ABDALLAH ET AL.-CONTINUOUS CULTURING OF T. ACOMYS

of parasitemia in the natural host Acomys cahirinus (Desmarest, 18 19). Parasitol. Res. 7 9 4 39-44 3. 2. Balber, A. E. 1983. Primary murine bone marrow cultures support continous growth of infectious human trypanosomes. Science 220: 421423. 3. Bioul-Marchand, M., Jadin, J., Steiger, R. F. & Boon, T. 1980. Multiplication of Trypanosoma cruzi in a mouse myocardial cell line. J. Parasitol., 66:1050-1052. 4. Brun, R. J. & Moloo, S. K. 1982. In vitro cultivation of animal infective forms of a West African Trypanosoma vivax stock. Acta Tropica, 39:135-141. 5. El-On, J. & Greenblatt, C. L. 1977. Trypanosoma lewisi, Trypanosoma acomys and Trypanosoma cruzi: a method for their cultivation with mammalian tissue. Exp. Parasitol., 41:3 1 4 2 . 6. Gray, M. A., Cunningham, I., Gardiner, P. R., Taylor, A. M. & Luckins, A. G. 1981. Cultivation of infective forms of Trypanosoma congolense from trypanosomes in the proboscis of Glossina morsitans. Parasitology, 82:8 1-85. 7. Hawke, C. J. 1985. Advances in trypanosomes culture. Parasitol. Today. 1:30-31. 8. Hirumi, H. & Hirumi, K. 1984. Continuouscultivation ofanimal infective bloodstream forms of an East African Trypanosoma congolense stock. Ann. Trop. Med. Parasitol., 78:327-330. 9. Hirumi, H., Doyle, J. J. & Hirumi, K. 1977a. African trypanosomes: cultivation of animal infective Trypanosoma brucei in vitro. Science, 196~992-994.

117

10. Hirumi, H., Doyle, J. J. & Hirumi, K. 1977b. Cultivation of bloodstream Trypanosoma brucei. Bull. Wld. Hlth. Org., 55:405-409. 1 1. Hoff, R. 1974. A method for counting and concentrating living Trypanosoma cruzi in blood lysed with ammonium chloride. J. Parasitol., 60527-528. 12. Mohamed, H. A., Maraghi, S., Wallbanks, K. R. & Molyneux, D. H. 1988. In vitro cultivation of Herpetosoma trypanosoma on embryonic fibroblastsand in semidefined cell free medium. J. Parasitol., 74:421-426. 13. Molyneux, D. H. 1976. Biology of trypanosomes of the subgenus Herpetosoma. In: Lumsden, W. H. R. & Evans, D. A. (ed.), Biology of the Kinetoplastida. Academic Press, New York, San Francisco, London, pp. 285-325. 14. Vincendeau, P., Guillemain, B., Dauloude, S. & Ripert, C. 1986. In vitro growth of Trypanosoma musculi: requirements of cells and serum free culture medium. Int. J. Parasitol., 16:387-390. 15. Yabu, Y. & Takayanagi, T. 1987. The effective long-term culture and cloning system for Trypanosoma gambiense bloodstream forms in vitro. Parasitol. Res., 73:38 1-383. 16. Zweygarth, E., Ahmed, J. S. & Rehbein, G. 1983. Cultivation of infective forms of Trypanosoma brucei evansi in a continuous culture system. Z. Parasitenkude, 69: 131-133.

Received 12-27-88: accepted 10-18-89

J. Protozool., 37(2), 1990, pp. 117-123 0 1990 by the Society of Protozoologists

Analysis of Chlamydomonas reinhardtii Histones and Chromatin RODNEY L. MORRIS,* LAURA R. KELLER,** ALFRED ZWEIDLER*** and PETER J. RIZU)* *Biology Department, Texas A&M University, College Station, Texas 77843, **Department of Biological Science, Florida State University, Tallahassee, Florida 32306 and ***Institute for Cancer Research, 7701 Burholme Avenue, Philadelphia, Pennsylvania 191 I 1 ABSTRACT. Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants. Key words. Amino acid analysis, HPLC, nuclear proteins, nucleosome, peptide mapping.

N

UCLEAR DNA o f eukaryotes is packaged into polynucleosome fibers by the specific interaction with five types of histones. Both higher plants and higher animals have similar histones, indicating that all types o f histones have evolved before their evolutionary divergence. Although there are abundant D N A binding proteins i n prokaryotes, their relationship t o t h e histones o f higher plants and animals is not clear [29]. Comparative studies o f the DNA-associated proteins of lower eukaryotes might be expected t o reveal transition forms between prokaryotic DNA binding proteins and the histones o f higher plants a n d animals. A particularly important group of organisms for such comparative studies is the diverse group of lower eukaryotes known as algae. Many o f these organisms are unicellular and, by several criteria, apparently represent a primitive form between prokaryotes and eukaryotes. For example, the group o f unicellular algae known as dinoflagellates do have a nucleus and condensed chromosomes, but do n o t contain nucleosomes or histones [ 181. In

contrast, histone-like proteins have been reported in Chforella [9], Vofvox [3], Chlamydomonas [22], Euglena [ 101, Porphyridium [ 11, Olisthodiscus [20], and the chrysophyte-like endosymbiont alga o f the dinoflagellate Peridinium balticum [2 I]. However, only for Euglena, Olisthodiscus, and the Peridinium endosymbiont have the histones been characterized beyond the point of demonstrating that they are acid soluble proteins with electrophoretic mobilities similar t o vertebrate histones. Although the presence in Chlamydomonas of acid soluble proteins with some physical properties similar t o histones has been reported previously [22], the chemical nature o f these proteins and their organization have remained uncharacterized. We have taken advantage of a cell wall mutant of Chlamydomonas reinhardtii and previously developed nuclear isolation procedure [ 1 I], t o characterize the histones a n d chromatin structure for this important organism i n more detail. A preliminary report o f this work has been presented [Morris, R. L., Keller, L. R. & Rizzo, P. J. 1985. J. Cell Biol., 101:204a].

118

J. PROTOZOOL., VOL. 37, NO. 2, MARCH-APRIL 1990

Fig. 1. Electron micrograph of C. reinhardtii chromatin. Chlamydomonas chromatin spreads were prepared from whole cells. The opaque flagellarroot/basal body connector structure [33] in the lower right ofthe low magnification field is found in tight association with Chlamydomonas nuclei, and provides evidence that the spread chromatin nearby is indeed from Chlamydomonas nuclei. Bar = 1 pm; inset bar = 0.1 pm.

MATERIALS AND METHODS Culture conditions and isolation of nuclei. A cell wall-less strain of C. reinhardtii, CW 92 [6], was used for these experiments. The cells were grown and nuclei isolated as described by Keller et al. [ 111. Nuclei for protein analysis were washed once with a solution containing 70% acetone, 1 mM dithiothreito1 (DTT), 1 mM phenyl methyl sulfonyl flouride (PMSF), once with 100% acetone, 1 mM DTT, 1 mM PMSF, air-dried and frozen at -20" C for later extraction. In later preparations, 1% dithiodiglycol was also included in the acetone wash solutions. Nuclei for micrococcal nuclease digestions were treated as described below. Electron microscope spreads. Chromatin spreads of C. reinhardtii nuclei of strain CW 92 were prepared according to Miller & Beatty [ 151. Micrococcal nuclease digestions. The DNA repeat length of chromatin was determined using micrococcal nuclease essentially as described by Hewish & Burgoyne [8]. The Chlamydomonm or chick adult intestine epithelialnuclear pellets were washed with a buffer containing 10 mM Pipes pH 7.4, 10 mM MgCl,, 1 mM CaCl,, 1 M sucrose, 1 mM EDTA, 0.2 mM EGTA, 60 mM KCl, 15 mM NaCl, and then suspended in this buffer at a concentration of 1.5 x lo8 nuclei/ml. After digestion with micrococcal nuclease for 15 min at 37" C, the reaction was stopped by adding 4 volumes of 20 mM Tris-HC1 pH 7.4,5mM EDTA, 1% SDS. After adding NaCl to 1 M, the samples were extracted twice with phenol/chloroform, precipitated overnight in 70%

ethanol at -20" C, collected by centrifugation, and washed with 70% ethanol. Aliquots containing 0.1 OD,,, units of DNA were suspended in 10 ml of 1.5% Ficoll, 0.02 M EDTA, and 0.01% bromphenol blue and resolved on 2% agarose gels containing 0.04 M Tris-acetate, pH 8.0,2 mM EDTA. The gels were stained with ethidium bromide and photographed. Histone extraction. The histones were extracted from isolated nuclei with 1 M CaCl,, 20 mM Tris-HC1 pH 7.4, 1% thiodiglycol, 200 U/ml aprotinin, 1 microgram/ml leupeptin on ice for 10 min. After adding HC1 to 0.3 N, the acid insoluble material was removed by centrifugation at 10,000 g at 4" C for 5 min. The acid soluble proteins in the supernatant were precipitated by addition of trichloroacetic acid (TCA) to 20% (w/ v), and allowed to stand for 10 min on ice. The pellet was washed successively with 20% TCA containing 1% thiodiglycol, followed by acetone containing 0.2% HC1, 1% thiodiglycol, and two washes with acetone containing 1% thiodiglycol. Calf thymus histones for electrophoretic comparison were prepared in this laboratory from newborn calf tissue. Histone electrophoresis. The protein samples were analyzed by SDS gel electrophoresis using the system originated by Laemmli [13], as modified by Thomas & Kornberg [28] for histones. The 18% polyacrylamide gels with the dimensions 0.8 mm x 92 mm x 64 mm were run at 175 V for 90 min at room temperature, stained with 0.1% Coomassie Brilliant Blue R-250 in 50% v/v methanol, 9% v/v acetic acid for 1 h, and destained in 20% ethanol, 7% (v/v) acetic acid.

MORRIS ET AL.- CHLAMYDOMONAS HISTONES

119

Fig. 2. Micrococcal nuclease digestions of C. reinhardtii and chick intestinal epithelial cell nuclei. Chromatin digested with micrococcalnuclease was purified, and analyzed by electrophoresis on a 2% agarose gel. Lanes ELF contain chick DNA, lanes H-L contain Chlamydomonas DNA, lanes A, G, and M contain pBR 322 DNA digested with restriction endonuclease Hinf I. Lane identification: B = no nuclease; C = 0.9U/ml nuclease; D = 30 U/ml nuclease; E = 150 U/ml nuclease: F = 600 U/ml nuclease; H = no nuclease; I = 0.9 U/ml nuclease; J = 30 U/ml nuclease; K = 150 U/ml nuclease; M = 600 U/ml nuclease.

Peptide mapping. The SDS-gel peptide mapping of C. reinhardtii histones was performed as described by Cleveland et al. [5]. The histones were separated on a 0.5 mm thick SDS minigel system, stained for 15 min, destained 30 min, and the bands of interest were excised with a razor blade and stored in 50% methanol- 1Yo mercaptoethanol at - 20" C until used for peptide mapping. For peptide mapping, the bands were rehydrated in the enzyme buffer for 15 min and then placed vertically in the wells of a 0.8 mm thick SDS mini gel. Staphylococcus aureus V8 protease (Miles Laboratories) 1 mg/ml in the enzyme buffer was added to each well (2 to 5 rl) and electrophoresis was conducted at 20 milliamperes until the dye reached the top of the separating gel. The power was turned of for 30 to 60 min to allow digestion of the samples. Electrophoresis was resumed at 175 V until the dye reached the bottom of the separating gel. The gel was stained with Coomassie Brilliant Blue as above, except the staining solution contained 4% formaldehyde. After destaining, the gel was stained with silver by the method of Wray et al. [32]. High performance liquid chromatography(HPLC). Histones were dissolved in 0.2% trifluroacetic acid (TFA, Pierce), 20% acetonitrile and resolved on a 4.6 x 250 mm column of 5 micron Bakerbond WP Octyl resin (J. T. Baker Research Products) with a linear gradient from 24.5% acetonitrile/3.5% methanol, to 56%

acetonitrile/8% methanol in 0.1% acetic acid, 20 mM CaCI,, at 1 ml/min over a 50-min period. The peak fractions were concentrated in vacuum and identified by SDS polyacrylamide gel electrophoresis. Amino acid analyses were performed by the Amino Acid Analysis Facility of the Fox Chase Cancer Center, Philadelphia, PA. Aliquots of the HPLC fractions were dried and hydrolyzed in the vapor phase over constant boiling HCl containing a drop of phenol, at 110" C for 16 h. The amino acids were derivatized with PITC and analyzed by reverse phase HPLC according to the PicoTag method of Waters Chromatography Division of Millipore Corporation, essentially as described by Bidlingmeyer et al. [2]. Hydrolytic losses were corrected based on co-processed amino acid and lysozyme standards. Methionine and cysteine were also determined after performic acid oxidation for 1 h on ice. RESULTS Miller spreads. Chromatin of C. reinhardtii appears as beaded fibers when cells are lysed in low ionic strength buffer and spread for electron microscopy according to Miller & Beatty [ 151 (Fig. 1). This fundamental subunit structure of chromatin has been observed for all eukaryotes with the exception of dinoflagellates [ 191. When the Chlamydomonas chromatin was

120

J. PROTOZOOL., VOL. 37, NO. 2, MARCH-APRIL 1990

Fig. 4. Peptide mapping of C. reinhardtii bands 1 and 2 with calf thymus H la and H 1b for comparison. A = calf thymus histones; B = C. reinhardtii band 1; C = C. reinhardtii band 2; D = calf thymus Hla; E = calf thymus Hlb. Digestion was with V8 protease and the gel was silver stained. Migration was from top to bottom.

Fig. 3. Sodium dodecyl sulfate gel of C. reinhardtii CaCI,-HCl extract of nuclei. CT = calf thymus histones; CR = C. reinhardtii. Migration was from top to bottom, gel was Coomassie stained. digested with micrococcal nuclease (Fig. 2), the DNA was cleaved at regular intervals, indicating the presence of typical nucleosomes and therefore the four core histones necessary to form them. The nucleosome spacing was very similar to that of the chick cell nuclei co-processed as a control. The average spacing from three different determinations were 189 base pairs for C. reinhardtiinuclei and 184 base pairs for chick intestinal epithelia nuclei. The rate of digestion was also similar in the two cell types, indicating a similar higher order structure of the bulk chromatin. This in turn predicts the presence of proteins functionally equivalent to vertebrate H 1 histones. As noted also by Robreau & Le Gal [22], we found that the Chlamydomonas histones are difficult to isolate by the standard acid extraction techniques used for vertebrate histones. As in the case of Physarum [ 161, 1 M CaCl, is effective in solubilizing the histones even from crude nuclear pellets. In Fig. 3, the Chlamydomonas histones extracted with 1 M CaCI, are com-

pared with calf thymus histones on SDS-gels. The algal histone preparation contains seven major components with molecular weights between 1 1 kD and 23 kD, similar to vertebrate histone preparations. Bands 1 and 2, with electrophoretic mobilities slightly lower than mammalian H 1 , are the only components completely extracted with 5% perchloric acid (data not shown), a property typical for mammalian H1 [7]. Band 3 has an electrophoretic mobility between mammalian H1 and H3 and is partially soluble in 5% perchloric acid, properties previously observed for plant H2 histones [26]. Band 4 co-migrates with mammalian H3, and band 7 with mammalian H4. The double band 516 co-migrates with mammalian H2A. To study the relationship between the algal and mammalian histones further, we applied the SDS-gel peptide mapping method [5]. Figure 4 shows that the V8 protease fragments of bands 1 and 2 d o not show any homology with any mammalian histone. However, bands 516 are similar to each other and show some similarity to calf H2A (Fig. 5). The clearest results were obtained for bands 4 and 7 which have fragment patterns which are essentially identical to those of mammalian histones H3 and H4 respectively (Fig. 5, 6). For positive identification, it was deemed necessary to purify each protein band in the SDS-gel. A recently developed micromethod for histone analysis by reversed phase high performance liquid chromatography [2 I], was very effective in purifying six of the major components. The fractions were analyzed on SDSgels and Triton-acid-urea gels (not shown). The double band 5/6 is not resolved by HPLC and may therefore be due to posttranslational modification, such as phosphorylation. The amino acid composition of the purified C. reinhardtii histones are listed and compared to calf thymus histones in Table 1. Band 1 contains about 30% lysine and 20% alanine and is similar to the “very lysine-rich” mammalian H 1 histones.

MORRIS ET AL.- CHLAMYDOMONAS HISTONES

121

Fig. 5. Peptide mapping of C. reinhardtii bands 3,4, 5 , and 6 with calf thymus histones H2A, H2B, and H3 for comparison. A = C. reinhardtii band 3; B = calf H3; C = C. reinhardtii band 4; D = calf H2A; E = C. reinhardtii band 5 ; F = calf H2B G = C. reinhardtii band 6 .

Band 2 was not recovered in sufficient amounts to give reliable composition data. Band 3 is a “slightly lysine-rich” histone (ca. 20% lysine) with a low arginine content as is typical for mammalian H2B histones. Bands 5/6 are very similar in composition to mammalian H2As with a typically high content of alanine, leucine, lysine, arginine and glutamic acid. Band 4 is almost identical to mammalian H3, and band 7 to mammalian H4. Both are arginine-rich, with H3 containing in addition, a large amount of glutamic acid and the only cysteine, while H4 is rich in glycine. DISCUSSION In the search for the origin of the 5 histone types which occur in similar form in both higher plants and animals, we are studying the chromosomal proteins of primitive eukaryotic organisms. Of particular interest are the algae because some members of this varied group show certain similarities to prokaryotes and because some algae have features typical of higher plants (e.g. photosynthetic, autotrophic, sessile) while others can live as heterotrophic mobile protozoans. We have previously shown that the chrysophyte-like endosymbiont alga of the dinoflagellate Peridinium balticum has a full complement of histones similar to higher organisms [21]. Here we have taken advantage of a wall-less mutant to study the chromatin organization of the unicellular motile green alga C. reinhardtii. We found that the nucleosome structure of this primitive organism is surprisingly similar to that of higher animals and higher plants. The nucleosome spacing of Chlamydomonas with 189 base pairs is very similar to that of transcriptionally active somatic cell types of vertebrates and angiosperms [30].This is in contrast to the 220 base pair spacing found in three other unicellular algae, namely Euglena [14], Olisthodiscus [25], and the chrysophyte-like endosymbiont alga Of Peridinium balticum [24], and the 172 base paif Spacing in the unicellular red alga Porphyridium [ 11. One of the determinants of nucleosome spacing is the very

Fig. 6. Peptide mapping of C. reinhardtii band 7 with calf thymus histone H4. A = C. reinhardtii band 7; B = calfH4. Digestion was with V8 protease and the gel was silver stained. Migration was from top to bottom.

122

J. PROTOZOOL., VOL. 31, NO. 2, MARCH-APRIL 1990

Table 1. Amino acid composition of Chlamydomonas reinhardtii histonesa Amino acid

Band 1

Calf H 1

Band3

CalfH2B

Band4

Calf H3

Band 5/6

Calf H2A

Band 7

Calf H4

asx glx ser glY his

3.4 10.4 6.5 5.6 1.1 1.7 3.2 18.8 7.6 1.8 4.1 0.9 0.0 2.7 3.4 1.o 28.0 0.0

1.8 3.2 7.3 6.4 0.0 1.8 4.6 27.1 9.6 0.5 3.7 0.0 0.0 0.9 4.1 0.5 28.4 0.0

5.8 10.2 6.5 6.2 I .3 3.4

4.8 8.0 11.2 5.6 2.4 6.4 6.4 10.4 4.8 4.0 7.2 1.6 0.0 4.8 4.8 1.6 16.0 0.0

3.9 11.1 2.2 5.6 1.3 13.2 7.7 13.4 4.6 2.2 4.2 1.6 0.8 5.0 9.4 3.0 10.0 0.9

3.7 11.1 4.4 5.2 1.5 13.3 7.4 13.3 4.4 2.2 4.4 1.5 0.7 5.2 8.9 3.0 8.2 1.5

6.0 7.8 3.5 13.7 I .3 7.3 3.1 14.6 4.1 2.7 6.5 0.0 0.0 5.1 10.4 0.9 12.9 0.0

6.2 9.3 3.1 10.9 3. I 9.3 3.9 13.2 3.9 2.3 6.2 0.0 0.0 4.7 12.4 0.8 10.9 0.0

5.5 7.0 3.3 14.8 I .8 14.2 7.8 6.6 1.4 3.5 6.0 2.2 0.0 5.5 7.6 2.3 9.7 0.9

4.9 5.9 2.0 16.7 2.0 13.7 6.9 6.9

thr ala pro tYr val met CYs ile leu phe lYS

m-ly

5.8

13.1 5.4 2.6 6.1 1.4 0.0 4.3 4.9 2.0 21.1 0.0

1.o

3.9 8.8 1.O 0.0 5.9 7.8 2.0 9.8 1.o

C. reinhardtii bands refer to the designations given in Fig. 3. All values are in mole percent. Values for calf thymus histones were taken from Walker et al. [31]. a

lysine-rich H 1 histones [ 121. Thus the unusually large spacings found in avian erythrocytes and in sea urchin germ cells can be attributed to the presence of unusual H1 histones, while the unusually short spacing in yeast chromatin can be correlated with the absence of H1 histones. The Chlamydomonas Hls, although slightly larger than the mammalian forms, have very similar properties. The H2B of C. reinhardtii is larger than the mammalian H2B, and has a mobility in SDS gels that is lower than H3, a property shared with the H2Bs of many higher plants [27]. The H2A of C. reinhardtii is resolved into two closely spaced bands by both SDS and acid-urea gel electrophoresis, but not by reversed phase chromatography. This could be due to phosphorylation of H2A. The observation of multiple H2As in SDS gels has also been noted in higher plants [27]. However, unlike many plant H2As which have C-terminal additions [23], the C. reinhardtii H2A is similar in size to mammalian H2A. The H3 and H4 histones appear to be highly conserved in C. reinhardtii, similar to the situation in Volvox [ 171 and in higher plants [4]. ACKNOWLEDGMENTS This work was supported by National Science Foundation Grants PCM-83 16708 and DCB-8701389 to L.R.K., National Institutes of Health Grants CA- 15 135, CA-06927, RR-05539, and an appropriation by the Commonwealth of Pennsylvania to A.Z., and National Science Foundation Grants PCM-83 18233, and DCB-86 16022 to P.J.R. LITERATURE CITED 1. Barnes, K. L., Cragie, R. A., Cattini, P. A. & Cavalier-Smith, T. 1982. Chromatin from the unicellular red alga Porphyridium has a nucleosome structure. J. Cell Sci., 57: 15 1-1 60. 2. Bidlingmeyer, B. A., Cohen, S. A. & Tarvin, T. L. 1984. Rapid analysis of amino acids using pre-column derivatization. J. Chromatogr., 336:93-104. 3. Bradley, D. M., Goldin, H. H. & Claybrook, J. R. 1974. Histone analysis in Volvox. FEBS Lett., 41:219-222. 4. Chaubet, N., Chaboute, M., Philipps, G. & Gigot, C. 1987. Histone genes in higher plants: organization and expression. Dev. Gen., 8: 461-473. 5. Cleveland, D. W., Fischer, S. G., Kirschner, M. W. & Laemmli, U. K. 1977. Peptide mapping by limited proteolysis in sodium dodecyl

sulfate and analysis by gel electrophoresis. J. Biol. Chem., 2 9 1 1021106. 6. Davies, D. R. & Plaskitt, A. I97 I . Genetical and structural analyses of cell-wall formation in Chlamydomonas reinhardtii. Genet. Rex, Camb.. 17:33-43. 7. Delange, R. J. & Smith E. L. 197I . Histones: structure and function. Ann. Rev. Biochem.. 40:279-3 14. 8. Hewish, D. R. & Burgoyne, L. A. 1973. Chromatin structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear DNAase. Biochem. Biophys. Res. Commun., 52504-5 10. 9. Iwai, K. 1964. Histones of rice embryos and of Chlorella. In: Bonner, J. & Ts’O, P. (ed.), The Nucleo-Histones. Holden-Day, San Francisco, California, pp. 59-7 1. 10. Jardine, N. J. & Leaver, J. L. 1978. The fractionation of histones isolated from Euglena gracilis. Biochem. J.. 169:103-1 11. 11. Keller, L. R., Schloss, J. A., Silflow, C. D. & Rosenbaum, J. L. 1984. Transcription of (Y and j3 tubulin genes in vitro in isolated Chlamydomonas reinhardtii nuclei. J. Cell. Biol., 98: 1138-1 143. 12. Kuenzler, P. & Stein, A. 1983. Histone H5 can correctly align randomly arranged nucleosomes in a defined in vitro system. Nature, 3021548-550. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. 14. Magnaval, R., Valencia, R. & Paoletti, J. 1980. Subunit organization of Euglena chromatin. Biochem. Biophys. Res. Commun., 92: 1415-142 1. 15. Miller, 0.L., Jr. & Beatty, B. R. 1969. Visualization of nucleolar genes. Science, 164:955-957. 16. Mohberg, J. & Rusch, H. P. 1969. Isolation of the nuclear histones from the Myxomycete Physarum polycephalum. Arch. Biochem. Biophys., 134377489. 17. Mueller, K. & Schmitt, R. 1988. Histonegenesof Volvoxcarferi: DNA sequence and organization of two H3-H4 gene loci. Nuc. Acids Res., 16:4121-4136. 18. Rizzo, P. J. 1985. Histones in protistan evolution. Biosystems, 18:249-262. 19. Rizzo, P. J. 1987. Biochemistry of the dinoflagellate nucleus. In: Taylor, C. F. R. (ed.), The Biology of Dinoflagellates. Blackwell Scientific Publications, Oxford, Great Britain, pp. 143-1 73. 20. Rizzo, P. J., Bradley, W. & Moms, R. L. 1985. Histones of the unicellular alga Olisthodiscus luteus. Biochemistry, 24: 1727-1 732. 21. Rizzo, P. J., Moms, R. L. & Zweidler, A. 1988. The histones of the endosymbiont alga of Peridinium balticum. Biosystems, 21:23 1238. 22. Robreau, G. & Le Gal, Y. 1975. Isolation and partial charac-

MORRIS ET AL.-CHLAMYDOMONAS HISTONES

terization of Chlamydomonas reinhardtii chromatin. Biochimie, 57:703710. 23. Rodriguez, J. de A., Brandt, W. F. & von Holt, C. 1985. The amino acid sequence of wheat histone H2A1: a core histone with a C-terminal extension. Eur. J. Biochem., 150:499-506. 24. Shupe, K. & Rizzo, P. J. 1983. Nuclease digestion ofchromatin from the eukaryotic algae Olisthodiscus luteus, Peridinium balticum, and Crypthecodinium cohnii. J. Protozool., 30: 599-606. 25. Shupe, K., Rizzo, P. J. & Johnson, J. R. 1980. DNA repeat length of chromatin from the unicellular alga Olisthodiscusluteus. FEBS Lett., 115:221-224. 26. Spiker, S., Key, J. L. & Wakim, B. 1976. Identification and fractionation of plant histones. Arch. Biochem. Biophys., 176310-5 18. 27. Spiker, S. 1985. Plant chromatin structure. Ann. Rev. Plant Physiol., 36:235-253. . Kornberg, R. D. 1975. An octamer of histones 28. Thomas, J. 0.% in chromatin and free in solution. Proc. Natl. Acad. Sci. USA, 72:26262630.

123

29. van Holde, K. E. 1988. Chromatin. Springer-Verlag,New York, New York, p. 168. 30. van Holde, K. E. 1988. Chromatin. Springer-Verlag,New York, New York, p. 302. 31. Walker, J. M., Goodwin, G. H., Smith, B. J. & Johns, E. W. 1982. The chromosomal proteins. In: Neuberger, A. & Deenen, L. M. van (ed.), Comprehensive Biochemistry, Vol. 19B, pt. 2. Elsevier Biomedical Press, Holland, pp. 507-573. 32. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. 1981. Silver staining of proteins in polyacrylamide gels. Anal. Biochem., 118:197203. 33. Wright, R. L., Salisbury, J. L. & Jarvik, J. 1984. A nucleusbasal body connector in Chlamydomonas reinhardtii that may function in basal body location or segregation. J. Cell Biology, 101:1903-1912.

Received 7-19-89;accepted 10-10-89

J. Profozool.,37(2), 1990, pp. 123-128 0 1990 by the Society of F'rotozoob&s

Scanning Electron Microscopic Observation of Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa MASAHIRO FUJISHIMA,*' HISAHIRO SAWABE** and KENJI IWATSUKI*** *Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, **Central Technical Laboratory, Nippon Oil Co., Ltd. Yokohama 231 and ***Tsukuba Life Science Laboratory, Nippon Petrochemicals Co. Ltd. Tsukuba 300-26. Japan

ABSTRACT. To identify the surface features of Holospora obtusa during its differentiation from the reproductive short form to the infectious long form, bacteria of four different buoyant densities were isolated by Percoll density gradient centrifugation of homogenates of host cells or isolated macronuclei, and examined with a scanning electron microscope. Bacteria of buoyant density 1.09 g/ml were reproductive short forms as well as cells at various stages in the elongation process including fully elongated ones. Bacteria of buoyant densities 1.11 g/ml and 1.13 g/ml were premature Iong forms and those of 1.16 g/ml were mature infectious long forms. Bacteria of buoyant density 1.09 g/ml had an entirely rough surface while those of buoyant densities 1.1 1 g/ml and 1.13 g/ml were smooth and had wale-like stripes on their surface. A small tapered tip was observed at one end of the bacteria of buoyant density 1.13 g/ml. Bacteria of buoyant density 1. I6 g/ml had an entirely smooth surface, but one end always showed a rough surface; this locally differentiated surface of the special tip of the infectious long form may be responsible for both the nuclear and species specificities of the infectivity of H. obtusa. These observations indicate that the surface of H. obtusa changes during differentiation and the special tip develops in bacteria of buoyant density 1.13 g/ml. Key words. Endonuclear symbiont, Holospora obtusa, Paramecium caudatum, scanning electron microscopy.

T

HE gram-negative bacterium Holospora obtusa is a mac-

and the bacterium transforms to the infectious long form at the ronucleus-specific endonuclear symbiont, which multi- stage of buoyant density 1. I6 g/ml [Fujishima, M. & Nagahara, plies only in the macronucleus of the ciliate Paramecium cau- K. 1985. VII Int. Congr. Protozool., 301 abst., Fujishima, M. datum. This bacterium changes its morphology during the life & Kojima, K. 1987. Zool. Sci., 4:994 abst., 61. Bacteria at all cycle. When the host cell grows this bacterium also grows by these stages have been isolated by Percoll density gradient cenbinary fission of the reproductive short form (1-1.5 pm in length) trifugation from homogenates of host cells or isolated macronuin the host macronucleus. When the host starves and stops clei [Fujishima, M. & Nagahara, K. 1985. VII Int. Congr. Prodividing, however, the reproductive short form also stops di- tozool., 301 abst., Fujishima, M. & Terado, T. 1986. Zool. Sci., viding, elongates to 13-1 5 Mm and increases its buoyant density 3:997 abst., Kojima, K. & Fujishima, M. 1987. Zool. Sci., 4: from 1.09 g/ml to 1.1 1 g/ml, 1.13 g/ml and 1.16 g/ml, sequen- 994 abst., Fujishima, M. & Kojima, K. 1987. 4:994 abst. 51. tially. The elongation process occurs entirely at the stage of The fine structure of the reproductive short and infectious buoyant density 1.09 g/ml. Then, dispersed nucleoids which can long forms of H . obtusa by transmission electron microscopy be stained with the DNA specific fluorochrome, 4', 6-diami- (TEM) has been reported [6-10, 121. It has been found that the dino-2-phenylindole (DAPI) appear at the stage of buoyant den- infectious long form consists of three regions; a typical bacterial sity 1.1 1 g/ml. These DAPI-positive nucleoids then accumulate cytoplasmic region, a homogeneous electron-dense region and in about the middle of the bacterial cell and two small dotted a special tip which is located at one end of the bacterium and regions in the DAPI-positive part begin to show strong fluores- is composed of a homogeneous electron-translucent material. cence at the stage of buoyant density 1.13 g/ml. Finally, the The infectious long form always penetrates host macronuclear space between the two dotted DAPI-positive regions narrows membranes with this special tip 1st. Therefore, this tip seems to have an ability to distinguish between the two kinds of nuclei and enable the bacterium to penetrate only macronuclear membranes. To compare the surface structures of the special tip and I To whom correspondence should be addressed.