Relations between basic proteins and nucleic acids in Acanthamoeba

430

I. P. S. Agrell et al.

These blastocysts had obviously a diminished survival in vitro with time, as shown by the fact that few healthy nuclei were recovered at 4, 6 and 8 h after the pulse. Continuous labelling for 4 h, however, produced 90 % of labelled nuclei. It was immediately obvious that there was a considerable degree of asynchrony within the cells of each embryo. Most embryos had unlabelled, diffusely labelled and spotty labelled nuclei (figs 1b, 2). Characteristic blocks of heavy labelling were often seen in several nuclei, as exemplified in figs 1 b, 2. In his excellent review of heterochromatin, Brown 161 records the widespread belief that there is not autonomous genetical activity during early embryonic stages and therefore heterochromatin may fail to appear, or may appear gradually, during these early stages. Our results are contrary to this belief. While we did not obtain evidence of active DNA synthesis at the two-cel1 stage, we observed obvious blocks of late replication at all stages investigated beginning with the four-cell one. There is general agreement that these blocks of late replications are indeed characteristics of heterochromatin. The heterochromatic state is considered the visible manifestation of suppression of gene action [6] (but see Schmid [7], for a somewhat different view). The remarkable degree of asynchrony among cells of the same embryo and the appearance of discrete blocks of late replication that parallel those seen in cultured cells from adult mice [8] indicate that heterochromatization of chromosomal segments is already instrumental in directing and controlling differentiation at the early stages of development in the mouse. This work has been suouorted bv grants from the Swedish Medical Research ‘douncil, -ChR, Roma and by contract N. 023-63-2 BIOI Euratom-University of Pavia. Dr H. Strander, Stockholm has been of great help with discussion and advice on the technique of obtaining embryos. This is publication No. 477 of the Euratom Biology Division.

REFERENCES I. Mintz, B, J exptl zoo1 157 (1964) 85. 2. Izquierdo, L & Roblero, L, Experientia 21 (1965) 532. 3. Woodland, H R&Graham, C F, Nature 221 (1969) 327.

Exptl Cell

Res 55

4. 5. 6. 7. 8.

Edwards, R G &Gates, A H, J endocrinoll8 (1959) 292. Tarkowski, A K, Cytogenetics 5 (1967) 394. Brown, S W, Science 151 (1966) 417. Schmid, W, Arch Klaus-Stift Vererb Forsch 42 (1967) I. Tiepolo, L, Fraccaro, M, Hulten, M, Lindsten, J, Mannini, A & Ming, P, L, Cytogenetics 6 (1967) 51.

Received March 24, 1969

RELATIONS BETWEEN BASIC PROTEINS AND NUCLEIC ACIDS IN ACANTHAMOEBA I. P. S. AGRELL, KJELLSTRAND

G. K. A. ANDERSSON

and P. T. T.

Summary The nucleoproteins in the cells of Acunthamoeba are easily extracted with 0.12 M NaCl. A substantial part of the basic proteins seem to be associated to acid substances other than nucleic acids. The amount of basic protein in the amoeba cell decreases after the cell cultures have ceased to grow.

The mitotic activity of free living soil amoebae, Acanthamoeba sp. Neff, was found to be strongly affected by phytohaemagglutinin. The mitogenetic action of this substance appears to be due to removal of extracellular inhibitors, possibly basic peptides [l, 2, 3, 51. The intrinsic capacity for regulation of cellular division appears restricted in this organism, which may be due to aberrant nucleoprotein relations. A preliminary analysis of the solubility behaviour of basic proteins from the amoeba indicated the presence of a very high amount of non-DNA-bound basic proteins [5]. From these starting points a more thorough analysis of the nucleic acids and basic proteins in the amoebae seemedto be of interest. Material and methods The Acanthamoeba cells were cultivated in suspension cultures in Neff’s “optimal growth medium” [18]. 250 ml erlenmayer flasks were used, containing 200 ml medium. The temperature was kept at +22”C and each flask was aereated-at a rate of 15 l/h. Each culture was started at a population density of 15 cells/mm3, taken from stock cultures in late exDonential Dhase of growth. The DOPUlation doubling time under ‘these conditions was ‘45 h. The final cell density at stationary phase reached 800 cells/mm3. For the biochemical analysis samples were taken from the middle of exponential phase and from stationary phase, about 24 h after cessation of growth. After determination of the cell density in counting chambers, the amoebae

Basic proteins and nucIeic acids in Acanthamoeba

I

NaCl

:: b

12

I

NaCl

3

~

I

123 NaCl

suspension was concentrated by centrifugation and kept in pure acetone at - 20°C. Two separate sets of cultures were run. The analysis of the fresh acetone powder was initiated with two extractions with 0.12 M NaCl-citrate at pH 5.8, one extraction with Tris-buffer and a third extraction with NaClcitrate. Thereby a distinction between 0.12 M NaCl-soluble and NaCl-insoluble nucleoproteins was made. The separation of DNA and RNA was made by PCA-extraction and alkaline hydrolysis [16]. DNA was determined with diphenylamine [9] and RNA by its UVabsorption at 2600 A. The basic proteins in the fractions were anaiysed as their reinechate salts [14]. Only the basic proteins insoluble at pH 8.3 were considered. A qualitative analysis of the basic proteins was also made through polyacryl amide gel electrophoresis [ll]. The values obtained and reproduced as amount per cell have been corrected for the appearance of binucleate cells [12]. To obtain a certain comprehension of the localization of the nucleoproteins in the amoeba cell the biochemical analysis was supplemented with some cytochemical measurements made upon amoebae in exponential phase fixed to glass. The fixations used were acetate buffered formol, alcohol-acetic acid and alcohol-acetone. Staining was made with Feulgen [13], methyl green and methyl green + pyronin Y [19], azur B [15] and fast green [6]. The concentration of stain in each cell was measured microspectrophotometrically by moving a small measuring area, 1 p2, several times over each cell. Through continuously recording the extinction in this way a differentiation between cytoplasm, nucleolus and nucleus could be accurate obtained. A correction was made for the respectively degree of compression. The type of fixation did not notably alter the stainability. At least 20 cells in three series were measured in this manner. The smallness of the object prevented more accurate measurement of the nuclear volumes. Additionally, series of ammoniacal silver impregnations were made on formol fixed cells [8]. For comparison, white blood cells, liver cells and Tetrahymena cells were also fixed and stained in the above mentioned manners.

Results In fig. 1 the sequenceof extraction of DNA and RNA with 0.12 M NaCl is reproduced and in table 1 the respective values for NaCl-soluble and NaCl-insoluble nucleic acids and basic pro-

z

123 NaCl

b

43 1

Fig. 1. The amount of the nucleic acid during thrice repeated extraction with0.12 M NaCI from two sets of cultures. C = initial amount. (a) Stationary phase; (b) exponential phase. 0 - 0, RNA; o--o,DNA. Abscissa: Sequence of extraction; ordinate: ,ug/l O6 cells.

teins are collected. It is evident that the amount of DNA and RNA per cell does not change during the growth period. The sameRNA/DNAratio, 6.3, was obtained from the exponential as well as from the stationary phase. However, the total amount of pH 8.3-insoluble basic proteins per cell decreasesmore than 40 % after completed growth in stationary phase(table 1). A high amount of both RNA and DNA could be extracted with 0.12 M NaCl, about 85 % of the RNA and 75 % of the DNA (fig. 1, table 1). The course of the extraction curves in fig. 1 possibly indicates that RNA is more easily eluted from cells in exponential phase while the contrary may be true for DNA. Yet, after three extractions, table 1, the distribution of the nucleic acids according to their solubility is very much the same for both exponential and stationary phase. Possibly only the ratio soluble/ insoluble RNA is actually higher in exponential phase. The basic proteins behave differently in this respect (table 1). The ratio NaCl-soluble/-insoluble basic protein was 1.8 in exponential phase but only 1.2 in stationary phase. It should further be stressedthat the ratio basic proteins/nucleic acids was found to be very high as concerns the NaCl-insoluble fraction. The pattern of the basic proteins was aIso very different in the soluble and insoluble fractions (fig. 2), while these patterns showed only minor divergences when exponential and stationary phase were compared. Neither did the electrophoretic pattern for each type of basic protein, corresponding to F,, Fe,, Exptl

Cell Res 55

432

I. P. S. Agrell et al.

Table 1. The amounts of DNA, RNA and basic proteins and the ratio basic proteins/nucleic acids (BPINA) in the 0.12 M NaCl-soluble and insoluble fractions pug/1O6 cells Insoluble

Exponential phase Stationary phase

DNA RNA Bas. Prot. BP/NA-ratio DNA RNA Bas. Prot. BP/NA-ratio

0.17 0.37 6.75 0.17 0.60 4.80

12.5

6.3

Soluble 0.49 3.73 12.10 0.48 3.50 5.75 1.4

FZband F, respectively, change with growth or extraction. The cytochemical measurements gave the following results on amoebae in exponential phase. Only the nucleus is Feulgen-positive. The nucleolus is Feulgen-negative. The DNA-concentration was, according to the measurements, about X 8 lower than in the Tetrahymena nucleus, which is in agreement with the results from biochemical analysis. Neither with methyl green alone or in combination with pyronine Y could any observable nuclear stain be obtained. Only the cytoplasm and the nucleolus becamestainable with pyronine, in a concentration ratio of about 0.5 : 1. With fast green, nucleolus, nucleus and cytoplasm stained in a proportion of about 1 :0.7 :0.4. When the amount of fast green stainable material was calculated, concentration x volume, it was found that the cytoplasm + nucleolus should contain about x 12 more than the nucleus proper. With azur B the nucleolus, nucleus and cytoplasm was stainable in a proportion of about 1 :0.5 :0.7. Thus the cytoplasm + nucleolus should hold about x 25 more azur B stainable acid material than the nucleus. This observation should be compared to the biochemically found RNA/ DNA-ratio of only 6.3. Ammoniacal silver impregnation never gave any positive results with the amoebae, despite variation of the fixation, staining and washing times. In the other cell material tested simultaneously, an intense nuclear stain always developed. Exptl

Cell Res 55

F 2a

2.9

FZb

F3

b

Fig. 2. The pattern of the basic proteins in the 0.12 M NaCi-soluble and insoluble fractions. The sum of F,-F3 for exponential and stationary phase, respectively, in each of the fractions is taken as 100. Abscissa: (a) Insoluble: (b) soluble; ordinate: $6. n , exponential phase; z, sta: tionary phase.

Discussion The DNA and RNA content of the amoeba cells did not change during the growth period, when exponential and stationary phase were compared. Such a change should perhaps not be expected. On the other hand, when the amoebae ceasedto multiply at stationary phase,the amount of basic proteins decreasedmarkedly, more than 40 %. Thus the ratio basic proteins/nucleic acids decreases correspondingly. However, one may ask whether all the basic proteins actually represent nucleoproteins. Some observations indicate that this may not be the case.Thus the ratio basic proteins/nucleic acids is exceedingly high, up to 12.5. Such a high ratio does not seemlikely to apply to nucleoprotein. Further, by cytochemica1 means, it was demonstrated that the cytoplasmic/nuclear ratio of basic as well as acid substancesattained much higher values than the biochemically established RNA/DNA-ratio. These facts taken together indicate that a substantial fraction of the basic proteins is coupled to acid macromolecules other than nucleic acids, e.g. acid proteins or acid polysaccharides, as has been shown for other cellular material [4]. Consistent with this is also the facts that the amoeba cells show a strong PAS-reaction [7], and produce polysaccharides for the formation of their cellulose cysts [17]. The high solubility of the amoeba-DNA in 0.12 M NaCl may be due to a competetive replacement of the nucleic acid by

Versatile growth other acid macromolecules at the histones. In Tetrahymena cells the caseseemsdifferent. There the amount of basic protein is higher at stationary than at exponential phase [IO]. The major amounts of RNA, DNA and basic proteins were soluble in 0.12 M NaCI, which indicates that aberrant nucleoprotein conditions prevail in the amoeba cell. A deviating nucleoprotein state is also implied by the nonstainability of the amoeba nucleus with ammoniacal silver and methyl green despite retained fast green and Feulgen stainability. The ratio basic proteins/nucleic acids was comparatively normal in the NaCl-soluble fraction and the distributional pattern of the soluble basic proteins is similar to that of ribosomal protein. Thus, the soluble proteins may be principally RNAassociated. The pattern of basic proteins in the insoluble fraction was different and the ratio basic proteins/nucleic acids was exceptionally high. It may be presumedthat the insoluble fraction contains the greater amount of those basic proteins which exist in macromolecular combinations other than nucleoproteins. Valuable technical aid was given by Miss Brita Nilsson. The investigation was facilitated by grants from the Royal Physiographical Society.

REFERENCES I. Agrell, I P S, Exptl cell res 42 (1966) 403. 2. -Ibid 43 (1967) 691. 3. - Ibid 50 (1968) 687. 4. Agrell, I P S & Christensson, E, Nature 191 (1961) 284. 5. Agrell, I P S & Karlsson, B, Exptl cell res 48 (1967) 634. 6. Alfert, M, & Geschwind, I, Proc natl acad sci US 39 (1953) 991. 7. Barnes, W &Jensen, T, J protozooll45 (1967), Suppl. 11. 8. Black, M M & Ansley, H R, J histochem cytochem 14 (1966) 177 9. Burton, K, Biochem j 62 (1955) 315. 10. Christensson, E, Arkiv zoo1 19 (1967) 297. I I. Johns, E W, Biochem j 104 (1967) 78. 12. Kjellstrand, P, Exptl cell res 53 (1968) 37. 13. Leuchtenberger, C, Gen cytochem meth 1 (1958) 220. 14. Lindh, N 0 & Brantmark, B L Anal biochem 10 (1965) 415. 15. Mundkur, B & Brauer, B, J histochem cytochem 14 (1966) 94. 16. Munro, H N & Fleck, A, Analyst 91 (1966) 78. 17. Neff, R J, Benton, W & Neff, R H, J cell bio123 (1964) 66 A. r’s

691801

chamber for cell cultures

433

18. Neff, R J, Ray, S A, Benton, W F & Wilborn, M, Meth cell physiol 1 (1964) 55. 19. Pearse, AC E,Histochemistry, p. 826. London (1960). Received March 20, 1969

A VERSATlLE CHAMBER FOR THE GROWTH OF DUPLICATE MONOLAYER CELL CULTURES DIRECTLY ON MICROSCOPE SLIDES G. E. LEVENSON,

Histology-Embryology Department, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa 19104, USA’ and Strangeways Research Laboratory, Cambridge, UK

During studies on various cell types grown in monolayer cultures, the need arose for a culture vessel with an absolutely flat glass growth surface. Conventional dishesdo not have uniformly flat bottoms and cells tend to pool in the depressions. The inclusion of cover glassesin dishes to permit fixation and staining of cells has several disadvantages; cells become interposed between cover glass and the bottom of the dish thus interfering with inverted optics and visibility, and after histological processingit is very difficult to relocate microscopic fields that have been studied before fixation. The present system overcomes these problems and has additional advantages. (1) Both experimental and control cultures are on the sameslide and can be processedsimultaneously and identically. (2) The cells are cultivated on fresh surfaces. This prevents poor visibility due to scratching that frequently mars expensive dishes. (3) Cultures may be grown on membranes under the same conditions as on glass. (4) This uniform flat surface and the parallel glass-air-liquid interfaces provide excellent uniform optical conditions all across the chamber. (5) Entire cultures may be preserved for staining and subsequent study. (6) Large numbers of fixed cultures can be processedsimultaneously usingconventional slide carriers and staining dishes. 1 Author’s permanent address. Exptl

Cell Res 55