Ceil, Vol. 6, 161-169,

October

1975,

Copyright

Characterization the Dinoflagellate and Implications

t’ 1975

by MIT

of the DNA from Crypthecodinium Cohnii for Nuclear Organization

J. Ft. Allen, Thomas M. Roberts, Alfred R. Loeblich, III, and Lynn C. Klotz Departments of Biology, and Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138

Summary Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For thls reason they are thought by some to be evolutionary Intermediates. Dlnoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of ldentlcal appearlng, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dlnoflagellate Crypthecodinlum cohnll with respect to DNA per cell, chromosome counts, and renaturation klnetits. The renaturatlon kinetic results tend to refute extreme polyteny and polyploldy as the mode of nuclear organlzation. Thls organism contains 5560% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatln contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotlc traits. Introduction The dinoflagellates comprise a major group of the eucaryotic algae. They occur most commonly as free-living, photosynthetic, marine protists, but the group is sufficiently diverse to include endosymbiotic, parasitic, heterotrophic, and freshwatertaxa. Of the hundreds of species which have been collected and described, only a few have been the subject of cytological and ultrastructural study, and even fewer have been subjected to biochemical investigation (Loeblich, 1967). Cytology of free-living dinoflagellates reveals an unusual pattern of nuclear organization. The chromosomes are attached to the nuclear envelope which remains intact throughout the cell cycle and is presumed to mediate the mitotic distribution of numerous (25-280) rod-like, acentric chromosomes to daughter cells (Bouligand, Puiseux-Dao, and Soyer, 1968a; Soyer, 1969; Kubai and Ris, 1969) (see Figure 1). There is no conventional mitotic spindle; however, Kubai and Ris (1969) have shown evidence suggesting that nuclear division in Crypthecodinium cohnii is driven by the elongation of extranuclear microtubular elements within membrane-lined cytoplasmic tunnels running through the dividing nucleus. Even among

eucaryotes, dinoflagellates contain inordinately large amounts of DNA, from 3.8 pg per cell in C. cohnii to 200 pg in Gonyaulax polyedra (HolmHansen, 1969) compared to human cells, for example, which contain about 2.7 pg of DNA per haploid genome (McKusick, 1964). The chromosomal organization and architecture of free-living dinoflagellates is also unusual. Chromosomes remain condensed throughout the cell cycle and are presumed to replicate by longitudinal division to form Y-shaped, then V-shaped structures (Hall, 1925; Gras& et al., 1965; Leadbeater and Dodge, 1967; Kubai and Ris, 1969). Thin sections reveal that the chromosome fibrils are of procaryotic width (30-60 A) (Giesbrecht, 1962; Hailer, Kellenberger, and Rouiller, 1964; Dodge, 1965; Bouligand, Soyer, and Puiseux-Dao, 1968b) arranged in regular arched whorls similar to those seen in bacterial nucleoids (Giesbrecht, 1962; Kowallik, 1971) (see Figure 1B). Cytochemical tests fail to demonstrate the presence of RNA and basic proteins in the chromosomes of free-living dinoflagellates (Ris, 1962; Dodge, 1964). These findings have been summarized by Dodge (1965) and form the basis for his proposal that dinoflagellates may represent a separate mesocaryotic group intermediate between procaryotes and eucaryotes. Recently Rizzo and Nooden (1974a, 1974b) have shown that isolated nuclei of C. cohnii and Peridinium trochoideum contain a small amount of protein compared to DNA for a eucaryote, but a similar amount compared to DNA as found in bacterial nucleoids. One acidsoluble protein is present which is electrophoretitally similar to Zea mays histone IV. Very little characterization of dinoflagellate DNA has been reported. Franker (1970) has shown that DNA isolated from dinoflagellate endosymbionts of the coelenterate Anthopleura elegantissima consists of nuclear and cytoplasmic fractions separable by isopycnic ultracentrifugation, and that their nuclear DNA contains a small fraction of 5-methylcytosine. Rae (1973) chromatographically detected large amounts of 5-hydroxymethyluracil in the DNA of C. cohnii, and attributed the discrepancy between buoyant density and melting temperature (T,) to its presence. He also concluded that this base is not uniformly distributed throughout the genome. This paper presents the results of our characterization of DNA (DNA per cell, DNA renaturation kinetics) from the dinoflagellate Crypthecodinium cohnii and discusses its relevance to nuclear organization. Preliminary results on 2 other free-living dinoflagellate species, Gyrodinium resplendens and Gymnodinium sp., are also discussed.

Cell 162

Results DNA per Cell The results of DNA per cell determinations of log and stationary phase cultures are shown in Table 1. In all 3 species examined, the amount of DNA per cell was found to be approximately twice the stationary value during logarithmic growth. Mi-

Figure

1. Electron

and Light

Micrographs

of Dinoflagellate

crospectrofluorometric measurements (Ruth, 1966) of individual cells of G. resplendens made by A. Brown in our laboratory show similar results. The DNA per cell can vary substantially from species to species (a 5-10 fold difference between C. cohnii and G. resplendens) and in every case is greater than that for human cells.

Chromosomes

(A) Transmission electron micrograph of a longitudinal section of the dinoflagellate Cachonina presence of condensed fibrous chromosomes in the interphase nucleus located in the posterior (6) An enlargement of the nucleus shown in Figure 1A. Note the arched whorls of chromosomal (C) A squashed acetocarmine-stained nucleus of C. cohnii revealing 106 chromosomes. (D) A drawing of the squashed nucleus shown in Figure IC.

niei Loeblich, III (IUCC portion of the cell. fibrils.

1654).

Note

the

Characterization 163

of Dinoflagellate

DNA

Our findings that logarithmically growing cells have twice the amount of DNA as resting cells places some constraints on the possible patterns of DNA replication among free-living dinoflagellates. It is apparent that DNA synthesis must be initiated shortly following division and be completed early in the growth cycle, with the completion of other cellular events imposing restraints on division. These findings are not in agreement with the measurements of incorporation of 32P into alkali-insoluble material reported by Franker et al. (1974) on C. cohnii, although it should be noted that his studies were performed under culture conditions

Table

1. DNA

per Cell of Dinoflagellate

Species Gymnodinium

Crypthecodinium

Gyrodinium

sp.

cohnii

resplendens

which give rise to 4- and g-cell (in addition to 2-cell) division products which are never seen under the conditions of growth used here (Tuttle and Loeblich, 1975).

DNA Renaturatlon

Species

Density of Culture (Cells/ml)

DNA

7.6 x 104 (log) 2.34 x 105 (stationary)

15.1, 7.25,

2.0 x 106 (log) 3.2 X 106 (stationary)

7.25, 7.2. 7.4 4.4, 3.6, 3.25

6.8 x 103 (log) 7.0 x 104 (stationary)

55, 70, 74 22, 36.8, 40.1

Each value for DNA per cell is the average measurements.

per Cell

(pg)

of duplicate

15.6. 15.4 7.7, 7.5

or triplicate

For comparison, E. coli cells contain 4.15 x 10-j pg DNA per genome, and human nuclei contain 2.7 pg DNA per haploid genome (McKusick, 1964).

Cot Figure

2. The

Kinetics

of Renaturation

as Measured

Kinetics

The C,t curve representation of the renaturation of the C. cohnii DNA is shown in Figure 2, along with similar data for E. coli and human nuclear DNA controls run under the same conditions. C. cohnii DNA renaturation has been followed from 15% to 85% completion, with limiting factors being the inability to measure accurately low C,t points by optical methods and apparent DNA degradation for points requiring longer than 2 days of renaturation. The C. cohnii DNA seems to be composed of roughly 2 kinetic classes: a repeated class comprising S-60% of the DNA and a highly complex class comprising 40-45% of the DNA when renaturation is monitored at fragment lengths of 500-600 base pairs. The average complexity of the repeated class is about one fifth to one tenth that of E. coli. To calculate whether the highly complex C. cohnii DNA might be unique DNA, we use the following equation: C = G F/N, where C is the number of copies of the highly complex DNA, G is the DNA per cell, F is the fraction of, and N is the complexity of the highly complex DNA. Using G = 3.8 pg, F = 0.45, and N = 0.45 x (1800/

mole

by HAP Chromatography

Q-’ set of DNA

E. coli (o), C. cohnii (0) and human nuclei (m). The points are corrected to those expected for fragment lengths of 450 base pairs in 0.12 M phosphate buffer (pH 6.6) at 80°C. The closed triangle (7) represents the corrected percent of C. cohnii DNA bound at C,t 20 at fragment length of roughly 3000 base pairs. The line through the E. coli points is an ideal curve for second order kinetics of renaturation with a C,t, of 2.3. The C,t, of the fast renaturing 55-60% of the C. cohnii DNA has been marked at 0.50 and the C& of slow renaturing component has been marked at 1800. The data have been corrected as explained in Experimental Procedures. Note that the terminal value for the E. coli renaturation is 90% (quite generally, HAP renaturation reaches a terminal value of 92 53%). However, the C.t, of the reaction has been chosen at 50% (not 45%). We use this practice systemmatically, but it should be considered as contributing an error of up to roughly 15% to our calculations.

Cell 164

2.3) x 4.15 x IO-3 pg = 1.46 pg, we obtain C = 1 .17, a number of copies which is consistent with unique DNA. Given the inherent inaccuracy in determining high C,t values, however, this DNA component may be present in 1 to 3 copies per cell. The repeated component was further analyzed in the following ways: -The renatured DNA, first separated on hydroxylapatite (HAP), was thermally melted as shown in Figure 3. The renatured C. cohnii DNA can be seen to have roughly 25% hyperchromicity with a T, 65°C below that of the sheared native DNA. The lowered T, indicates that the repeats are not perfect copies of each other, but instead are about 10% mismatched, assuming a lowering in T, of 1 “C for each l-1.5% mismatch (Laird, McConaughy, and McCarthy, 1969). The 25% hyperchromicity is less than the native value of 36%. Such a decrease in hyperchromicity is just what is expected to arise from the formation of duplexes which are partially mismatched and which are shorter on the average than the expected two thirds of the original singlestranded length (of 500-600 bases) due to the interspersion of the renaturing repeated sequences with nonrenaturing unique sequences. The fact that this lower hyperchromicity is for DNA isolated at C,t = 100, well after single-strand ends of repeated DNA should have renatured, suggests that the single-strand ends, if they exist, consist of the highly complex DNA. This result may be compared with the E. coli renatured DNA shown in Figure 3, which has regained almost full sheared hyperchromicity at a point where the renaturation is 85% complete. -To gain information about the possible occurrence of interspersed repeats, DNA was sheared to much longer lengths by needle shearing with a 27 gauge needle and then renatured to C,t 20. With this procedure, the amount of DNA bound increased from 60% at 500 base pairs to 85% at needle-sheared (roughly 3000 base pairs) lengths. Even after correction for the “zero time binding” (Davidson et al., 1973) (5% at 500, and 32% at 3000 base pairs), there is a net increase from 56% to 76 &4% (6 determinations) renatured at C,t 20. The increased binding at longer fragment lengths is consistent with the idea that the repeated sequences are probably interspersed with unique DNA, forming large concatemers at 3000 base pairs but not at 500-600 base pairs when renatured, as appears to be the case with all other eucaryotes (Britten and Kohne, 1968) except perhaps certain fungi (Whitney and Hall, 1974). It is of interest that the amount of double-strand material in the C,t 20 needle-sheared DNA varied with temperature of both renaturation and HAP elution. The above figures hold for our standard condition: renaturation

and HAP elution at 60 f 1 “C. Raising either temperature to 65°C appeared to lower the percent bound from 85% (uncorrected for zero time binding) to 75% (uncorrected for zero time binding). Although we have no ready explanation, this may represent a continuum of stabilities on which temperature variation imposes different cutoff criteria. Recently it has been possible to prepare small amounts of radioactive DNA using an adenine auxotroph of C. cohnii (ade -1) grown in the presence of 3H-adenine (Allen et al., 1975). A preliminary experiment using needle-sheared, radioactive C. cohnii DNA (length roughly 3000 base pairs), which after renaturation to C,ts ranging from 2 to 20 was exposed to Sl nuclease (gift of A. Efstratiadis and prepared by the method of Britten, Graham, and Neufeld, 1974) indicates that the repeated DNA is interspersed with highly complex DNA with the repeated material composing some 30-35% of the total. Sizing of the Sl-resistant, double-stranded DNA in the analytical ultracentrifuge indicates that the repeated regions average 400-450 nucleotides in length. The length of highly complex regions interspersed with repeated DNA has yet to be determined. However, if one simply assumes that the DNA is homogeneous in composition with blocks

‘.411

TEMPERATURE

OC

Figure 3. Thermal Denaturation Profiles Measured at 260 nm for Native and Renatured DNAs from E. coli and C. cohnii in 0.12 M PB (0.18 M Na+). The T,s are marked by arrows. The following symbols are used: (a) sheared native E. coli DNA; (0) renatured double-stranded E. coli DNA isolated on HAP after renaturing to a C,t 6; (m) sheared native C. cohnii DNA; and (0) renatured double-stranded C. cohnii DNA isolated on HAP after renaturing to a C,t 100.

Characterization 165

of Dinoflagellate

DNA

of repeated DNA 425 base pairs long, alternating with highly complex blocks of uniform length, then it can be easily calculated that the highly complex blocks would be 1000 base pairs in length. Extensive use of Sl nuclease to determine the lengths of the repetitive sequences has been made by the Britten-Davidson group, for example, Davidson et al. (1974) who found that the blocks of repeats are not homogeneously distributed in the genome. Hence it is best to consider our assumption above as merely a computational device. -Finally, the renaturation of the repeated material was examined separately. A large quantity of sonicated C. cohnii DNA (about 450 base pairs) was renatured to C,t 20 and the double-stranded material separated on HAP. Figure 4 shows the renaturation of this material. The separated, repeated material renatures over a rather broad range of C,t values, indicating the presence of more than one kinetic class. Although this fact is not surprising, it was not easily resolved in renaturation measurements of unfractionated DNA. We have also made preliminary measurements on the renaturation of DNA from 2 other dinoflagellate species. G. resplendens has about 10 times as much DNA as C. cohnii. This fact, coupled with our

inability to follow renaturation for longer than 2 to 3 days on this particular DNA, has made measurement of the renaturation kinetics of G. resplendens unique DNA impossible. However, we have detected substantial amounts of repetitious DNA in the organism (data not shown). The DNA of Gymnodinium sp. is unusual when compared to the other two dinoflagellates in that a substantial percentage (20-40%) of the DNA renatures faster than we can measure (CJ 2 x 10-j). The renaturation kinetic measurements do show, however, more slowly renaturing, repeated sequence fractions in addition to the very rapidly renaturing fraction. The data on both G. resplendens and Gymnodinium sp. is not complete enough to draw quantitative conclusions, except that repetitive sequences exist and that considerably more complex sequences exist in addition. Discussion Renaturation kinetics on DNA give a very clear-cut distinction between procaryotic and eucaryotic genomes in that procaryotes possess extremely small amounts of repeated sequence DNA as compared with eucaryotes (Britten and Kohne, 1968). More-

Cot mole 0-l set Figure

4. The Renaturation

Kinetics

of the Portion

of Sonicated

C. cohnii

DNA Which

Binds

HAP at C,t 20.

Sonicated C. cohnii DNA was allowed to renature at 60°C in 0.12 M PB to C,t 20. The single- and double-stranded portions were separated by HAP chromatography, and the double-stranded portions were dialyzed into 0.12 M PB and allowed to renature once more at 60°C. The time course of this second renaturation as measured on HAP is marked by the closed circles (0); the kinetics of renaturation of E. coli DNA measured by HAP are shown by open circles (0) as a reference. The E. coli DNA curve is drawn as an ideal second order curve, while the C. cohnii DNA curve is hand-fitted to the points. The 4 E. coli points were run merely to insure that the particular batch of HAP being used was behaving as expected in regard to the C,tr,z of E. coli, and to point out that renaturation of E. coli DNA terminates with only 90% of the DNA bound. The percent binding to HAP (R) has been corrected for zero time binding in the manner described in Experimental Procedures.

Cell 166

over, the repeated sequences of the eucaryotes studied thus far (organisms as diverse as toads and sea urchins) have been found to be interspersed with unique DNA (Davidson et al., 1973; Graham et al., 1974). Perhaps the only eucaryotes which differ from this pattern are the yeasts and Drosophila. Yeasts have very little repeated DNA (from 5 to 15%) which is arranged in large blocks of repeats and not interspersed with the unique DNA (Whitney and Hall, 1974). For Drosophila the blocks of repeats are longer and further apart (Manning, Schmid, and Davidson, 1975; for review of this general topic see Lewin, 1975). In regard to renaturation kinetics, then, C. cohnii DNA has a typical amount and complexity of repeated sequences for a eucaryote; our results thus far indicate that the DNA is interspersed in a manner similar to most other eucaryotes (Davidson et al., 1974). While the DNA renaturation kinetic results indicate the presence of l-3 copies of the highly complex sequences, nitrosoguanidine-induced mutation frequencies of C. cohnii are consistent with some genes being present in a single copy or haploid condition (Roberts et al., 1974). It should be noted that the DNA studied in all our experiments is whole cell DNA, not nuclear DNA. We have no information about the amount of cytoplasmic DNA in dinoflagellates, but believe it to be small, since Rizzo and Nooden (1972) determined that the nucleus of log phase C. cohnii contains 6.9 pg DNA, and we find 7.3 pg for log phase cells. Furthermore, our data suggest the interspersion of repetitive and nonrepetitive DNA sequences, that is, that these sequences are linked. Hence the repetitive sequences must be nuclear, since the nonrepetitive sequences are obviously nuclear. As may be seen in Figure 2, the renaturation of dinoflagellate DNA is both qualitatively and quantitatively different from bacterial DNA. It appears, therefore, that from the standpoint of DNA sequence distribution and repetition, dinoflagellates are eucaryotic. The quantitative data on C. cohnii allow us to make some definite statements on dinoflagellate nuclear organization. The large number of morphologically similar chromosomes in dinoflagellates (an example, the 99-l 10 rod-shaped chromosomes in C. cohnii) suggests that the nucleus might be polyploid. The large dimensions, permanently condensed state, and the appearance of the whole mounted chromosomes in the electron microscope have led Haapala and Soyer (1973) to propose a polytene model for the dinoflagellate chromosomes consisting of 500 to 1000 strands of DNA. If polyploidy were an important feature of the dinoflagellate genome, and if all chromosomes were identical, then the least repeated sequences would

occur as at least 100 copies. From the renaturation kinetics 40-45% of C. cohnii DNA is repeated l-3 times. If the nucleus were 100 fold polyploid and each chromosome composed of nonrepetitive DNA, the curve represented by the dashed line in Figure 5 would result. It is less easy to refute genomic models involving progressively lower degrees of polyploidy using renaturation kinetics alone. If the chromosomes were polytene in the sense of being 500 to 1000 identical copies of a DNA strand in each chromosome, the renaturation kinetic curve shown as the dotted line in Figure 5 would result. To draw this dotted line, we have assumed that the DNA polytenized is nonrepeated. Laird et al. (1974) have shown that in the case of D. melanogaster polytene chromosomes not all of the DNA is polytenized. The renaturation kinetic data on C. cohnii cannot be used to rule out this type of differential polytenization; that is, the highly complex DNA could represent the unpolytenized DNA, and the repeated DNA could represent those sequences which have polytenized. The thermal denaturation curve of the isolated, renatured, repeated DNA shown in Figure 3 indicates that the repeats are not perfect, whereas one would expect perfect repeats in the case of polytenization. These imperfect repeats are, instead, more typical of the repetitious DNA sequences of higher eucaryotes (Britten and Kohne, 1968).

I

2 2 9 i+ *

I

I

I

I

I

I

I

.I

I 10 loo Cot mole t-’ set

I

1

3040506070aogoI& 1

.Ol

I

loo0

lO,OcQ

Figure 5. Graphical Comparison of the Renaturation Kinetics Expected for Several Models of Dinoflagellate Nuclear Organization and the Experimental Results Obtained for Whole-Cell DNA from C. cohnii The dashed line (-----) represents the kinetics expected for a 100 fold polyploid state in C. cohnii, assuming each DNA sequence is present only once per chromosome. The dotted line(. .) shows the expected curve if each chromosome were 700 fold polytene, where the sequences polytenized contain no measurable repeated DNA, as is the case for bacterial genomes. The solid line (-) shows the experimentally observed kinetics of renaturation.

Characterization 167

of Dinoflagellate

DNA

The existence of highly complex DNA, the nature of the repetitive sequences in C. cohnii (highly mismatched), and haploid mutation frequency, all argue against the dinoflagellate nucleus being highly polytene or polyploid. Higher eucaryotes have the following 2 traits: repeated DNA sequences interspersed in DNA of higher complexity, and the existence of chromatin composed on approximately equal amounts of histones and DNA. These 2 eucaryotic traits could have evolved as an ensemble or separately. Our DNA renaturation kinetic data showing higher eucaryotic DNA sequence properties, coupled with data on the procaryotic amounts of basic protein in dinoflagellates (Rizzo and Noodgn, 1974a, 197413) and ultrastructural observations revealing chromatin fibrils of procaryotic dimensions (Haller et al., 1964), indicate that repeated DNA and the existence of eucaryotic chromatin evolved separately. Our data indicate that the dinoflagellates diverged from the higher eucaryotic lineage before the evolution of eucaryotic chromatin, but after the evolution of repeated DNA, provided this repeated DNA is of the same origin as that in higher eucaryotes. An alternative explanation is that they arose from a but along a line divergent from common “pool,” the typical eucaryotic lineage. The chromosome structure of dinoflagellates is probably not a degenerate organization derived from a more typical higher eucaryote because of the long (Precambrian) evolutionary history of dinoflagellates (assuming dinoflagellate affinities for some Precambrian acritarchs) (Loeblich, 1974). If the evolution of the several traits of higher eucaryotes occurred at different times in evolution, as the results discussed above indicate, it is not surprising that extant organisms showing a combination of higher eucaryotic traits and primitive procaryotic traits exist. Whether such organisms constitute a mesocaryote (an evolutionary intermediate) is open to question; that is, are they on a direct evolutionary line, or have they diverged at some early time to form a highly evolved but distinct organization from higher eucaryotes? Experimental

Procedures

Cell Culture, Harvest, and DNA lsolatlon Cultures of 2 marine species, Crypthecodinium cohnii (Seligo) Chatton in Grass& 1952, Indiana University Culture Collection (IUCC) No. 1649 and Gyrodinium respendens Hulburt, 1957, IUCC No. 1655, and one freshwater species, Gymnodinium sp. strain number 160. were grown axenically without agitation in 2.8 I Fernbath flasks. C. cohnii was grown in medium MLH (Tuttle and Loeblich, 1975) at 27°C and 2 ft candles. G. resplendens was grown in medium GPM (Loeblich, 1975) at 20°C and 200 ft candles. Gymnodinium sp. was grown in medium DL-1 [2.9 mM NaNO,, 1 mM CaCl2, 1 mM MgS04, 0.22 mM disodium glycerophosphate, 0.22 mM KCI, 10 mM morpholine ethane sulfonate (MOPS buffer)], 5 ml PIV metals/l (5 ml of PIV metals contains 6.2 mg trisodium

N-hydroxyethyl ethylene diamine triacetic acid, 0.965 mg FeCIX * 6H20, 0.18 mg MnCI?. 4H20, 0.052 mg ZnCl*, 0.02015 mg CoC12. 6H20, and 0.063 mg Na2Mo0.,* 2H20), pH 7.2 (with HCI and NaOH) at 10°C and 200 ft candles. Aliquots of cells were counted in a Model ZS, Coulter Counter, and cells were harvested by centrifugation at 800 x g, 0-5’C in the GSA rotor of the RCP-B Sorvall centrifuge. C. cohnii and Gymnodinium sp. were lysed in a French pressure cell at 4000 lb/in2 in 0.15 M NaCI, 0.1 M Nal EDTA (pH 7.2). G. resplendens was lysed in a NaCI:EDTA solution by adding sodium dodecyl sulfate to 2% or sodium N-lauroyl sarcosinate to 1%. NaClO., was added to 1 M and incubated 15 min at 20°C. The lysates were extracted with 1-2 vol 24:l chloroform:2-pentanol, and the emulsion was separated by centrifugation at 10,000 x g for 10 min at 0.5”C. DNA was precipitated by addition of 1.2 vol 100% ethanol and wound on a glass rod. After the precipitated DNA was redissolved in 0.015 M NaCI, 0.05 M EDTA (pH 7.2), it was digested first with pancreatic RNAase (Worthington) 100 ug/ml, then with pronase (Calbiochem B grade) 200 ug/ml at 37°C for 2 hr. After enzyme treatment, the DNA solution was extracted with chloroform:2-pentanol, until no protein could be seen at the interface of the centrifugally separated phases, then precipitated with an equal volume of ethanol. The purified DNA was redissolved in 0.005 M sodium phosphate buffer (PB) (pH 6.88), then exhaustively dialyzed into 0.12 M PB in the cold. 1 O-20 mg of C. cohnii or Gymnodinium sp. DNA of high purity, as judged by optical and isopycnic criteria, could be prepared at one time. G. resplendens DNA purified as above was judged to be about 85% pure; however, attempts to further purify the DNA by passage over hydroxylapatite (HAP) and by ultracentrifugation were unsuccessful. Micrococcus lysodeikticus, E. coli, and human nuclear DNAs were prepared as above after lysis with detergent. DNA per Cell Measurements of DNA per cell of the 3 species studied were performed using the reaction of diaminobenzoic acid with deoxyribose (Kissane and Robins, 1958; as modified by Holm-Hansen, Sutcliffe, and Sharp, 1968). Cells were extracted with methanol, cold 5% trichloroacetic acid, ethanol, then hydrolyzed at 60°C with 1 .O N HCI, rather than perchloric acid, as suggested by Hinegardner (1971). Fluorescence was measured in an Aminco-Bowman fluorimeter (excitation wavelength was 405 nm; emission wavelength was 505 nm). Excitation and emission spectra from dinoflagellate samples were identical to spectra obtained from the reactions of diaminobenzoic acid with purified human nuclear DNA and E. coli DNA. The technique is sensitive to 0.5-l .O ug DNA, and measurements were reproducible. Molecular Weight Determination by Sedimentation Velocity The molecular weight of double- and single-stranded DNA for renaturation experiments was determined from the neutral (0.12 M PB at pH 6.88) or alkaline (0.9 M NaCI, 0.1 M NaOH) sedimentation using a Beckman Model E ultracentrifuge according to Studier (1965). DNA Renaturatlon Klnetlcs Renaturation was performed according to the method of Britten and Kohne (1968). Sonically sheared DNA samples in 0.12 M PB to 0.6 M PE were heated for 5 min at 100°C to 110°C. then quickly cooled to 60 * 1 “C and allowed to renature at this temperature. At various times samples were diluted to 0.12 M PB (if necessary) and assayed for degree of renaturation by passage over hydroxylapatite (HAP) columns at 60 i 1 “C. HAP was prepared as discussed by Bernardi (1971). Single strands were eluted in 0.12 M PB and double-stranded material in 0.6 M PB. Amounts of DNA in singleand double-stranded fractions were assayed by measurement of optical density at 260 nm. All fractions were corrected for scatter at 260 nm due to HAP by subtracting out the absorbance at 320

Cell 168

nm, as suggested by Davidson et al. (1973). To correct for the different extinction coefficients of single- and double-stranded material, raw absorbance data for double strands were multiplied by a factor of 1 .I4 (1.14 = g , the approximate ratio of hyperchromicites of doubleand single-stranded material measured from room temperature to 100°C). DNA recovery from HAP was 92 +3% for all DNA except that of Gymnodinium sp. Lengths of DNA fragments used were measured either by neutral or alkaline sedimentation velocity centrifugation. Average fragment size varied from 350 to 650 base pairs in different DNA samples. All renaturation profiles have been corrected to those expected at 450 base pairs using an Ll/z dependence for renaturation rate. All renaturation measurements made in 0.6 M PB have been corrected to those expected in 0.12 M PB for graphing purposes by multiplying actual C,ts by 6.5 (Wetmur and Davidson, 1988). The measured raw percent DNA renatured has been corrected for zero time binding as follows: zero time binding is the amount of DNA binding HAP when DNA O.D. at 260 nm = 0.2, the lowest practical concentration we could work with, was passed over HAP as quickly as possible following denaturation and cooling to 60°C (roughly 1 min). Thus zero time corresponds to a C,t = 2 x IO-3 moles phosphate X I set-I. The graphed percent renatured (R) is thus: R = 96 binding

to HAP - 96 zero

time binding

Bernardi, G. (1971). In Methods in Enzymology, 210, L. Grossman and K. Moldave. eds. (New York: Academic Press), pp. 95-139. Bouligand, Y.. Puiseux-Dao, Acad. Sci. 266, 1287-1289. Bouligand, Y., Soyer, soma 24, 251-287. Britten,

M.-O.,

The observed zero time bindings were 3 f2% for E. coli DNA, 7 13% for C. cohnii DNA, and 15 f2% for human nuclear DNA. The exact nature of the DNA binding at zero time is uncertain. In the case of E. coli, the binding may well be artifactual (Alberts and Doty. 1968), since Wilson and Thomas (1974) saw no such zero time binding in their investigations. In the human placental DNA, some of the binding is probably palindromic (approximately 3-50/o), and some may well be the main band rapidly renaturing DNA [Hearst et al. (1974) find 7.7% of main band DNA with a kinetic complexity of 35 base pairs or fewer], while a few percent may be artifactual. In the case of the dinoflagellates, the matter clearly needs further study using radioactively labeled DNA to distinguish between rapidly renaturing DNA and palindromic sequences. As a check on the hydroxylapatite data, renaturation of C. cohnii and G. resplendens DNA was followed optically on a Gilford 2000 recording spectrophotometer. Expected deviations (Rau and Klotz. 1975) from second order kinetics make exact interpretation of these results difficult, and for this reason they are not shown. They appear, however, to be in good qualitative agreement with the HAP data.

Soyer,

M.-O.

and Puiseux-Dao,

R. J., and Kohne,

D. E. (1968).

(1968a).

S. (1968b).

Science

CRH

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The authors wish to acknowledge their debt to Professor S. Latt, Department of Pediatrics, Harvard Medical School, for the gift of human placental nuclei; William A. Muller for assisting with fluorescence measurements; Alan Brown for microspectrofluorimetric measurements; Ft. C. Tuttle for assisting with radioactive labeling of DNA and for advice on dinoflagellate nutrition; Donald Rau for discussion of DNA renaturation kinetics; Professor J. W. Hastings for the use of instruments; and Professor Paul Doty for his generous support and encouragement. The research was supported by the NIH. T.M.R. was supported by a National Science Foundation predoctoral fellowship. J.R.A. was partially supported by the Maria Moors Cabot Foundation for Botanical Research, Harvard University.

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