeruleus macronuclear DNA bands as a single, homogeneous component, showing no satellite bands. The ultracentrifugation curve, published by Mandel & Muzyka (1971) for Stentor shows no satellite either. However, in some species, satellite DNA is difficult to demonstrate, since its base composition is quite similar to that of the main band (Tobia et al. 1972).

(ii) Molecular weight
Estimation of the molecular weight of eukaryotic DNA is difficult. The content in nucleic acids is greater than in viruses and bacteria and it is almost impossible to isolate eukaryote DNA without degradation: mechanical shearing and other factors such as specific site intracellular nucleases may occur and explain, for example, the heterogeneity observed in measurements on electron micrographs. Two basically different molecular-weight classes of macronuclear DNA, according to the mode of formation of the macronucleus, have been reported in ciliates. It was thus interesting to see if Stentor DNA was more closely related to one class than to the other. The molecular weight of the DNA from hypotrichs is low. In Stylonychia Prescott et al. (1973) reported that all the macronuclear DNA molecules consist of short pieces, ranging from 0-2 to 2-2 fan, whereas micronuclear DNA molecules are many times larger. The authors conclude that the DNA in the macronucleus of this ciliate may represent free genes. Very low molecular weight has also been found recently in Oxytricha and Euplotes (Wesley, 1975). T. pyriformis seems to have another molecular organization. Murti (cited by Raikov & Ammermann, 1974) demonstrated that the macronuclear DNA molecules are much longer in this ciliate (about 35 /tm corresponding to a M.W. of about 6 x 10 7 daltons) than in Stylonychia. The molecular weight of the DNA of T. pyriformis GL, calculated by Miyagishi & Andoh (1973) after sucrose gradient centrifugation, is heterogeneous, ranging between 33 and 40S (3 x 10 7 -10 8 daltons); however, S is affected by the form of the molecule and aggregation effects. In P. multimicronucleatum syngen 2, continuous stretches of over 80 /tm (M.W. higher than 10 8 ) have been measured on electron micrographs (Barnett & Zadylak, 1974). 8. coeruleus macronuclear DNA did not possess many short molecules like those observed in Stylonychia. Its average molecular weight of about 4 x 1 0 ' daltons (and more for the largest molecules measured on electron micrographs), allows one to place Stentor DNA closer to that of Tetrahymena than of hypotrichs.

(iii) Benaturation experiments
Analysis of the rate of reassociation provides two useful pieces of information: the relative proportion of repeated and unique sequences present in the haploid genome and the size and complexity of this genome (Britten & Kohne, 1968). In free-living protozoa, very few species have been studied to date by this technique. Stentor genome appears relatively simple, reiterated sequences representing only 10-15% of the total. In comparison, Flavell & Jones (1970) estimated that about 5 % of the DNA of T. pyriformis is repetitive, and Allen & Gibson (1972) estimated, from reannealing profiles for the same species, that about 5-30% is repetitive, depending on the strain. Results from Yao & Gorovsky (1974) suggest that T. pyriformis DNA contains few highly repetitive sequences. Like T. pyriformis, P. aurelia DNA contains few repetitive sequences, estimated at about 15% of the total (Allen & Gibson, 1972). In Stylonychia, a large number of repeated sequences have been found in the micronuclei Prescott et al. 1973). During formation of the macronucleus, the repeated sequences are preferentially lost, thus the macronucleus does not contain a measurable amount of repeated DNA.
The genome size of. Stentor (6 x 10 10 daltons) is not very large compared to that of other ciliates, being in the range reported by Allen & Gibson (1972) for T. pyriformis (4 x 10 10 to 15 x 10 10 daltons) and P. aurelia (about 20 x 10 10 daltons).
In conclusion, by its structure and biochemical characteristics the macronucleus of Stentor appears closer to the model proposed for T. pyriformis and P. aurelia than to the one proposed for Stylonychia and other hypotrichs. gestions from Dr. M. Crippa and Dr M. Jacobs-Lorena of the Laboratory for molecular embryology are gratefully acknowledged. This work was supported by the Fonds National Suisse de la Recherche Scientifique (grant no.3.603.71). We also thank Dr Ph. Dubois of the D.E.S. in Geneva for his help.