290

CALCIUMIONAND Tetrahymena

3. Kidder GW, Dewey VC, Parks RE. 1951. Studies on the inorganic requirements of Tetrahymena. Physiol. Zool. 24, 69-75. 4. Kinoshita H, Dry1 S, Naitoh Y. 1964. Relation between the magnitude of membrane potential and ciliary activity in Paramecium. J . Fac. Sci. Uniu. Tokyo. Sect. 4, 10, 303-9. 5. Nanney DL, McCoy JW. 1976. Characterization of the

Tetrahymena pyriformis complex. Trans. A m . Microsc. SOC.95, 664-82. 6. Ogawa Y. 1968. The apparent binding constant of GEDTA for calcium at neutral pH. J . Biochem. 64, 255-7. 7. Soldo AT, Van Wagtendonk WJ. 1969. The nutrition of Paramecium aurelia, Stock 299. J . Prototool. 16, 500-6.

J. Prototool., 26 2 ) , 1979, pp. 290-294 0 1979 by

the iociety of Protozoologuts

RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii PETER J. RIZZO Biology Department, Texas A&M University, College Station, Texas 77843

SYNOPSIS. Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to M e with optima at .- 0.01 and 0.02 M MgC12, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCL concentrations. In the presence of 0.01 M MgCL the optimum (NI-L)&Od concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [8H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated 3 pmoles of rH]UMP/pg DNA at 25 C for 15 min, and N 80% of this activity was inhibited by the eukaryotic RNA polymerase I1 inhibitor, a-amanitin (20 pg/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with a-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).

-

Index Key Words: Crypthecodinium cohnii; isolated nuclei; RNA synthesis; a-amanitin-sensitive RNA polymerase.

D

INOFLAGELLATES are eukaryotic organisms that differ from other eukaryotes in that they possess several bacterial features in the organization of their chromosomes. These unusual or “primitive” features of dinoflagellate chromosomes include a type of mitosis in which a centromere and conventional mitotic spindle are absent ( 17), and meiosis which appears to be a onedivision process (2, 15). In electronmicrographs of ultrathin sections, dinoflagellate chromosomes have a characteristic transverse banding which has been the subject of much discussion (3, 10, 11, 19, 28, 33). Dinoflagellate nuclei contain numerous (usually 100-200) chromosomes (6, 20), and extremely high amounts of DNA (1, 16, 25). The numerous identical-appearing chromosomes (6, 20) are attached to the nuclear envelope during division (17, 33) and remain condensed throughout the entire cell cycle (6, 17, 3 3 ) . Although these permanently condensed “dinokaryotic” chromosomes superficially resemble metaphase chromosomes, they do not produce the differential crossbanding of metaphase chromosomes (Q-, G- or C-bands) (12), and comparison of whole mount electronmicrographs of dinoflagellate chromosomes (11, 14) with whole mounts of typical metaphase chromosomes ( 7 ) , reveals significant differences in the structural details. The organization of the DNA within dinoflagellate chromosomes has several eukaryotic features including the presence of satellite DNA ( 2 3 ) and repeated sequences interspersed with nonrepeated sequences ( 1) . Another unique feature of dinoflagellate DNA has recently been reported; it contains substantial amounts of hydroxymethyluracil, a base not present in significant amounts in other eukaryotes (24). Dinoflagellate chromosomes differ from those of typical eukaryotes in the absence of histones. Although a histone-like

protein is present in log-phase chromatin from 2 dinoflagellate genera, this protein does not correspond to any of the 5 vertebrate histones (26). The absence of histones in dinokaryotic nuclei is undoubtedly the basis for failure to detect nucleosomes in them by electron microscopy ( 14). Since histones presumably repress RNA synthesis in other eukaryotes, a question arises concerning the mechanism by which gene repression is exerted on a gross level in dinoflagellates. The occurrence of prokaryotic traits in an organism with eukaryotic organelles (including nucleoli), has led many investigators to suggest the possibility that the organization of the dinoflagellate genome is intermediate between that of prokaryotes and eukaryotes (1, 6, 14, 20, 33). An important aspect of the dinoflagellate nucleus that has not yet been investigated is the endogenous RNA polymerase activity. I n the present report, optimal conditions are described for the assay of endogenous RNA polymerase in isolated nuclei of Crypthecodinium cohnii (Seligo). MATERIALS AND METHODS Culture Conditions and Isolation of Nuclei

An axenic stock culture of Crypthecodinium cohnii was kindly provided by Dr. J. R. Allen (Department of Biochemistry and Biophysics, Oregon State University, Corvallis) . The stock cultures were maintained in 5 ml of MLH medium ( 3 4 ) , and transferred to “low-form” flasks containing one liter of MLH in which the glucose and acetate were reduced 10-fold. The cultures were incubated at 2 7 C in the dark for 72 h. Nuclei were isolated

291

TRANSCRIPTION IN DINOFLAGELLATE NUCLEI

\

0

I 5

I 10

Divdmf cltion

I

I

U

10

bM1

In all experiments the assays were performed as described in Materials and Methods. Fig. 1. Effect of the divalent cations Mg2t and Mn" on the endogenous nuclear RNA polymerase activity of Crypthecodinium cohnii. M e supplied as MgCL (no MnCIo) ; Mn2+ supplied as MnCL (+ 1.67 mM MgCL from TGMED) ,

as described previously (25) with slight modifications. Cells were harvested by centrifugation for 5 min at 3,290 g in the Sorvall GS3 rotor and suspended in isolation medium (90 ml isolation medium/liter of culture). The isolation medium, adapted from Honda et al. (18), consisted of 0.25 M sucrose, 5% (w/v) Dextran 40, 2.5% (w/v) Ficoll, 5 mM CaCI,, 0.5 mM dithiothreitol and 0.01 M Tris-HC1 ( p H 7.5). Immediately before cell disruption 10 pl of a 0.01 M stock solution of phenylmethylsulfonylfluoride (PMSF) in ethanol was added/rnl of isolation medium, to give a final concentration of 0.1 M PMSF. Cells were disrupted in a Biosonik ultrasonicator (Bronwill Scientific), as described previously (25). Nuclei were sedimented from the homogenate by centrifugation at 1,935 g for 5 min in the Sorvall SS-34 rotor, and homogenized in isolation medium containing 0.05% (v/v) Triton X-100 using a PotterElvehjem homogenizer with a loose-fitting Teflon pestle. The homogenate was layered over a 2-step discontinuous gradient consisting of the following: 7 ml of 2.4 M sucrose, 10% (w/v) Dextran-lo, 5 mM MgCI, and 0.01 M Tris-HC1 (pH 7.5), overlayered with 40 ml of 2.2 M sucrose, 5 mM CaCl, and 0.01 M Tris-HC1 ( p H 7.5). The upper % of the 2.2 M sucrose layer was stirred, and the tubes were centrifuged at 48,000 g for 1 h. The inside walls of the tubes were wiped with a cotton swab and the nuclear pellet was suspended, with the aid of a glass rod, in 1-2 ml of buffer containing 0.05 M Tris-HC1, p H 7.9, 50% glycerol (v/v), 5 mM MgCl,, 0.1 mM EDTA and 0.5 mM dithiothreitol (TGMED). Nuclei were assayed for endogenous RNA polymerase activity and stored at -20 C. Under these conditions the preparations could be stored for several weeks without substantial loss of activity. Analytic Procedures RNA Polymerase Assay.-Endogenous nuclear RNA polymerase activities were measured by adding 25 pl of nuclei (10-30 pg of DNA) to 50 pl of reaction mixture. The final concentrations of reaction mixture components were: Tris, 0.08 M ( p H 7.9); MgCI,, 0.01 M; dithiothreitol, 2 mM; (NH4),S04, 0.025 M;

0

o

I

I

I

I

I

am

a1

0.l

03

Q1

EWaW

M

Fig. 2. Effect of (NR)SSOn concentration on the activity of endogenous nuclear RNA polymerase from Crypthecodinium cohnii.

ATP, CTP and GTP, 0.5 mM each; unlabeled UTP, 0.025 mM; 2.5 pCi of [3H]UTP (12 Ci/mmole); and 16.7% (v/v) glycerol in a final volume of 75 pl. The assay tubes were incubated at 25 C for 15 min and 50 pFGI aliquots were placed onto DE-81 filters, which were then immersed in 0.5 M Na2HP04. The filters were washed as described by Roeder (29) and radioactivity of the spots assayed by liquid scintillation in toluene fluor at 25% efficiency. Since [3H]UMP incorporated into RNA is counted more efficiently than is unincorporated [3H]UTP under the above assay conditions (29), previously examined filters were treated overnight with 1 ml of NCS solubilizer along with a known amount of [3H]UTP and assayed again by liquid scintillation. The relative counting efficiency was then determined by comparing the counts obtained before and after solubilization of the r3H]RNA and the known amount of [3]UTP. One picomoIe of incorporated [3H]UMP represents 519 CPM under these conditions. DNA Determination.-DNA was estimated by the diphenylamine colorimetric method, as described by Burton ( 4 ) . Chemicals Unlabeled nucleoside triphosphates, dithiothreitol, PSMF, sucrose (Grade 1), spermine-HC1 (as tetrachloride), and spermidine-HC1 (as trichloride) were purchased from Sigma [3H]UTP from Schwarz/Mann; a-amanitin from Henley and Co.; NCS solubilizer from Amersham/Searle; DE-81 filters from Whatman; and Dextran and Ficoll from Pharmacia. RESULTS Isolation of Nuclei.-Several modifications in the original isolation procedure (25) were necessary to allow its adaptation for RNA polymerase studies. A replacement of Mg2+ for Ca2+ 10% Dextran-10 cushion was necesions in the 2.4 M sucrose sary because calcium inhibits RNA polymerase activity. Since magnesium was not as effective as calcium in stabilizing nuclear integrity, this replacement was done only for the cushion. Dithiothreitol was included to stabilize endogenous RNA polymerase

+

292

TRANSCRIPTION IN DINOFLAGELLATE NUCLEI TABLE 1. Effect of uarious additions on the endogenous RNA polymerase activity of isolated nuclei from Crypthecodinium cohnii. Additive None* RNAse A (5 pg/ml) t DNAse (5 pg/ml) DNAse (20 pg/ml)* Actinomycin-D (50 pg/ml) i Actinomycin-D (100 pg/ml) KOH§ a-Amanitin (20 pg/ml) lI

*

%

Cpd2.5. PI nuclei

Incorporation

13,267 43 7 1,081

100 3.3 8.1

608 4.6 46 7 3.5 310 2.3 656 4.9 19 2,521 RNA polymerase activity was assayed as described in Materials and Methods IM & M ) . t Assay was conducted as described, except that after incubaton 1 vol. of a 10 pglml solution of RNAse was added to the reaction tubes followed immediately by incubation for 30 min at 37 C. Aliquots were then placed on filters and processed. *The nuclei and substance to be tested were combined in the assay tube and incubated on ice for 10 min. The reaction was started with the addition of the reaction mixture. § Assay was conducted as described in M & M, except that the completed reaction was made 0.3 N in KOH, incubated at 37 C for 30 min, neutralized with HCI and placed on filters for counting. ll Assay was conducted as described in M & M, except that the reaction mixture contained a-amanitin. The data represent averages from 7 different preparations of nuclei.

*

I

I

I

1

I

I

5

m

15

20

15

33

TIME

(min)

Fig. 3. Time courses of RNA synthesis at various temperatures in isolated nuclei of Crypthecodiniun cohnii.

activity, and PMSF was incorporated to reduce proteolytic activity. Since cleaner preparations of nuclei were obtained if the crude nuclear pellet was homogenized in isolation medium containing 0.05% Triton X-100, 0.1% Triton was omitted from the 2.2 M sucrose solution. Optimal Conditions for R N A Synthesis.-The reaction was stimulated by Mg2+ but inhibited by Mn2+. The response to increasing concentrations of MgCI, resulted in a biphasic curve, with optima at 0.01 M and 0.02 M. A typical curve is shown in Fig. 1. Magnesium concentrations between 0.02 and 0.05 M always resulted in progressive inhibition. It is also shown in Fig. 1 that a dramatic inhibition is obtained when increasing amounts of MnCI, are included in the reaction mixture. The inhibition was obtained using MnCI, concentrations ranging from 0.1 to 10 mM. When increasing concentrations of ( NH,),SO, were included in the reaction mixture, a peak of activity was observed at 0.025 M, followed by increasing inhibition (Fig. 2 ) . On microscopic examination, the nuclei after the assay was completed were found to be lysed at this ionic strength. It is possible that the sharp salt activation peak is merely a result of nuclear lysis. The time course for R N A synthesis at 25, 30 and 37 C is shown in Fig. 3. Incorporation at 25 and 30 C was similar for 15 min, while incorporation a t 37 C was much lower after 15 min and had leveled off after 30 min. A greater amount of incorporation of labeled triphosphates into RNA at 25 than at 37 C has also been observed in isolated nuclei of mammalian cells (22, 31). The data given in Figs. 1-3 were then used to determine the assay conditions given in Materials and Methods. Nature of the Reaction and Product.-The incorporation of [3HJUMPby C. cohnii nuclei was sensitive to actinomycin D and dependent on the presence of undegraded DNA (Table 1). The product of the reaction was also sensitive to ribonuclease and

-

KOH digestion (Table 1) . When the bicyclic octapeptide aamanitin was included in the reaction mixture at a final concentration of 20 pg/ml, 81% of the RNA polymerase activity was inhibited (Table 1) . In the presence of increasing amounts of a-amanitin, the inhibition was rapid at lower concentrations and leveled off at 20 pg/ml (Fig. 4). Partial inhibition by this compound is characteristic of nuclear RNA polymerase activity in other eukaryotes ( 3 0 ) . The omission of one or more ribonucleoside triphosphates greatly reduced the incorporation, the amount of reduction being dependent on the triphosphate omitted-omission of C T P caused the greatest, while omission of GTP caused the least reduction (Table 2 ) . Although these differences are reproducible, the

-

-

-

oa 0

1

2

n

m

.(-amanitin

[pg/ml)

Fig. 4. Effect of a-amanitin concentration on the activity of endogenous RNA polymerase from isolated nuclei of Crypthecodinium cohnii.

TRANSCRIPTION I N DINOFLAGELLATE NUCLEI

293

TABLE2. Effect on polymerase activity from isolated nuclei of Crypthecodinium cohnii of omitting nucleoside triphosphates.*

Reaction mixture

Cpm/25 pl nuclei ~

Complete -ATP -CTP -GTP -ATP, -CTP, -ATP, -ATP.

%

Incorporation

~

GTP GTP CTP CTP, GTP

100 9.5

7,173 682 89 1,984 963 78 146 1 18

1.2 27.7 13.4

1.1

2 .o 1.7

-

rp.rmkm 0

u

0

0

* Assay was carried out as described in M & M.

I

2

I

0

I

I@

10

Polvmim

reason for them is not known at the present time. Possible explanations include different ribonucleoside triphosphate pools in the nuclei, or contaminants in the chemicals used. I t has been shown that RNA synthesis in nuclei isolated from animal ( 3 5 ) and plant (27) tissues can be enhanced considerably (2-fold or more) by including exogenous DNA in the reaction mixture. Only a slight enhancement of RNA polymerase activity was observed when various amounts of native and denatured calf thymus DNA were included in the reaction mixture in the present study (Table 3 ) . Because polyamines have been shown to stimulate RNA polymerase from a variety of organisms including prokaryotes (5, 9 ) and higher eukaryotes (21, 321, it was of interest to see if polyamines would also stimulate RNA polymerase activity in dinoflagellate nuclei. Spermidine-HC1 and spermine-HC1 were therefore tested for stimulatory activity, and the results are summarized in Fig. 5. I t can be seen that the nuclei responded differently to the 2 polyamines-spermine produced a marked inhibition, while spermidine caused a slight stimulation which gradually fell back to control levels with increasing concentration. The endogenous RNA polymerase activity/U DNA is similar to that of typical eukaryotes (Table 4). This result is important with regard to the mechanism for gene repression on a gross level in the absence of histones (see Discussion). DISCUSSION Conditions for RNA Synthesis.-Results from a number of laboratories indicate that the chromosomes of dinoflagellates differ from those of other eukaryotes in their structure, chemical composition, and behavior during mitosis and meiosis. Their

Fig. 5. Effect of the polyamines spermidine-HC1 and spermineHC1 on the activity of endogenous RNA polymerase from isolated nuclei of Crypthecodinium cohnii. The polyamines and nuclei were combined in the assay tubes and the reaction was started by the addition of the reaction mixture.

prokaryotic nature (along with the eukaryotic nucleolus) suggests that the endogenous nuclear RNA polymerase activity of these organisms may be different from that in other eukaryotes. In the present report, the conditions required to obtain active endogenous RNA polymerase activity in isolated nuclei of C. cohnii are described. The reaction is dependent on the presence of all 4 nucleoside triphosphates and undegraded DNA and is inhibited by actinomycin D. The reaction product is sensitive to ribonuclease and K O H digestion. I t is concluded from these results that the observed activity represents the incorporation of [3H]UMP into RNA with the aid of one or more endogenous DNA-dependent RNA polymerases. An interesting observation made in the present study is the inhibition of RNA polymerase activity by Mn2+. Although it has been previously reported that MnC12 failed to stimuIate IZNA polymerase activity in isolated nuclei of HeLa cells treated with actinomycin D ( 3 1 ) , inhibition of nuclear RNA synthesis by MnC1, concentrations as low as 0.1 M has not been reported for a eukaryote. Multiple R N A Polymerases.-In contrast to the situation found in prokaryotes, multiple forms of DNA-dependent RNA polymerase with different intranuclear locations have been detected in all eukaryotes examined thus far (see Ref. 30 for a recent review). Briefly, polymerase I is located in the nucleolus, synthesizes rRNA precursor, and is insensitive to a-amanitin, while polymerase I1 is located in the nucleoplasm, synthesizes mRNA precursor, is stimulated by manganese, and is inhibited by low concentrations of a-amanitin. A 3rd class of eukaryotic RNA

TABLE 3. Effect of exogenous calf thymus D N A on R N A synthesis in isolated nuclei from Crypthecodinium cohnii.* Cpm/25 fil nucleit

DNA added (pg/assay tube)

Native

Dena t ured

1.25 2.5 5.0 10.0

8,061 9,028 9,820 9,260

8,888

8,973 9,009 10,236

*Assay was carried out as described in M & M, except that various Concentrations of calf thymus DNA were included in the reaction mixture. Denatured DNA was obtained by boiling for 5 min, followed by rapid cooling on ice. The data are averages of 3 separate experiments using 3 different preparations of nuclei. t Nuclei incorporated 7,469 Cpm without exogenous DNA.

TABLE 4 . Incorporation of [$H]UMP intp R N A in nuclei isolated from various organisms. Source of nuclei

pmoles of UMP incorp./ pg of DNA

Incubation

References

Rat liver 2.7 lo’, 37 c 35 Tobacco leaf 1.2 lo., 37 c 13 Hen oviduct 5.5 15’, 25 C a Mouse myeloma 1.5 15’, 25 C 22 Crypthecodinium cohnii* 3.0 15’, 25 C present study *Nuclei were isolated and assayed for endogenous RNA polymerase activity as described in M & M.

294

TRANSCRIPTION I N DINOFLAGELLATE NUCLEI

polymerases (polymerase 111) is also located in the nucleoplasm, synthesizes tRNA, and i s inhibited by very high concentrations of a-amanitin. I t is not known if multiple RNA polymerases are present in dinoflagellates. However, the data given in Fig. 4, which reflect incomplete inhibition by a-amanitin at a concentration known to inhibit RNA polymerase I1 completely and polymerase I11 partially in other eukaryotes (30), suggests this possibility. On the other hand, it is also possible that only one form of RNA polymerase is present, with a partial sensitivity to a-amanitin. Solubilization of the enzyme and chromatography on DEAE columns will be necessary to answer this question. Although problems exist concerning enzyme stability, various methods for solubilizing RNA polymerase from isolated nuclei and whole cells are currently being explored. Sensitivity to a-Amanitin: Prokaryotic vs Eukaryotic Nature of RNA Polymerase.-Prokaryotes and eukaryotes differ considerably with regard to the properties of their RNA polymerases. The present finding that C. cohnii nuclei contain a large percentage of RNA polymerase activity sensitive to a-amanitin suggests that dinoflagellate RNA polymerases are eukaryotic in this characteristic. I t is noteworthy, however, that this inhibition is atypical in that the polymerase activity is inhibited also by low concentrations of Mn2+, including those concentrations known to stimulate the activity of these enzymes in other eukaryotes. Since a-amanitin inhibits those RNA polymerases that in all other eukaryotes prefer Mn*+ over Mg2+ ( 3 0 ) , it seems possible that real differences exist between RNA “polymerase 11”-type enzymes in C. cohnii and the polymerase I1 found in other eukaryotes. Mechanisms for Eukaryotic Gene Repression without Histones. -The general interpretation of in vitro studies on RNA synthesis in other eukaryotes is that most of the nuclear DNA is inaccessible to RNA polymerase due to nonspecific repression by histones. As shown in Table 4, the endogenous RNA polymerase activity/U DNA in C. cohnii nuclei is similar to that of other eukaryotes. One possible explanation for this similarity in activity is that there is in dinoflagellates a mechanism for course control analogous to that mediated by histones in other eukaryotes. ACKNOWLEDGEMENTS

I thank Mrs. Sherri Sutton Symank for help with some of the experiments, and Dr. J. R. Wild for a critical reading of the manuscript. LITERATURE CITED 1. Allen JR, Roberts TM, Loeblich AR 111, Klotz LC. 1975. Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization. Cell 6, 161-9. 2. Beam CA, Himes M, Himelfarb J, Link C, Shaw K. 1977. Meiosis in Crypthecodinium cohnii. Genetics 87, 19-32. 3. Bouligand Y, Soyer M - 0 , Puiseux-Dao S. 1968. La structure fibrillaire et l’orientation des chromosomes chez les Dinoflagell6s. Chromosoma (Berlin) 24,25 1-87. 4. Burton K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62,315-22. 5. Chow CT. 1977. Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum. Can. J . Microbiol. 23, 534-58. 6. Dodge JD. 1966. The Dinophyceae, in Godward MBE, ed., T h e Chromosomes of the Algae, Edward Arnold, New York, pp. 96-115.

7. DuPraw EJ. 1970. DNA and Chromosomes, Rhinehart and Winston, New York, pp. 132-71. 8. Ernest MJ, Schutz G, Feigelson P. 1976. RNA synthesis in isolated hen oviduct nuclei. Biochemistry 15, 824-9. 9. Giorno R, Stamato T, Lydersen B, Pettijohn D. 1975. Transcription in vitro of DNA in isolated bacterial nucleoids. J. Mol. Biol. 96,217-37. 10. Grass6 P, Hollande A, Cachon J, Cachon-Enjumet M. 1965. Nouvelle interpretation de I’ultrastructure du chromosome de certains Pbridiniens. C.R. Acad. Sci. Paris, 260, 1743-7. 11. Haapala OK, Soyer M-0. 1973. Structure of dinoflagellate chromosomes. Nature N e w Biol. 244, 195-7. 12. 1974. Absence of loneitudinal differentiation of dinoflagellate (Prorocentrum miclns) chromosomes. Hereditas 78, 141-5. 13. Hamilton RH, Kunsch U, Temperli A. 1972. Simple rapid procedures for isolation of tobacco leaf nuclei. Anal. Biochem. 49.48-57. i4. Hamkalo BA, Rattner JB. 1977. The structure of a mesokaryote chromosome. Chromosorna (Berlin) 60,39-47. 15. Himes M, Beam C. 1975. Genetic analysis in the dinoflagellate Crypthecodinium (Gyrodinium) cohnii : evidence for unusual meiosis. Proc. Natl. Acad. Sci. U S A 72,4546-9. 16. Holm-Hansen 0. 1969. Algae: amounts of DNA and organic carbon in single cells. Science 163,87-8. 17. Kubai D, Ris H. 1969. Division in the dinoflagellate Gyrodinium cohnii (Shiller) . I. Cell Biol. 40,508-28. 18. Honda SI, Hongladarom R, Laties GG. 1966. A new isolation medium for plant organelles. J. Exp. Bot. 17, 460-72. 19. Livolant F, Giraud M-M, Bouligand Y. 1978. A goniometric effect observed in sections of twisted fibrous materials. Biol. Cellulaire 31, 159-68. 20. Loeblich AR 111. 1976. Dinoflagellate evolution: speculation and evidence. J . Protorool. 23, 13-28. 21. Karpetsky TP, Hieter PA, Frank JJ, Levy CC. 1977. Polyamines, ribonucleases, and the stability of RNA. Mot. Celt. Biochem. 17,89-99. 22. Manluff WF Jr, Murphy EC, Huang RCC. 1973. Transcription of ribonucleic acid in isolated mouse myeloma nuclei. Biochemistry 12,3440-6. 23. Rae PMM. 1973. 5-HydroxymethyluraciI in the DNA of a dinoflagellate. Proc. Natl. Acad. Sci. U S A 70, 1141-5. 24. - 1976. Hydroxymethyluracil in eukaryote DNA: a natural feature of the Pyrrophyta (dinoflagellates). Science 194, 1062-4. 25. Rizzo PJ, NoodCn LD. 1973. Isolation and chemical composition of dinoflagellate nuclei. J . Protozool. 20, 666-72. 26. , - 1974. Partial characterization of dinoflagellate chromosomal proteins. Biochim. Biophys. Acta 349,41527. 27. ,Pedersen K, Cherry JH. 1978. Transcription and RNA polymerase stimulatory activity in nuclei isolated from soybean. Plant Sci. Lett. 12, 133-43. 28. Roberts TM, Tuttle RC, Allen JR, Loeblich AR 111, Klotz LC. 1974. New genetic and physiocochemical data on the structure of dinoflagellate chromosomes. Nature 248, 466-7. 29. Roeder RG. 1974. Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. J . Biol. Chem. 249,241-8. 30. ___ 1976. Eukaryotic nuclear RNA polymerases, in Losick R, Chamberlain M, eds., R N A Polymerase, Cold Spring Harbor, New York, pp. 285-329. 31. Sarma MH, Feman ER, Baglioni C. 1976. RNA synthesis in isolated HeLa cell nuclei. Biochim. Biophys. Acta 418, 29-38. 32. Singh VK, Sung SC. 1972. Effect of spermidine on DNA-dependent RNA polymerases from brain cell nuclei. J. Neurochem. 19,2885-8. 33. Soyer M-0, Haapala OK. 1974. Division and function of dinoflagellate chromosomes. 1. Microsc. 19, 137-46. 34. Tuttle RC, Loeblich AR 111. 1975. An optimal growth medium for the dinoflagellate Crypthecodinium cohnii. Phycologia 14, 1-8. 35. Yu F-L. 1975. An improved method for the quantitative isolation of rat liver nuclear RNA polymerases. Biochim. Biophys. Acta 395,329-36.

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