Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver

Molecular and Biochemical Parasitology, 12 (1984) 249-260 Elsevier

249

MBP 00454

EVIDENCE FOR MAJOR DIFFERENCES IN RIBOSOMAL SUBUNIT PROTEINS FROM PLASMODIUM BERGHEI AND RAT LIVER

F R E D E R I C K W. MILLER* and J U D I T H ILAN**

Department of Anatomy and Developmental Biology Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, U.S.A. (Received 30 December 1983; accepted 9 March 1984)

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their n u m b e r and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace a m o u n t s of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal R N A and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins. Key words: Plasmodium berghei; Ribosomal proteins; Two-dimensional polyacrylamide gel electrophoresis

INTRODUCTION

Malaria plasmodia remain poorly understood in terms of their molecular biology. Little is known specifically about the parasite's translational apparatus. Studies have shown that plasmodial ribosomes are typically eukaryotic in terms of their monomeric and subunit sedimentation rates [1,2], ultrastructure [3], and susceptibility to inhibitors in cell-free protein synthesizing systems [4]. However, the ribosomes of

*Present address: Clinical Center, 9N240, N I A D D K , National Institutes of Health, Bethesda, M D 20205, U.S.A. **Present address: Department of Reproductive Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, U.S.A. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

250

Plasmodium species differ from those of their hosts in ribosomal RNA size [5-11], stability [11], base composition [2,10-13], and genomic D N A units [14]. Because of the limited knowledge of malaria ribosomes and the potential for the development of new antimalarials, a detailed study of ribosomal RNA and ribosomal proteins was initiated in the conveniently maintained rodent parasite, Plasmodium berghei. We have previously shown that an unusually large RNA molecule is associated with the parasite's small ribosomal subunit, while a uniquely hydrolyzed RNA species is contained in the large ribosomal subunit [11]. Here, we report the first enumeration and partial characterization of malaria ribosomal proteins, and show that major differences exist between these parasite proteins and those of the host. MATERIALS AND METHODS

Preparation of ribosomes. The NYU2 strain of P. berghei was maintained by serial transfer in 30-60 day old Sprague-Dawley female rats. All animals were fed ad libitum and sacrificed by ether anesthesia. All solutions and siliconized glassware were autoclaved and all operations were performed at 4°C unless otherwise indicated. Ultrastructurally-intact, viable parasites and purified malarial ribosomes, derived from polysomes active in protein synthesis and free from host ribosome contamination, were obtained as previously described [11]. Purified host liver polysomes were obtained from uninfected female rats by the method of Hoffman and Ilan [15].

Preparations of ribosomal proteins. Purified ribosomal subunits were obtained from polysomes as previously described [11] using 10-40% (w/v) linear sucrose (ribonuclease free) gradients centrifuged for 10 h in a Spinco SW 27 rotor at 26000 rpm at 28°C. The fractions containing the 40 S subunits and those containing the 60 S subunits were pooled and concentrated by vacuum dialysis, and were then precipitated with ethanol [16]. Extraction and electrophoresis of RNA was by the method of Miller and Ilan [11]. Proteins were extracted from polysomal suspensions or ethanolprecipitated ribosomal subunits by the method of Barritault et al. [17]. Protein concentrations were determined using bovine serum albumin as the standard [18]. Aliquots of acetone-precipitated proteins were radioiodinated [19] and passed through a Biogel P-4 column, previously saturated with bovine serum albumin and equilibrated with first dimension sample buffer (8 M urea, 10 mM dithiothreitol, 2% (v/v) Triton X-100, 2.5% (v/v) acetic acid). Peak excluded fractions of ribosomal proteins were pooled, reduced, and alkylated prior to electrophoresis [20].

Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins. Reduced and alkylated ribosomal proteins were precipitated with acetone and resuspended in first dimension sample buffer including 15% (v/v) glycerol. Samples were heated for 3 min in a boiling water bath just prior to electrophoresis. Proteins were fractionated in the first dimension on the basis of charge and hydrophobicity by Method III of Hoffman

251 and Dowben [21]. Gels were rimmed from their tubes and equilibrated in 100 ml of second dimension sample buffer (63 mM Tris HC1, pH 6.8, 1.2% lithium dodecyl sulfate, 10 mM dithiothreitol) for 40 minutes prior to second dimension electrophoresis. Equilibrated first dimension gels were anchored on top of 15% (w/v) acrylamide, 0.4% (w/v) bisacrylamide [22] slab gels (140 × 160 X 2 mm) by covering them with hot second dimension sample buffer containing 1% agarose. Lithium dodecyl sulfate was substituted for sodium dodecyl sulfate so that electrophoresis could be performed at 4°C and 0.25% linear polyacrylamide was included to prevent cracking when the slab gels were dried. For the second dimension, electrophoresis was towards the anode at 4 W per gel (constant power) until the bromphenol blue tracking dye entered the separation gel. The power was then increased to 9 W per gel until the dye left the slab gel (about 10-12 h later). Gels were fixed in 45.4% methanol, 9.2% acetic acid for 1 h on a shaker and stained in the above solution plus 0.25% (w/v) coomassie brilliant blue R for 1 h. Gels were destained over 2 days with 2-3 changes of 5% methanol, 7.5% acetic acid. For autoradiography, gels were photographed, dried, and exposed to Kodak XR-5 X-ray film which was developed by standard techniques. RESULTS

Ribosomal RNA analysis. Ribosomal RNA was isolated from aliquots of the pooled subunit fractions and analyzed by sodium dodecyl sulfate electrophoresis [11] to determine the extent of cross contamination by the other subunit. No contamination of parasite ribosomes by host ribosomes was detected by ribosomal RNA analysis. Electrophoresis of the subunit ribosomal RNA showed contamination of less than 1% of malarial or liver 40 S ribosomal subunits by 60 S subunits (data not shown). However, more contamination of 60 S ribosomal subunits by 40 S subunits was detected regardless of the fraction collected under the 60 S peak. This was probably due to contamination of 60 S ribosomal subunits by small quantities ofundissociated 80 S monoribosomes. The contamination of 60 S ribosomal subunits by 40 S subunits was estimated to be 2% and 4% in P. berghei and rat liver respectively, and was considered acceptable for subunit ribosomal protein analysis. Malaria ribosomal subunit proteins. The solubility and two-dimensional electrophoretic mobility of ribosomal proteins were not altered by radioiodination as shown by coelectrophoresing aliquots of stainable amounts of P. berghei ribosomal subunit proteins and trace amounts of the same labeled proteins. Fig. 1 shows a representative electrophoretogram and autoradiogram resulting after coelectrophoresis of such a mixture of P. berghei ribosomal proteins isolated from 40 S subunits. Only a small amount of stainable and labeled material remained at the origin of the first dimension gels and streaked into the slab gels. None of the ribosomal proteins migrated toward the anode in first dimension gels (data not depicted). Thus, all of the proteins extracted

252

B

O

t~ Fig. 1. Comparison of unlabeled and 12q-labeled P. berghei40 S ribosomal subunit proteins by two-dimensional electrophoresis. Unlabeled (100 lag) and labeled (1 X 106 dpm) malarial small ribosomal subunit proteins were mixed and coelectrophoresed as described in Materials and Methods. The direction of migration of proteins in the first dimension was from left to right, in the second dimension from top to bottom. The stacking gels, which contained no protein, have been removed from this and all other electrophoretograms. Proteins that are clearly present on the gels or on autoradiograms exposed for longer periods, but which did not stain or label intensely enough to reproduce photographically, are encircled with a dashed line. Ribosomal proteins were numbered on the basis of increasing molecular weight and first dimension migration distance. The numbers designated for each ribosomal protein from the small subunit are preceeded by an 'S' and those from the large ribosomal subunit are preceeded by an "L'. Arrows indicate the relative migration of the following markers: bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; chymotrypsinogen, 25.7 kDa; and cytochrome C, 12.5 kDa. A, stained gel; B, autoradiogram (12 h exposure).

f r o m p a r a s i t e a n d h o s t r i b o s o m e s w e r e d i s p l a y e d in the t w o - d i m e n s i o n a l e l e c t r o p h o retic s y s t e m a n d e a c h o f t h e s e p r o t e i n s is r a d i o i o d i n a t e d by the c h l o r a m i n e - T m e t h o d . T h i r t y r i b o s o m a l p r o t e i n s f r o m P. berghei 40 S s u b u n i t s c a n be r e p r o d u c i b l y r e s o l v e d by this t w o - d i m e n s i o n a l e l e c t r o p h o r e t i c s y s t e m (Fig. 1), a n d r a n g e in a p p a r e n t m o l e c u l a r size f r o m 11.7-40.7 k D a w i t h a n u m b e r a v e r a g e m o l e c u l a r size o f 24.3 k D a ( T a b l e I). A s i m i l a r c o e l e c t r o p h o r e s i s o f t25I-labeled a n d u n l a b e l e d P. berghei 60 S r i b o s o m a l s u b u n i t p r o t e i n s is s h o w n in Fig. 2. T h e s e r i b o s o m a l p r o t e i n s are 36 in n u m b e r a n d r a n g e in m o l e c u l a r size f r o m

12.0-42.6 k D a ( T a b l e I). A l t h o u g h s o m e o f these

r i b o s o m a l p r o t e i n s are l a r g e r t h a n t h o s e f r o m the p a r a s i t e ' s s m a l l r i b o s o m a l s u b u n i t , t h e i r n u m b e r a v e r a g e m o l e c u l a r size o f 22.2 k D a is a c t u a l l y less t h a n the n u m b e r a v e r a g e m o l e c u l a r size o f P. berghei 40 S r i b o s o m a l s u b u n i t p r o t e i n s . T h e r e a p p e a r s to

253 be no detectable contamination by parasite 40 S ribosomal proteins. Close analysis of these gels and of the differences in apparent molecular weights shows that no ribosomal protein is shared by the two subunits. P. berghei ribosomal protein L2 can occasionally be resolved into two components, designated L2a and L2b, when 60 S subunit ribosomal proteins are analyzed, while L2b was not usually present in two-dimensional electrophoretograms of total polysomal proteins from P. berghei. All malarial 60 S ribosomal subunit proteins appear to be radioiodinated, but like ribosomal proteins of the 40 S ribosomal subunit, they do not stain with the same relative intensity as they are labeled.

Comparison of parasite and host ribosomal subunit proteins. A representative coelectrophoresis of stainable amounts of rat liver 40 S ribosomal proteins and trace amounts of ~25I-labeled P. berghei ribosomal proteins from the comparable subunit is depicted in Fig. 3. Thirty proteins can be resolved from rat liver 40 S ribosomal subunits ranging in apparent molecular size from 9.2-37.5 k D a with a number average molecular size of 21.7 kDa (Table I). These results are in good agreement with other studies of rat liver ribosomal proteins despite different methods of analysis and limitations in the technique [16,23-25]. A close study of many gels, electrophoresed to maximize resolution of selected groups of ribosomal proteins, demonstrated that while the mobilities of ribosomal proteins relative to molecular weight markers varied some-

A

iiiiiiiiiiiii~ ~i~i~iiiiiiiiiii!i iiii:i!i!iii!i:i

Fig. 2. Two-dimensional electrophoretogram of unlabeled (150 lag) and ~25I-labeled(1.5 X 106 dpm) malarial 60 S ribosomal subunit proteins. (See legend to Fig. 1.) A, stained gel; B, autoradiogram (12 h exposure).

40.7

36.5

35.3

34.5

33.3

33.0

31.6

31.5

31.5

31.3 30.8

27.3

25.1

23.1 23.0

21.3

20.2

$2

$3

$4

$5

$6

$7

$8

$9

S 10 S 11

S 12

S13

S14 S15

S16

S17

19.9-20.6

20.8-21.6

22.6-23.6 22.3-23.6

24.8-25.5

27.0-27.6

30.9-31.7 30.9-31.1

31.2-31.7

31.2-31.7

31.5-31.7

32.5-33.3

32.9-33.6

34.3-34.7

34.6-35.7

36.4-36.5

38.8-41.7

18.8

21.0

21.6 21.2

22.4

27.1

28.8 28.3

29.1

29.1

29.5

30.6

30.7

32.1

32.6

35.8

37.5

M mean

M mean

M range

Rat liver

P. berghei

S1

Protein

Small ribosomal subunit

18.2-19.4

20.4-21.5

21.2-22.0 20.7-21.7

21.7-23.2

26.6-27.7

28.0-29.9 28.0-28.4

28.4-30.3

28.4-30.3

28.8-30.7

30.3-31.0

30.3-31.4

31.4-33.1

31.8-33.9

35.7-36.2

36.6-38.3

M r range

41.5-42.8 39.5-40.6 32.6-32.8 32.0-32.3 29.4-30.0 29.4-30.0 27.9-28.3 25.1-27.2 24.6-25.1 24.4-24.9 24.1-24.6 23.6-24.1 22.2-22.8 22.0-22.5 21.3-21.9 21.1-21.7 20.8-21.5

42.1 40.0 32.7 32.1 29.7 29.7 28.1 26.5 24.8 24.7 24.4 23.9 22.6 22.2 21.6 21.4 21.0

L2b L3

LI6 LI7

L15

L13 L14

Lll LI2

L10

L9

L7 L8

L6

L5

L4

42.0-43.3

42.6

M range

M r mean

P. berghei

L2a

L1

Protein

Large ribosomal subunit

Apparent molecular weights ()< 10-3) of P. berghei and rat liver ribosomal proteins a

TABLE I

25.7 24.5

26.6 26.4

27.1

25.3-25.9 24.1-24.7

26.3-26.9 25.9-26.9

26.9-27.3

26.9-27.5

26.9-27.5

27.2 27.2

28.2-28.9 26.9-24.5

28.6-29.3

29.6-30.0 28.6-29.6

31.1-31.8

31.8-32.6

40.7-43.1 33.8-35.1

43.0-46.8 42.0-45.3

M r range

28.6 27.2

28.8

29.8 28.9

31.4

32.1

42.7 34.3

45.2 44.2

M mean

Rat liver

17.6-18.8 16.4-17.7 16.2-17.6 16.0-17.4 15.8-17.4 15.4-16.7 14.4-15.9 12.7-14.0 11.1-12.4

19.9-20.6 19.3-20.4 18.3-19.2 17.8-18.8 16.9 16.5 16.0 15.5

13.9 13.7 11.4 10.3 10.0 9.2

15.2 14.3 14.2

Based upon 3 independent determinations.

18.2 17.1 16.9 16.7 16.6 16.0 15.2 13.3 11.7

$22 $23 $24 $25 $26 $27 $28 $29 $30

a

20.1 19.8 18.7 18.2

S18 S19 $20 $21 14.8-15.5 13.9-14.5 13.7-14.5 13.4-14.1 13.2-13.9 10.9-11.6 9.9-10.6 9.6-10.3 8.7- 9.4

16.4-17.1 16.0-16.8 15.6-16.3 15.0-15.8 L18 L19 L20 L21a L21b L22 L23 L24 L25 L26 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L40 L41 17.8-18.5 17.8-18.5 17.8-18.5 16.7-17.4 16.2-16.9 15.6-16.3 14.6-15.3 14.0-14.7 13.9-14.6 13.9-14.6 13.9-14.6 12.6-13.4 12.4-13.1 11.7-12.6

18.0 18.0 18.0 17.1 16.5 15.8 14.9 14.3 14.2 14.2 14.2 13.0 12.8 12.1

20.6-21.2 19.2-19.8 18.2-19.1 18.0-18.7

20.8 19.5 18.6 18.2

16.5 16.2 15.0 14.9 14.9 14.2 13.8 12.6 12.5 12.3 12.2 11.3 10.9 10.3 10.3

24.2 23.9 23.2 22.7 21.9 20.8 19.4 17.9 17.1 17.0

23.8-24.4 23.6-24.1 22.7-23.6 22.5-23.0 21.6-22.2 20.4-21.0 19.0-19.6 !7.6-17.9 16.6-17.6 16.6-17.3 16.2-16.9 15.8-16.5 14.7-15.4 14.6-15.1 14.6-15.1 13.9-14.6 13.4-14.1 12.3-13.0 12.1-12.9 11.9-12.6 11.9-12.6 11.0-11.7 10.6-11.3 9.99-10.6 9.88-10.6

256

Fig. 3. Comparison of P. berghei and rat liver small ribosomal subunit proteins by two-dimensional electrophoresis. Unlabeled (130 p.g) rat liver 40 S and ~2SI-labeled(1 X 106 dpm) malarial 40 S ribosomal subunit proteins were mixed and coelectrophoresed. (See legend to Fig. 1.) A, stained get; B, autoradiogram (13 h exposure).

what from gel to gel (Table I), within an individual gel, each parasite and host ribosomal protein displayed the same relative position to the others and in most cases had a distinct electrophoretic mobility. Because of the partial overlap in electrophoretic migration of some of these proteins and the limitations of the method, we cannot exclude the possibility that a few o f the parasite and host ribosomal proteins have the same electrophoretic mobility. Malarial and liver ribosomal proteins from the large ribosomal subunit were coelectrophoresed in a similiar m a n n e r (Fig. 4). Rat liver proteins isolated from 60 S subunits were resolved into 41-43 c o m p o n e n t s (L2 and L21 each appearing as two separate spots in some subunits preparations). The apparent molecular size of these ribosomal proteins was 10.3-45.2 k D a with a n u m b e r average molecular size of 22.4 k D a (Table I). Certain parasite ribosomal proteins seem to be ' h o m o l o g o u s ' to certain liver ribosomal proteins. F o r example, liver ribosomal proteins L1, L2a, L2b, L3, L4 and L5 have electrophoretic similarities to P. berghei L I , L2a, L2b, L4, L3, and L5, respectively. However, although some parasite and host subunit ribosomal proteins possess overlapping molecular weight estimates (Table I), most of these ribosomal proteins demonstrated a distinguishable first dimension or second dimension electrophoretic mobility under the conditions of this study. Even though the two-dimensio-

257

A

Fig. 4. Coelectrophoresis of unlabeled rat liver (200 tag) and 12SI-labeledmalarial (1 X 106 dpm) large ribosomal subunit proteins. (See legend to Fig. 1.) A, stained gel; B, autoradiogram (13 h exposure). nal electrophoretic mobility of a protein is a composite result of many of its structural features, neither the coincidence nor difference in mobility by another protein are sufficient to judge the true homology of such proteins, which can only be determined by amino acid sequence data. By adding the apparent molecular weight of subunit ribosomal RNA [ 11] to the sum of apparent molecular weights of ribosomal proteins - assuming that each ribosomal protein is represented once per subunit - a total subunit molecular weight was calculated. The ribosomal RNA and ribosomal proteins from P. berghei small ribosomal subunits are larger than those from the corresponding liver subunit, giving a significantly higher calculated subunit molecular weight of 1.63 X 106 for the parasite small subunit versus 1.35 X 106 for the host. Conversely, ribosomal RNA and ribosomal proteins from plasmodial large ribosomal subunits are smaller than those from rat liver large subunits resulting in a relatively lower particle molecular weight (2.30 X 106 compared to 2.76 X 106). DISCUSSION The ribosomal proteins of P. berghei small and large ribosomal subunits were resolved by two-dimensional electrophoresis into 30 and 35-36 species, respectively. Because of different methods of ribosome isolation, ribosomal protein extraction, and electrophoresis, it is difficult to compare these data with the limited information available about other protozoan ribosomal proteins. It has been reported that Tetra-

258

hymenapyriformis contains 30-40 and 35-46 different ribosomal proteins in the small and large ribosomal subunits respectively, depending upon methodology and the strain used for study [26-28]. Euglena gracilis is another protozoan which seems to possess more ribosomal proteins than those described for P. berghei here: 33-36 proteins in 40 S ribosomal subunits and 37-43 proteins in 60 S ribosomal subunits [29], all of which have been reported to range in molecular weight from 10 000-104 000 [30]. These molecular weight estimates are higher than those determined by one-dimensional studies for E. gracilis subunit ribosomal proteins [31] or by two-dimensional electrophoresis for most other eukaryotic ribosomal proteins. Because of different methods of electrophoresis, the numbering of ribosomal proteins employed in this study does not correlate with that of other workers [16,23-25]. Nonetheless, the number of rat liver ribosomal proteins resolved, the range of apparent molecular weights, and the number average molecular weights of ribosomal proteins reported in this study correlate fairly well with other such determinations in the literature. Several sets of parasite and host ribosomal proteins appear to be approximately the same size, but differ in first dimension electrophoretic mobility. Examples include P. berghei $7-10, $23-24, L9-10, L22-24, L30-32, and rat liver $8-9, L8-9, L10-13, L25-26, L29-31, and L36-37. This has been reported for other ribosomal proteins [24,25,32] and may represent secondary modifications of the same proteins which could alter their charge or hydrophobicity. The finding that both parasite and host ribosomal protein L2 was usually present as a single spot in polysomal preparations, but was often resolved into 2 spots, L2a and a slightly smaller component L2b, in 60 S subunit preparations, has also been reported for other ribosomal proteins [25,33]. This phenomenon probably represents an artifactual modification of L2 during ribosomal subunit isolation and may be the result of specific proteolysis as described for large ribosomal subunit proteins from protozoa [28] to mammals [33,34]. Although addition o f a protease inhibitor (1 mM phenylmethylsulfonyl fluoride) to all polysomal and ribosomal protein isolation solutions just prior to use did not alter the electrophoretograms for either P. berghei or liver (results not shown), we cannot exclude the possibility that some of the differences between parasite and host ribosomal proteins might be due to selective limited proteolysis. Since all the erythrocytic stages of the parasite have been used as the source of ribosomes in these studies, the ribosomal components we describe reflect an average of all these stages. It is possible that differences exist in ribosomal protein content among the erythrocytic stages as well as the non-erythrocytic stages of the parasite, reflecting different translational demands in varying intracellular environments or during different cellular activities. Ribosomal proteins might also vary among the species and strains of malaria parasites and may be useful for definitive biochemical cataloging of plasmodial isolates as in the case for T. pyriformis [26].

259 ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y N A T O g r a n t S A 5 - 2 - 0 5 B (1255) 1506 (75) A G , USPHS Grant A5011673, and USPH Training Grant HD-0020-18. REFERENCES 1

2 3 4 5 6 7 8 9 10 11 12 13

14 15 16

17 18 19

Cook, R.T., Rock, R.C., Aikawa, M. and Fournier, M.J. (1971) Ribosomes of the malarial parasite, Plasmodium knowlesi. I. Isolation, activity, and sedimentation velocity. Comp. Biochem. Physiol. 39B, 897-911. Sherman, I.W. and Jones, L.A. (1977) The Plasmodium lophurae (avian malaria) ribosome. J. Protozool. 24, 331-334. Aikawa, M. and Cook, R.T. ( 1971) Ribosomes of the malarial parasite P. knowlesi. II. Ultrastructural features. Comp. Biochem. Physiol. 39B, 913-917. Sherman, I.W. (1976) The ribosome of the simian malaria Plasmodium knowlesi. II. A cell-free protein synthesizing system. Comp. Biochem. Physiol. 53B, 447-450. Tokuyasu, K., Ilan, J. and Ilan, J. (1969) Biogenesis of ribosomes in P. berghei. Milit. Med. 134, 1032-1038. Warhurst, D.C. and Williamson, J. (1970) Ribonucleic acid from Plasmodium knowlesi before and after chloroquine treatment. Chem. Biol. Interact. 2, 89-106. Trigg, P.I., Shakespeare, P.G., Burt, S.J. and Kyd, S.I. (1975) RNA synthesis inP. knowlesimaintained both in vivo and in vitro. Parasitology 71, 199-209. Hyde, J.E., Zolg, J.W. and Scaife, J.S. (1981) Isolation and characterization of ribosomal RNA from the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 4, 283-290. Dame, J.B. and McCutchan, T.F. (1983) Cloning and characterization of a ribosomal RNA gene from Plasmodium berghei. Mol. Biochem. Parasitol. 8, 263-279. Vezza, A.C. and Trager, W. (1981) Preliminary characterization of the major RNA species from Plasmodiumfalciparum. Mol. Biochem. Parasitol. 4, 149-162. Miller, F. and llan, J. (1978) The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis. Parasitology 77, 345-365. Sherman, I.W., Cox, R.A., Higginson, B., McLaren, D.J. and Williamson, J. (1975) The ribosomes of the simian malaria, Plasmodium knowlesi. I. Isolation and characterization. J. Protozool. 22, 568-572. Cox, R.A., Godwin, B. and Hastings, J.R.B. (1976) Spectroscopic evidence for the uneven distribution of adenine and uracil residues in rRNA of Drosophila melanogaster and of P. knowlesi and its possible evolutionary significance. J. Biochem. 155, 465-475. Dame, J.B. and McCutchan, T.F. (1983) The four ribosomal DNA units of the malaria parasite Plasrnodium berghei. J. Biol. Chem. 258, 6984-6990. Hoffman, W.L. and Ilan, J. (1974) Purification on hydroxyapatite of liver ribosomes and polysomes from unfasted mice. Biochim. Biophys. Acta 366, 199-214. Sherton, C.C. and Wool, I.G. (1972) Determination of the number of proteins in liver ribosomes and ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis. J. Biol. Chem. 247, 4460-4467. Barriault, D., Expert-Bezangon, A., Gurin, M.F. and Haynes, D. (1976) The use of acetone precipitation in the isolation of ribosomal proteins. Eur. J. Biochem. 163, 131-135. Bramhall, S., Noack, N., Wu, M. and Lowenberg, J.R. (1969) A simple colorimetric method for determination of protein. Anal. Biochem. 31, 146-148. Leister, D.E. and David, I.B. (1974) Physical properties and protein constituents of cytoplasmic and mitochondrial ribosomes of Xenopus laevis. J. Biol. Chem. 249, 5108-5118.

260 20 21 22 23 24

25

26 27 28

29 30 31 32 33 34

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