Isolation and characterization of RNA of Entamoeba histolytica

Molecular and Biochemical Parasitology, 12 (1984) 261-272

261

Elsevier MBP 00456

ISOLATION AND CHARACTERIZATION OF RNA OF ENTAMOEBA HISTOL YTICA

R I C H A R D A. A L B A C H , V I R A P O N G P R A C H A Y A S I T T I K U L and G A R Y M. HEEBNER*

Department of Microbiology and Immunology, University of Health Sciences/The Chicago Medical School, North Chicago, IL 60064, U.S.A. (Received 20 December 1983; accepted 12 March 1984)

RNAs were isolated from Entamoeba histolytica with a high salt sodium dodecylsulfate-diethylpyrocarbonate technique. Majority species of 25 S, 17 S and 4 S RNAs were detected after sucrose gradient centrifugation. An additional 5 S R N A was detected by polyacrylamide gel electrophoresis. The molecular weights of these RNAs as determined by completely denaturing polyacrylamide gel electrophoresis were 1.31 × 106 (25 S), 0.803 >( 106 (17 S), 4.0 )< 104 (5 S) and 2.5 >( 104 (4 S). The 25 S R N A was labile and dissociated under mild denaturing conditions (between 37°C and 55°C) into 17 S and 16 S RNAs with molecular weights of 0.700 )< 106 and 0.614 )< 106, respectively; under completely denaturing conditions an additional 5.8 S RNA with a molecular weight of 4.8 X 104 was detected. Evidence is presented which suggests that the lability of the 25 S RNA is the result of an in vivo cleavage rather than one which is generated during RNA isolation. Key words: Entamoeba histolytica; RNA extraction; Nicked 25 S RNA; R N A molecular weights

INTRODUCTION

Numerous reports exist concerning isolation and characterization of RNA, especially rRNA in protozoa. In some protozoa, the large rRNA is apparently an intact polynucleotide [ 1-3]. However, in many cases, as is also found in certain other lower eukaryotes [4-7], the large rRNA is labile (= nicked) and dissociates under denaturing conditions into two fragments of approximately equal size, comparable in size to the rRNA found associated with the smaller subunit (see [8,9] for review). Although the nick in the large rRNA is usually approximately centrally located, it is not in Plasmodium berghei [10]. Barker [ 11] and Barker and Swales [ 12] allude to the paucity of published informa-

*Present address: GCA/Precision Scientific, 3737 W. Cortland, Chicago, IL 60647, U.S.A. Abbreviations: DEP, diethylpyrocarbonate; M M H , methylmercuric hydroxide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecylsulfate. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

262 tion on RNAs of the genus Entamoeba. These authors point out that this is due, in part, to substantial difficulties in isolating undegraded RNA from Entamoeba species. However, they did characterize RNA isolated from both cysts and trophozoites of E. invadens in the presence of carrier RNA. Trophozoites possess two ribosomal RNAs ranging in size from 18 to 20 S and 24 to 28 S, with molecular weights of 0.7 X 10 6 and 1.4 X 106, respectively. In vivo autoradiographic evidence of labile rRNA in E. histolytica was reported by Albach et al. [13]. This was based on rapidity with which long-term [SH]uridine labeled cytoplasmic RNA turned over. Hence, we were not surprised when our initial attempts to isolate intact rRNA from E. histolytica using a variety of techniques failed. In this paper, we report the development of a high salt sodium dodecylsulfate (SDS)-diethylpyrocarbonate (DEP) technique for isolation of amebal RNA and the characterization of 25 S, 17 S, 5 S (and 4 S) RNAs. The 25 S RNA is labile and dissociates into two large fragments (17 and 16 S) and a more rigidly bound smaller 5.8 S fragment. The molecular weights of these RNAs have been estimated. Evidence is also presented which suggests that the lability of the 25 S RNA is most likely the result of an in vivo cleavage rather than one which is generated artifactually during RNA isolation. Preliminary data obtained were presented at the 9th Seminar on Amebiasis [14]. MATERIALS AND METHODS

Cultivation and harvesting of amebae. Entamoeba histolytica, strain HK-9, was cultured axenically in TYI-S-33 medium [15]. Early log phase amebae from 48 h cultures were harvested by centrifugation, pooled and washed in cold calcium and magnesium free phosphate buffered saline. Amebae used in RNA extractions were at least 95% viable as determined by Trypan blue exclusion. Isolation of ribosomes. Ribosomes were isolated from amebae by a modification of the procedure described by Morgan et al [16] for isolation of ribosomes from cysts of E. invadens. Ribosomes (75 S) and subunits (55 and 35 S) recovered from 10-30% sucrose gradients contained approximately equal amounts of RNA and protein. RNA extraction. After extensive evaluation of various extraction procedures, three modifications of the SDS-DEP technique of Solymosy et al [17] were developed: (1) addition of NaC1 in high concentration to the extraction buffer; (2) the concomitant addition of both SDS and D E P to the cell suspension just prior to cell disruption, and (3) incubation temperatures of around 30°C rather than 37°C. The 'high salt' extraction buffer contained the following ingredients: 0.05M Tris-HC1, p H 7.5, 0.5 M NaCI and 5 mM MgSO4. Pooled washed amebae (approx. 2-4 X 10 7 cells) were suspended in extraction buffer at an approximate concentration of 1 X 10 6 cells m l -l and chilled (0-4°C). SDS was added to yield a final concentration of 1% (w/v) followed by

263 immediate addition of 0.02 ml D E P ml -~ cell suspension. Cells were rapidly disrupted in the cold using a Ten Broeck homogenizer, Dounce homogenizer, a n d / o r by shaking in screw-capped tubes. Results obtained with amebae using this extraction procedure were independent of the mode of cell disruption. The amebal homogenate was incubated 5 min at 30°C with occasional stirring followed by quick-cooling on ice. (In our preliminary report [14], the incubation was at 25°C.) The mixture was centrifuged 5 min at 8 000 × g, and the supernatant retained. NaCI was added at room temperature to yield a final concentration of 0.1 g ml -~, the mixture incubated 5 min at 30°C, then quick-cooled on ice for 5 min. The amebal homogenate was centrifuged at 4°C for 10 min at 10 000 × g, the supernatant recovered and recentrifuged at 4°C for 20 min at 23 000 × g. RNA was precipitated with cold ethanol, centrifuged and dissolved in one of several buffers, contingent on subsequent handling: phosphate buffered saline for sucrose gradient analysis, TAB or E buffer for polyacrylamide gel electrophoresis (PAGE). TAB buffer consisted of 0.04 M Tris-acetate (pH 7.8), 0.02 M sodium acetate and 1 m M disodium EDTA; E buffer (pH 8.2) consisted of 0.05 M boric acid, 5 m M sodium borate and 10 mM sodium sulfate. Isolated RNA (absorbance maximum at 260 nm) was pancreatic RNase sensitive and DNaseI resistant. Protein determinations (Coomassie Blue assay [18]) revealed that the RNA was free of detectable protein (sensitivity level less than 1 lag protein ml-~).

Sucrose gradient centrifugation. Linear sucrose (Sigma, RNase free) gradients (5-20% w/v) were prepared using an Instrumentation Specialties C o m p a n y (ISCO) model 570 automatic gradient former. Sucrose gradients containing RNA samples (1-10 A260 units) were centrifuged in a Beckman model L2-50 preparative ultracentrifuge (SW 27.1 swinging bucket rotor) at 21 000 rpm for 19 h at 4°C. Following centrifugation, gradients were fractionated and scanned at 254 nm using an ISCO model 640 density gradient fractionator coupled with an ISCO model UA-5 absorbance monitor. S values were estimated according to the method of Martin and Ames [19] using standard marker RNAs. Gradient forming and fractionating equipment was purged with 3% hydrogen peroxide and rinsed with sterile distilled water before each use. Polyacrylamide gel electrophoresis (PAGE). Horizontal slab gels were formed in a modified Bethesda Research Laboratories (BRL) gel casting tray. Non-denaturing 2.4% polyacrylamide-0.5% agarose gels were prepared in TAB or E buffer using a modification of the technique described by Poulsen [20]. Denaturing 2.4% polyacrylamide-0.5% agarose gels containing 10 mM methylmercuric hydroxide (MMH) were prepared in E buffer as described by Sebo and Schmit [21]. All electrophoretic separations were carried out using a BRL model H3 horizontal bed electrophoresis unit as a 'submerged gel' system. Before electrophoresis, gels were pre-electrophoresed to remove any free acrylamide, catalysts or other charged impurities which may have remained in the gels. Conditions for electrophoretic separations were as described in 'Results' and legends to the figures. After electrophoresis, gels were either

264

stained with toluidine blue 0 and scanned at 620 nm, or scanned directly (unstained) at 254 nm on quartz plates (Wilmad Glass Co., N J). Individual RNAs were isolated from polyacrylamide gels and re-electrophoresed under non-denaturing or denaturing conditions in a second gel matrix.

Molecular weight estimations. Molecular weights of isolated RNA were determined via denaturing gel electrophoresis as described by Sebo and Schmit [21]. Standard marker RNAs used in molecular weight determinations were: mouse liver cell 18 S (0.700 X 106) rRNA; E. coli 23 S (0.996 X 106) and 16 S rRNA (0.529 X 106); and rat liver cell 5 S (4.12 X 104) and 5.8 S (5.34 X 104) rRNA. RESULTS

RNA isolation. Inconsistent results were obtained with isolated ribosomes and subunits. However, when RNA was isolated from intact amebae, major species of 4, 17 and 25 S RNAs were consistently detected. Using a modification of the original SDS-DEP technique (without added 'high salt' and incubation temperatures of 25°C), 17 and 25 S RNA were recovered in an approx. 2:1 ratio on sucrose gradients [14]. When the high salt SDS-DEP technique was used, 4, 17 and 25 S RNAs were also detected; 17 and 25 S were recovered in a ratio close to one. To facilitate better resolution of these RNAs, isolated amebal RNA was fractionated via non-denaturing P A G E (Fig. 1). In addition to the three majority species of RNA, a 5 S RNA which was not detected previously and a small, approximately 16 S peak were also observed. The ratios of 17

28S

18S

25s

.4

17S

.2

~es

I

/

0

4S

J

I

7

i

I

I

2

4

6

8

10

distance

migrated

I

(cm)

Fig. 1. Electrophoresis of amebal R N A in a 2.4% polyacrylamide-0.5% agarose gel in TAB buffer at 20 V for 24 h, 4°C. Direction of migration is from left to right. Arrows indicate position of marker 18 and 28 S R N A electrophoresed under identical conditions.

265

S:25 S RNA and small amounts of 16 S RNA suggested that some degradation a n d / o r dissociation of the 25 S occurred. That we were dealing with dissociation and not degradation was evidenced by recovery of all A260 absorbing material added to gels in high molecular weight form; none of it was in low molecular weight (acid soluble) form. Efficient inactivation of amebal RNase was confirmed through coincubation experiments [22] and coextractions by subjecting mouse liver cell RNA and amebae to the RNA extraction procedure. Examination of RNA extracts via P A G E and acid precipitation revealed no degradation of mouse liver rRNA; i.e., all material was recovered in high molecular weight form. To examine stability of the amebal RNAs, RNA extracts were incubated at various temperatures and subsequently separated using non-denaturing P A G E (Fig. 2). The large 25 S RNA was stable at temperatures up to approx. 33°C (Fig. 2b). At 37°C, the amount of 25 S RNA detected decreased with a concomitant increase in the 16 and 17 S peaks (Fig. 2c). At 55°C, the 25 S RNA disappeared with a further increase in the 16 and 17 S RNAs (Fig. 2d). Also, treatment at 55°C for 5 min resulted in the appearance

E g:

,q, tn

e c i.

0

0

2

4

distance

6

8

migrated

10 (cm)

Fig. 2. Electrophoresis of amebal R N A incubated for 5 min at 0°C (a); 33°C (b); 37°C (c); 55°C (d). Electrophoresis was in a 2.4% polyacrylamide-0.5% agarose gel in TAB buffer at 20 V for 24 h, 4°C. Direction of migration is from left to right.

266 of 5.8 S RNA which was not observed at lower incubation temperatures. Such dissociation products were stable upon heating at 55°C for at least 30 min as evidenced by complete recovery of such products in high molecular weight form.

Denaturing PAGE of RNA (molecular weight estimations). To fractionate and determine molecular weights of small amebal RNAs (viz., 4, 5 and 5.8 S RNA) isolated, RNA was electrophoresed under complete denaturing conditions in M M H gels for a relatively short time period; viz. 6 h, 4°C (Fig. 3). The approx. 5 and 5.8 S RNAs were detected in approximate equimolar amounts and had average molecular weights of 4.0 X 104 and 4.8 X 104, respectively. The 4 S RNA had a molecular weight of approximately 2.5 X 104. To determine molecular weights of large amebal RNAs (16, 17 and 25 S), isolated RNA was fractionated via M M H denaturing gels for 24 h at 4°C. Scans of gels revealed an absorbance profile as shown in Fig. 4. Intact amebal 25 S RNA completely dissociated under these conditions and was absent from such profiles. Three RNA species were observed. Low molecular weight RNAs (that is, 4, 5 and 5.8 S species) migrated out of gels and were not detected. This was due to the increase in voltage used during denaturing electrophoresis (80 V) as compared to non-denaturing electrophoresis (20 V). The three RNAs detected were in the 16 to 17 S range of sucrose gradients and had the following molecular weights: 17 S, 0.803 X 106 (-F 0.017); 17 S, 0.700 X 106 (-F 0.016); and 16 S, 0.614 X 106 ( ± 0.013). These data are means of approx. 20 separate experiments, done in triplicate; values in brackets represent ± 1 standard error.

c

~.

.4

53.4 kDa

~°~ .2 ~

1il.2 kDa

v)a~=

40 kDn t

m

\

•

\

0

15S) 4 8 kOa

2 4 distance

;

4S

i

j

10

migrated(cm)

Fig. 3. Electrophoresis under complete denaturing conditions of amebal RNA in a 2.4% polyacrylamide0.5% agarose gel ( 10 mM MMH) in E buffer at 80 V for 6 h, 4°C. Direction of migration is from left to right. Arrows indicate position of marker 53.4 and 41.2 kDa RNA electrophoresed under identical conditions.

267

7 0/ 0

kDa

996 kDa /

529

kDa

E G

.4

700

ul

kOa

OI

117S

1----- 614kDa (16S)

e c (g JD

.2

O m JD t~

0

I

t

i

2

4

6

distance

8

migrated

1'0 (cm)

Fig. 4. Electrophoresis under complete denaturing conditions of amebal RNA in a 2.4% polyacrylamide0.5% agarose gel ( 10 m M M M H ) in E buffer at 80 V for 24 h, 4°C. Direction of migration is from left to right. Arrows indicate position of marker 996, 700 and 529 k D a RNA electrophoresed under identical conditions.

Origin of RNA dissociation products. To determine which of the three RNAs observed in MMH gels (Fig. 4) were dissociation products of 25 S RNA, intact 25 S RNA was first recovered from non-denaturing gels then subjected to P A G E again under both non-denaturing (Fig. 5) and denaturing (Fig. 6) conditions. Additionally, the other majority RNA species observed in such non-denaturing gels were likewise examined for lability. Non-denaturing gels. Mid points of individual amebal RNAs isolated from non-denaturing gels and subjected to PAGE a second time under non-denaturing conditions showed the following (Fig. 5a-e). Most of the 25 S RNA was recovered intact; small amounts of 16 and 17 S RNAs were also observed (Fig. 5a). In contrast to the unstable 25 S RNA, other isolated amebal RNAs (17, 16, 5 and 4 S) did not display any lability; UV absorbance profiles were unchanged by a second electrophoretic separation (Fig. 5b-e). No 5.8 S RNA was detected after electrophoresis of isolated amebal 25 S RNA under non-denaturing conditions (Fig. 5a).

Denaturing gels. To determine molecular weights of the dissociation products, mid points of peaks were cut from non-denaturing gels and electrophoresed a second time under complete denaturing conditions. Fig. 6 depicts typical UV absorbance profiles obtained from these experiments. E. coli rRNAs were recovered from gels and re-electrophoresed as 'controls' and to provide molecular weight markers (Fig. 6d-e). As shown in Fig. 6a, amebal 25 S RNA dissociated, forming 17 and 16 S RNAs with

268

(n)

(o) 25S

4s

(b}

(f)

23s

175

(g)

(©)

16S

16S

. i

i

i

(d)

i

~

•

~

io

$S

o

2

~

e

8

lO

distance

migrated (cm)

Fig. 5. Electrophoresis of individual amebal RNAs. RNAs were first separated in non-denaturing gels; individual RNAs were recovered and electrophoresed again in non-denaturing gels (2.4% polyacrylamide0.5% agarose gels at 20 V for 24 h, 4°C). The following individual RNAs are shown: 25 S RNA (a); 17 S RNA (b); 16 S RNA (c); 5 S RNA (d); and 4 S RNA (e). E. coli marker rRNAs are also shown: 23 S RNA (f) and 16 S RNA (g). Direction of migration is from left to right. e s t i m a t e d m o l e c u l a r weights o f 0.700 X l0 s a n d 0.614 X 106, respectively. Since e l e c t r o p h o r e t i c s e p a r a t i o n was at 80 V for 24 h (cf. to 20 V e m p l o y e d for n o n - d e n a t u ring gels; see legends to the figures), the 5.8 S R N A ran off the gels. The '17 S' p e a k recovered f r o m n o n - d e n a t u r i n g gels was resolved into two c o m p o n e n t s : a large 0.803 X 106 D a p e a k a n d a very small 0.700 X 106 D a p e a k (Fig. 6b). In contrast, 16 S R N A m i g r a t e d as a single species (0.614 X 106 D a ) u n d e r d e n a t u r i n g c o n d i t i o n s (Fig. 6c). C o m p a r i s o n o f these d a t a i n d i c a t e d t h a t the 0.803 X 106 D a R N A c o r r e s p o n d e d to the true small 17 S R N A (most likely r i b o s o m a l - see Discussion) while the 0.700 X 106 D a R N A a n d 0.614 X 106 D a R N A were d i s s o c i a t i o n p r o d u c t s o f 25 S R N A . Since the 25 S R N A c o m p l e t e l y d i s s o c i a t e d in d e n a t u r i n g gels, its m o l e c u l a r weight (1.31 X 106) was e s t i m a t e d by a d d i n g the two large b r e a k d o w n p r o d u c t s . A l t h o u g h the 5.8 S R N A was also a d i s s o c i a t i o n p r o d u c t o f the 25 S R N A , it was not used for e s t i m a t i o n of the 25 S R N A Mr.

269

a)

/

700

kDa

d)

996

kDa

E c

(el

¢b) 803

'

'

kDa

' 529

'

'

kDa

0 c Q

~-.J

0

0 m

614

o

2

,i

2

4

6

8

10

kDa

e

a distance

~o migrated(cm)

Fig. 6. Electrophoresis under complete denaturing conditions of individual amebal RNAs. RNAs were first separated in non-denaturing gels; individual RNAs were recovered and electrophoresed again in denaturing gels (2.4% polyacrylamide-0.5% agarose gels (10 m M M M H ) in E buffer at 80 V for 24 h, 4°C). The following individual amebal RNAs are shown: 25 S R N A (a); 17 S RNA (b); and 16 S RNA (c). E. cold marker rRNAs are also shown: 23 S R N A (d) and 16 S R N A (e). Direction of migration is from left to right.

DISCUSSION

We are reporting, for the first time, some characteristics of the majority species of RNAs of E. histolytica isolated with a high salt SDS-DEP technique. Initial attempts to isolate amebal RNA using a variety of techniques, including phenol and the original SDS-DEP technique [17], failed. Modifications which included incorporation of various RNase inhibitors, such as proteinase-K and chloroquin phosphate, an apparent amebal RNase inhibitor [23], in the extraction medium prior to cell disruption also resulted in inconsistent recovery of intact RNA [24]. RNA was present in the 4-6 S range of sucrose gradients a n d / o r degraded to low molecular weight material. Difficulty in extracting intact RNA in amebae is due, in part, to the presence of a very potent and resistant RNase in Entamoebae which survives phenol extraction [11]. In addition to abundant soluble RNase in E. histolytica [23], an RNase which is intimately bound to ribosomes and subunits has also been reported by us in these amebae [22]. Morgan et al. [16] also reported that cysts of E. invadens contain an RNase and that ribosomes of such cysts are 'extraordinarily sensitive to its action'. These are probable reasons for difficulties encountered in extracting intact rRNA from isolated ribosomes. Comparable such problems prompted Shine and Dalgarno [7] to extract rRNA directly from whole cells of insects (i.e., to decrease the possibility

270

of introducing nicks in exposed regions of RNA during ribosome preparation). We also found this to be necessary. A high salt SDS-DEP procedure was eventually developed which results in good yields of RNA from whole amebae. The high salt concentration (0.5 M NaCI) stabilizes the nicked RNA [25] and aids in inhibiting RNase. Majority RNA species of 25, 17 and 4 S, in addition to a 5 S RNA are detected. The 25 S is labile and dissociates into two loosely bound 17 and 16 S fragments and a more rigidly bound 5.8 S fragment. Since similar data (three large size and two small size rRNAs) have been reported in a free-living amebae [26], this may prove to be a characteristic of the Sarcodinids. Using procedures not unlike those used by Cordingley and Turner [27], we find no evidence for the existence of other unusual small RNAs as has been found in trypanosomes [9,27]. Further experiments using high percentage gels (e.g., 10%) will be necessary to rule out possible existence of such RNAs. Although these RNAs were isolated from pelleted amebae and not ribosomes, we are confident that at least the 17 and 25 S RNAs (and dissociation products) are of ribosomal origin (very likely the 5 S RNA also): (I) they represent approx. 80% of the total RNA; (2) the same size RNAs (17 and 25 S) have been isolated from RNPs of ribosomal origin, although not in the same amounts as those recovered from intact cells; (3) they are labeled with [3H]uridine after long-term labeling experiments, and are not pulse labeled [28]; and (4) the 25 S RNA dissociates into three species as occurs in the large rRNA of many other protozoa. That the three components of amebal 25 S RNA (that is, 17, 16 and 5.8 S) are primary nicks and not the result of artifacts introduced during isolation (i.e., secondary nicks) is supported by RNA co-extraction experiments using amebae and mouse liver cells RNA. Complete recovery of intact mouse liver cell RNA from these extracts indicates effective inhibition of amebal RNase during initial steps of the high salt S D S - D E P extraction procedure. Furthermore, Cordingley and Hames [29] suggest that a criterion for in vivo existence of such cleavages should be by demonstration that the cleavage products exist under conditions when high molecular weight RNA (e.g., pre rRNA, hnRNA or mRNA) is also isolated. Such experiments have been done by us [28]. In that paper, we reported isolation of intact high molecular weight nuclear RNA with several isolation procedures, including the high salt SDS-DEP technique and guanidine-HC1; 25 S RNA was still nicked. The events involved in the production of the in vivo primary nicks in large rRNA of lower eukaryotes vary from species to species (see [4] for review). In some cells, the nick in large rRNA is produced within the nucleus, prior to formation of complete ribosomes. However, in other cells, the large rRNA is nicked in the cytoplasm, after formation of ribosomal subunits. The significance of discontinuous large rRNA in lower eukaryotes is also presently unknown. Several hypotheses have been proposed. For example, the nick may be the result of aging of ribosomal subunits. Alternatively, such RNA cleavage might be a prerequisite event in producing functionally active ribosomal subunits. Although its significance in E. histolytica is also not known, we

271

have preliminary evidence that the large rRNA is more labile in amebae from older cultures. ACKNOWLEDGEMENT

This work was supported, in part, by BRSG Grant RR-5366 awarded by the Biomedical Research Grant Program Division of Research Resources, National Institutes of Health. REFERENCES 1 2 3 4 5 6 7

8 9 10 11

12 13 14 15 16 17

Hyde, J.E., Zolg, J.W. and Scaife, J.G. ( 1981) Isolation and characterization of ribosomal RNA from the h u m a n malaria parasite Plasmodiumfalciparum. Mol. Biochem. Parasitol. 4, 283-290. Sherman, I.W. and Jones, L.A. (1977) The Plasmodium lophurae (avian malaria) ribosome. J. Protozool. 24, 331-334. Trigg, P.I., Shakespeare, P.G., Burr, S.J. and Kyd, S.I. (1975) Ribonucleic acid synthesis in Plasmodium knowlesi maintained both in vivo and in vitro. Parasitology 71, 99-209. Davis, F.C. and Mullersman, R.W. (1981) Processing of the ribonucleic acid in the large ribosomal subunits of Urechis caupo. Biochemistry 20, 3554-3561. Ishikawa, H. (1973) Primary and secondary nicks in the ribosomal R N A of insects. Biochem. Biophys. Res. C o m m u n . 54, 301-307. Payne, P.I. and Loening, U.E. (1970) RNA breakdown accompanying the isolation of pea root microsomes. Biochim. Biophys. Acta 224, 128-135. Shine, J. and Dalgarno, L. (1973) Occurrence of heat dissociable ribosomal R N A in insects: The presence of three polynucleotide chains in 26 S R N A from cultured Aedes aegypti cells. J. Mol. Biol. 75, 57-72. Castro, C., Hernandez, R. and Castaneda, M. (1981) Trypanosoma cruzi ribosomal RNA: internal break in the large-molecular-mass species and number of genes. Mol. Biochem. Parasitol. 2, 219-233. Hernandez, R., Nava, G. and Castaneda, M. (1983) Small-size ribosomal R N A species in Trypanosoma cruzi. Mol. Biochem. Parasitol. 8,297-304. Miller, F.W. and Ilan, J. (1978) The ribosomes of Plasmodium berghei: Isolation and ribosomal ribonucleic acid analysis. Parasitology 77, 345-365. Barker, D.C. (1976) Differentiation of Entamoeba patterns of nucleic acids and ribosomes during encystment and excystation. In: Biochemistry of Parasites and Host-Parasite Relationships, (Van den Bossche, H., ed.), pp. 253-260, Elsevier/North Holland-Biomedical Press, Amsterdam. Barker, D.C. and Swales, L.S. (1972) Characteristics of ribosomes during differentiation from trophozoite to cyst in axenic Entamoeba sp. Cell Diff. 1,297-306. Albach, R.A., Booden, T. Boonlayangoor, P. and Downing, S. (1977)Entamoebahistolytica: Autoradiographic analysis of nuclear sites of R N A synthesis. Exp. Parasitol. 42, 248-259. Heebner, G.M. and Albach, R.A. (1982) High salt SDS-DEP technique for isolation of'intact' rRNA from Entamoeba histolytica. Arch. Invest. Med. (Mex). 13 (suppl. 3), 23-28. Diamond, L.S., Harlow, D.R. and Cunnick, C.C. (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72, 431-432. Morgan, R.S., Slayter, H.S. and Weller, D.L. (1968) Isolation of ribosomes from cysts of Entamoeba invadens. J. Cell. Biol. 36, 45-51. Solymosy, R., Lazar, G. and Bagi, G. (1970) An improved version of the diethyl pyrocarbonate method for extracting ribosomal nucleic acids. Anal. Biochem. 38, 40-45.

272 18 19 20 21 22 23 24 25 26 27 28 29

Spector, T. (1978) Refinement of the coomassie blue method of protein quantitation. Anal. Biochem. 86, 142-146. Martin, R.G. and Ames, B.N. (1961) A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Biol. Chem. 236, 1372-1379. Poulson, R. (1973) Isolation, purification and fractionation of RNA. In: The Nucleic Acids, (Stewart, P.R. and Letham, D.S., eds.), pp. 243-261, Springer-Verlag, New York, NY. Sebo, T.J. and Schmit, J.C. (1982) Analytical gel electrophoresis of high molecular weight RNA in acrylamide-agarose gels containing methylmercuric hydroxide. Anal. Biochem. 120, 136-145. Prachayasittikul, V. and Albach, R.A. (1982) RNA depolymerase in Entamoeba histolytica: soluble vs. ribosomal. Arch. Invest. Med. (Mex). 13 (suppl. 3), 29-35. Ahzar, S. and Mohan Rao, V.K. (1975) Ribonuclease activity ofEntamoeba histolytica. Zbl. Bakteriol. Hyg., I. Abt. Orig. A. 230, 270-278. Boonlayangoor, P., Otten, M., Booden, T. and Albach, R.A. (1978) Isolation and sedimentation analysis of RNA from Entamoeba histolytica. Arch. Invest. Med. (Mex). 9 (suppl. 1), 121-128. Bostock, C.J., Prescott, D.M. and Lauth, M. (1971) Lability of 26 S ribosomal RNA in Tetrahymena pyroformis. Exp. Cell Res. 66, 260-262. Stevens, A.R. and Pachler, P.F. (1972) Discontinuity of 26 S rRNA in Acanthamoeba castellani. J. Mol. Biol. 66, 225-237. Cordingley, J.S. and Turner, M.J. (1980) 6.5 S RNA; preliminary characterization of unusual small RNAs in Trypanosoma brucei. Mol. Biochem. Parasitol. 1, 91-96. Prachayasittikul, V. and Albach, R.A. (1983) Rapidly labeled nuclear RNA in Entamoeba histolytica. J. Protozool. 30, 5A. (Abstract No. 18). Cordingley, J.J. and Hames, B.D. (1977) Specific cleavage of ribosomal RNA in Dictyostelium discoideum ribosomes. FEBS Lett. 82, 263-266.