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A comparative study of the ribonucleic acids of three species of trypanosomatids
Comp. Biochem. Physiol., 1978, Vol, 59B, pp. 1 to 4. Pergamon Press. Printed in Great Britain
A COMPARATIVE STUDY OF THE RIBONUCLEIC ACIDS OF THREE SPECIES OF TRYPANOSOMATIDS NYOIA M. MORALES* AND JOHN F. ROBERTS Department of Zoology, North Carolina State University, Raleigh, North Carolina 27607, U.S.A.
(Received 9 May 1977) Abstract--1. The ribosomal RNAs of Crithidiafasciculata, Leishmania tropica, and Trypanosoma brucei have sedimentation values of 25-27S, 18-20S and 5S. 2. The molecular sizes of the two heavier components are 1.24-1.28 M (106 daltons) and 0.84-0.89 M as determined by polyacrylamide gel electrophoresis. 3. The RNA of the three species studied shows a tendency to interact forming a complex that is very difficult to separate by sucrose gradient centrifugation. 4. The 25-27S RNA is very unstable. 5. The 18-20S RNA has a molecular size higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba.
INTRODUCTION Ribosomal R N A (rRNA) seems to have increased in size with evolution, the values remaining fairly constant within each group. Bacterial rRNA has sedimentation values of 23 and 16S, with molecular weights (MW) of 1.1 and 0.55 x 106 daltons (M) respectively. Sedimentation coefficients of rRNA from lower animals and plants are 25 and 16-18S, with MWs of 1.3 and 0.7 M (Attardi & Amaldi, 1970). M W larger than 0.7 M have been reported for Amoeba and Euqlena (Loening, 1968). Higher animals have a ribosomal component of 17-18S with a M W of 0.7 M and a 28S component that apparently increases in M W from the lower to the higher animal forms (1.4-1.7 M), (Attardi & Amaldi, 1970). Studies of the rRNA of the trypanosomatid Crithid?afasciculata revealed sedimentation values comparable to those of plants and lower animals, but polyacrylamide gel electrophoresis showed a ribosomal component with an unusually high molecular size, similar to those reported for Amoeba and Euolena (Loening, 1968). These findings led us to compare the r R N A of members of the family Trypanosomatidae from three different genera, namely, C. fasciculata, Leishmania tropica and Trypanosoma brucei. MATERIALS AND METHODS
Buffers 1. 0.01M TRIS-HC1, pH 7.6; 0.01M EDTA; 0.25M sucrose. 2. 0.01M TRIS-HC1, pH 7.5; 0.03M NH4Cl; 0.3mM MgC12. 3. 0.01 M Sodium Acetate, pH 5.2; 0.05 M NaC1; 0.1 mM MgCl2.
* Present address: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Trian$1e Park, North Carolina 27709, U.S.A.
C.B.P. 59/1 a ~
Organisms and culturing conditions C. fasciculata was grown in 21. flasks, at room temperature, with shaking, in a liquid medium containin 0.5% casein hydrolysate, 0.5% yeast hydrolysate, 0.01% liver extract (1:20), 0.0015% heroin in 50% triethanolamine and 1.5% sucrose (all w/v). L. tropica was grown at 26°C in a diphasic medium consisting of a blood agar base overlaid with an equal amount of Lock's solution (0.8% NaCl, 0.02% KCI, 0.02% CaC12, 0.03% KH2P04, 0.25% glucose). The solid base consisted of 3.7% brain heart infusion, 1% neopeptone, 2.5% bactoagar and 30% human blood. T. brucei was kept frozen under liquid N2; the organisms were thawed rapidly at 37°C and inoculated in rats. The trypanosomes were separated from the blood according to the method of Lanham (1968). All cells were collected by centrifugation for 10 rain at 1000g and washed with buffer 1.
Isolation of RN A RNA was isolated by the phenol method of Scherrer (1969), the Diethyl Pyrocarbonate (DEP) method described by Poulson (1973) or by the sodium dodecylsulfate (SDS) method as follows. Cells were resuspended in 10-12 vol of buffer 2, containing 0.5% bentonite; SDS was added to 1% final concentration and the cells disrupted by homogenization with a Sorvall omnimixer. The lysate was incubated at 37°C, for 5 rain, chilled and centrifuged for 10 min at 3020g; 1 ml quantities of the clear supernatant were placed on sucrose gradients for centrifugation. The RNA could be recovered from the gradients by precipitation with 2 vol of ethanol after adjustment of the NaCl concentration to 0.1 M.
Sucrose gradient centrifugation Forty milliliters, 5-50% sucrose gradients in buffer 3 were overlaid with 1 ml samples containing between 200 and 2000/~g RNA and centrifuged in the Beckman SW 25.2 rotor at 75,465 g~v for 17 hr at 4°C. One milliliter fractions were collected from the bottom of the tube and the absorbance (A) at 260.m of the fractions recorded. S values were estimated according to Martin & Ames (1961), using rat liver RNA as the standard.
2
NYDIA M. MORALESAND JOHN F. ROBERTS
Polyacrylamide gel electrophoresis The method employed was that described by Peacock '& Dingman (1967). A plastic spacer was placed on top of the 2.5% slab gel and 10 pl samples containing between 30 and 60pg RNA in 10% sucrose overlaid. Electrophoresis was carried out in the cold at 10 mA for 4 hr. The apparent molecular size was estimated from the electrophoretic mobility, using rat liver RNA of 1.75 and 0.7 M as reference values (Groot et al., 1970). RESULTS
0.8
27S
~ 0.6 o
19S
0.4
/
C. fasciculata R N A RNA isolated by either the phenol or the DEP methods could not be resolved when centrifuged on 5-20~o or 10--30~ gradients. Centrifugation on 5-50~ gradients resolved the RNA into 24.9_ 0.2, 17.8 + 0.1 and 5.2 + 0.2S (mean + SE for 5 determinations). The molecular size estimated by polyacrylamide gel electrophoresis was 1.24 and 0.84 M, for the heavier ribosomal components; there were two lighter bands migrating with the rat liver 5S RNA. The 1.24 M RNA is unstable giving 0.73 and 0.57 M bands (Fig. 1). For further details see Morales & Roberts (1977). L. tropica R N A RNA isolated by the DEP method and centrifuged on 5-50~ sucrose gradients showed two large peaks of 5S and 17S, with a shoulder of approximately 26.9S on the 17S peak; presence of EDTA in the gradient or pronase treatment of the RNA prior to centrifugation did not improve the resolution. RNA prepared by the SDS method, in either the presence or absence of 0.59/o bentonite resolved into 27.4 + 0.2, 19.0 + 0.3 and 4.9 + 0.2S components (means _ SE for 3 determinations), when centrifuged on 5-50% sucrose gradients (Fig. 2). Polyacrylamide gel electrophoresis resulted in separation of the RNA obtained with the DEP method, molecular sizes thus estimated were 1.28 M, 0.89 M and 0.62 M for the three heavier bands (mean for 5 b
a
c
d
e
f
g
h
i
1.75M
0.7
M
~ m
_,~m' ,,
m--
m m ~
m
mm
m m
~
m
m
m
---~-
~
m~
mmmm
m m m
m
m
| m - -
m
m
Fig. 1. Electrophoretic analysis of trypanosomatid and rat liver RNAs in a 2.5~ polyacrylamide slab gel. a--Rat liver RNA. b--C. fasciculata RNA. c--C. fasciculata RNA from the 25S region of a sucrose gradient, d--C. fasciculata RNA from the 18S region of a sucrose gradient, e--C. fasciculata RNA from the 5S region of a sucrose gradient. f~C. fasciculata RNA incubated for 3 rain at 52°C. g--C. fasciculata 25S RNA incubated for 10 rain at 52°C. h--T. brucei RNA. i--L. tropica RNA.
0.2
\
,,,/. I
"4 I
I
I0
t
20 50 Fraction
L
)
4O
Fig. 2. Sucrose gradient analysis of L. tropica rRNA. RNA prepared by the SDS method was placed on a 40 ml 5-50% sucrose gradient and centrifuged for 17 hr at 4°C and 75,465 g,v in the Beckman SW 25.2 rotor. determinations The SE varied between 0.003 and 0.004). As in the case of C. fasciculata, there were also two light bands migrating with the rat liver 5S RNA (Fig. 1l Although 27S RNA was not seen in the samples prepared by the DEP method and analyzed by sucrose gradient centrifugation, the intensity of the 1.28 M band obtained from these preparations with electrophoresis is greater than the intensity of either the 0.89 M or 0.62 M bands, suggesting the absence of extense degradation of the 27S RNA and indicating a possible interaction of the 27 and 19S RNAs, that is not negated by the presence of the chelating agent, EDTA. T. brucei R N A RNA isolated by the SDS-bentonite method and centrifuged on sucrose gradients resolved into 25.4 + 0.1, 19.6 + 0.3 and 4.5 + 0.09S (means + SE for 4 determinations) peaks (Fig. 3). Polyacrylamide gel electrophoresis of RNA prepared by phenol extraction separated into 1.28, 0.85 and 0.58 M components (means for 6 determinations), plus two lighter bands, migrating close to the 5S RNA (Fig. 1). Occasionally a 0.72 M band was also present. All bands were resistant to DNase and pronase, but sensitive to RNase. Degradation of the 1.28 M RNA occurred easily either during the extraction or during the preparation of the samples for electrophoresis with resulting enhancement of the 0.72 and 0.58 M bands. DISCUSSION High molecular size rRNA isolated from different species of Trypanosomatids, by different methods, showed a tendency to interact, forming a complex that was very difficult to be resolved with sucrose gradient centrifugation. C. fasciculata RNA could be separated only through the use of 5-509/0 gradients.
Study of the ribonucleic acids serve size classes, The three species of trypanosomarids studied showed a 25-27S RNA and an 18-20S 1.5 RNA, which compare with values obtained with other protozoa (Barker & Swales, 1972; Chi & Suyama, 20S 1970; Krawiec & Eisenstadt, 19701 Gel electrophoresis revealed three high molecular size bands E ranging from 1.24-1.28, 0.844).89, and 0.57-0.62 M. In E 0 some occasions, a band of 0.72 M could also be 1.0 oJ detected. Electrophoretic results obtained with RNA <~ from different regions of a sucrose gradient (see Morales & Roberts, 1977, and Fig. l) and with RNA samples previously incubated at elevated temperatures, indicate that the 1.24-1.28 M RNA corresponds to the 25-27S RNA and that it easily undergoes 0.5 degradation to give the 0.72 and 0.57-0.62 M bands. ! The fact that the 0.844).89 M RNA was only found in the region of the sucrose gradient corresponding / to 18S RNA and that none of the degradation products of the 25S RNA has this molecular size indiI I I I cates that this RNA corresponds to the 18S RNA. l 2O 30 40 T. brucei 1.28 M RNA seemed to be more unstable I0 than the corresponding RNA of the other two species Fraction studied. No 0.72 M component was detected in L. troFig. 3. Sucrose gradient analysis of T. brucei rRNA. Cen- pica RNA; in the case of C. fasciculata and T. brucei trifugation conditions as described in Fig. 2. the 0.72 M band was absent in some preparations and was more evident after heat treatment of the samples. This suggests that this band is a product of degradation and does not correspond to the 0.7 M rRNA L. tropica RNA isolated by the DEP method, and of other eukaryotes. The instability of heavy rRNA T. rangeli (results not shown) isolated by the phenol has been reported for several organisms such as: method, could not be resolved even in 5-50~ gra- Tetrahymena (Bastock et al., 1971), Euglena (Loening, dients. The fact that the same preparation could be 1968), Amoeba (Loening, 1968; Stevens & Pachler, separated by electrophoresis, and that with the SDS 1972) and insects (Shine & Dalgarno, 1973). It has method three peaks were obtained even in the been suggested that the instability is due to the presabsence of bentonite, rules out the possibility of ence of "hidden nicks" in the RNA molecule, the degradation of the heavier peak and banding of the resulting strands being held together by hydrogen products with the lighter peak. The interaction is bonding. The presence of such nicks probably indiprobably not an artifact introduced by the method cates some spatial configuration of the RNA within of isolation alone (since rat liver RNA isolated by the ribosomes, that leaves certain nucleotides exposed the same methods could be resolved with typical to the action of nucleases. Lava-Sanchez & Puppo sucrose gradients) but a property inherent to the (1975) have done experiments that support this idea. RNA of the trypanosomatids studied. The interaction In Musca carnaria the 26S RNA (1.42 M) breaks into is probably not due to cation bridges, since EDTA 0.74 M and 0.68 M components upon incubation for in the gradients failed to improve the resolution. Pro- 3 min at 60°C. Newly formed 26S RNA (first 3 hr nase treatment of the RNA before ultracentifugation labeling) is heat stable, suggesting that the "nicking" did not improve the resolution either, suggesting that occurs in the cytoplasm. Incubation of 26S RNA and the aggregation is not due to the presence of proteins. ribosomes obtained during the first three hours of RNA prepared by the SDS-bentonite method from labeling, in the presence of 0.01-1 ng/ml RNase any species could always be separated into 3 com- resulted in random breakage of the 26S RNA; ponents by sucrose gradient centrifugation. The basic whereas the 26S RNA extracted from the ribosomes difference between this method and the other methods had only 3 nicks. employed was that instead of precipitating the RNA The 1.24-1.28 M RNAs compare to the heavier with ethanol overnight, the RNA extract was immedi- component of plants and some protozoa (Loening, ately placed on a gradient and separated, probably 1968), but the 0.82-0.9 M RNA has a considerably not allowing enough time for the aggregation to higher molecular size than the 0.7 M reported for the occur. The importance of time in the formation of second heavy component of rRNA of most eukarthe complex is also suggested by the fact that a C. yotes. It compares, however, with values obtained for fasciculata RNA sample prepared by the DEP this second heavy component in Amoeba and Euglena method that had been previously analyzed by sucrose (Loening, 1968). The information now available gradient centrifugation, could no longer be separated reveals the presence of this 0.8-0.9 M rRNA species by ultracentrifugation after several months of storage in Protozoa of the Classes Mastigophora and Sarcoin the freezer, although gel electrophoresis still dina; the members of the Class Cilliata (Paramecium, showed the usual bands, indicating absence of major Tetrahymena) having a 0.7M RNA comparable to degradation. that of other eukaryotes (Attardi & Amaldi, 1970; As already mentioned, rRNA seems to have in- Loening, 1968). It is intriguing that although most creased in size with evolution, with a tendency to pre- biological evidence suggests the emergence of higher
!
V
4
NYDIA M. MORALESAND JOHN F. ROBERTS
animals from flagellates, it is the cilliates that have R N A that compares to the R N A of higher animals. The study of the R N A from more species of protozoa might clarify the evolutionary significance of the sizes of rRNA. REFERENCES
ATTARDI G. & AMALDI F. (1970) Structure and synthesis of ribosomal RNA. A. Rev. Biocher~ 39, 183-226. BARKER D. C. & SWALESL. S. (1972) Characteristics of ribosomes during differentation from trophozoite to cyst in axenic Entamoeba sp. Cell Differentiation 1, 297-306. BASTO~I~C. J., PRESCOTTD. M. ~--LAUTH M. 0971) Lability of 26S rRNA in Tetrahymena pyriformis. Expl Cell Res. 66, 260-262. Cm J. C. H. & SUVAI~L,~Y. (1970) Comparative studies on mitochondrial and cytoplasmic ribosomes of Tetrahymena pyriformis. J. molec. Biol. 53, 531-556. GROOT P. H. E., A~aJ C. & BORSTP. (1970) Variation with temperature of the apparent molecular weight of rat liver mitochondrial RNA, determined by gel electrophoresis. Biochent biophys. Res. Commun. 41, 1321-1327. KRAWIEC S. & EISENSTADTJ. M. (1970) Ribonucleic acids from the mitochondria of bleached Euglena gracilis. Biochim. biophys. Acta 217, 132-141. LANHAM S. M. (1968) Separation of trypanosomes from the blood of infested rats and mice by anion exchangers. Nature, Lond. 218, 1273-1274.
LAVA-SANCHEZP. A. & PuvvO S. (1975) Occurrence in vivo of "hidden breaks" at specific sites of the 26S ribosomal RNA of Musca carnaria. J. molec. Biol. 95, 9-20. LOENING U. E. (1968) Molecular weights of ribosomal RNA in relation to evolution. J. molec. Biol. 38, 355-365. MARTIN R. G. d?¢.AMESB. N. (1961) A method for determining sedimentation behaviour of enzymes: application to protein mixtures. J. molec. Biol. 236, 1372- 1379. MORALES N. M. & ROBERTSJ. F. (1977) The ribonucleic acids of Crithidia fasciculata. Submitted to J. Protozool. PEACOCKA. C. & D1NGrCtANW. (1967) Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827. POULSONR. 0973) Isolation, purification and fractionation of RNA. In The Ribonucleic Acids. (Edited by STEWART P. R. & LETHAN D. S.), pp. 243 259. Springer Verlag, New York. SCHERRERK. (1969) Isolation and sucrose gradient analysis of RNA. In Fundamental Techniques in Virology. (Edited by HABEL K. & SALZMON. P.), pp. 413-432. Academic Press, New York. SHINE J. & DALGARNOL. (1973) Occurrence of heat dissociable rRNA in insects: the presence of 3 polynucleotide chains in 26S RNA from cultured Aedes aegypti cells. J. molec. Biol. 75, 57-72. STEVENS A. R. & PACHLER P. F. (1972) Discontinuity of 26S rRNA in Acanthamoeba castellani. J. molec. Biol. 66, 225-237.
A COMPARATIVE STUDY OF THE RIBONUCLEIC ACIDS OF THREE SPECIES OF TRYPANOSOMATIDS NYOIA M. MORALES* AND JOHN F. ROBERTS Department of Zoology, North Carolina State University, Raleigh, North Carolina 27607, U.S.A.
(Received 9 May 1977) Abstract--1. The ribosomal RNAs of Crithidiafasciculata, Leishmania tropica, and Trypanosoma brucei have sedimentation values of 25-27S, 18-20S and 5S. 2. The molecular sizes of the two heavier components are 1.24-1.28 M (106 daltons) and 0.84-0.89 M as determined by polyacrylamide gel electrophoresis. 3. The RNA of the three species studied shows a tendency to interact forming a complex that is very difficult to separate by sucrose gradient centrifugation. 4. The 25-27S RNA is very unstable. 5. The 18-20S RNA has a molecular size higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba.
INTRODUCTION Ribosomal R N A (rRNA) seems to have increased in size with evolution, the values remaining fairly constant within each group. Bacterial rRNA has sedimentation values of 23 and 16S, with molecular weights (MW) of 1.1 and 0.55 x 106 daltons (M) respectively. Sedimentation coefficients of rRNA from lower animals and plants are 25 and 16-18S, with MWs of 1.3 and 0.7 M (Attardi & Amaldi, 1970). M W larger than 0.7 M have been reported for Amoeba and Euqlena (Loening, 1968). Higher animals have a ribosomal component of 17-18S with a M W of 0.7 M and a 28S component that apparently increases in M W from the lower to the higher animal forms (1.4-1.7 M), (Attardi & Amaldi, 1970). Studies of the rRNA of the trypanosomatid Crithid?afasciculata revealed sedimentation values comparable to those of plants and lower animals, but polyacrylamide gel electrophoresis showed a ribosomal component with an unusually high molecular size, similar to those reported for Amoeba and Euolena (Loening, 1968). These findings led us to compare the r R N A of members of the family Trypanosomatidae from three different genera, namely, C. fasciculata, Leishmania tropica and Trypanosoma brucei. MATERIALS AND METHODS
Buffers 1. 0.01M TRIS-HC1, pH 7.6; 0.01M EDTA; 0.25M sucrose. 2. 0.01M TRIS-HC1, pH 7.5; 0.03M NH4Cl; 0.3mM MgC12. 3. 0.01 M Sodium Acetate, pH 5.2; 0.05 M NaC1; 0.1 mM MgCl2.
* Present address: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Trian$1e Park, North Carolina 27709, U.S.A.
C.B.P. 59/1 a ~
Organisms and culturing conditions C. fasciculata was grown in 21. flasks, at room temperature, with shaking, in a liquid medium containin 0.5% casein hydrolysate, 0.5% yeast hydrolysate, 0.01% liver extract (1:20), 0.0015% heroin in 50% triethanolamine and 1.5% sucrose (all w/v). L. tropica was grown at 26°C in a diphasic medium consisting of a blood agar base overlaid with an equal amount of Lock's solution (0.8% NaCl, 0.02% KCI, 0.02% CaC12, 0.03% KH2P04, 0.25% glucose). The solid base consisted of 3.7% brain heart infusion, 1% neopeptone, 2.5% bactoagar and 30% human blood. T. brucei was kept frozen under liquid N2; the organisms were thawed rapidly at 37°C and inoculated in rats. The trypanosomes were separated from the blood according to the method of Lanham (1968). All cells were collected by centrifugation for 10 rain at 1000g and washed with buffer 1.
Isolation of RN A RNA was isolated by the phenol method of Scherrer (1969), the Diethyl Pyrocarbonate (DEP) method described by Poulson (1973) or by the sodium dodecylsulfate (SDS) method as follows. Cells were resuspended in 10-12 vol of buffer 2, containing 0.5% bentonite; SDS was added to 1% final concentration and the cells disrupted by homogenization with a Sorvall omnimixer. The lysate was incubated at 37°C, for 5 rain, chilled and centrifuged for 10 min at 3020g; 1 ml quantities of the clear supernatant were placed on sucrose gradients for centrifugation. The RNA could be recovered from the gradients by precipitation with 2 vol of ethanol after adjustment of the NaCl concentration to 0.1 M.
Sucrose gradient centrifugation Forty milliliters, 5-50% sucrose gradients in buffer 3 were overlaid with 1 ml samples containing between 200 and 2000/~g RNA and centrifuged in the Beckman SW 25.2 rotor at 75,465 g~v for 17 hr at 4°C. One milliliter fractions were collected from the bottom of the tube and the absorbance (A) at 260.m of the fractions recorded. S values were estimated according to Martin & Ames (1961), using rat liver RNA as the standard.
2
NYDIA M. MORALESAND JOHN F. ROBERTS
Polyacrylamide gel electrophoresis The method employed was that described by Peacock '& Dingman (1967). A plastic spacer was placed on top of the 2.5% slab gel and 10 pl samples containing between 30 and 60pg RNA in 10% sucrose overlaid. Electrophoresis was carried out in the cold at 10 mA for 4 hr. The apparent molecular size was estimated from the electrophoretic mobility, using rat liver RNA of 1.75 and 0.7 M as reference values (Groot et al., 1970). RESULTS
0.8
27S
~ 0.6 o
19S
0.4
/
C. fasciculata R N A RNA isolated by either the phenol or the DEP methods could not be resolved when centrifuged on 5-20~o or 10--30~ gradients. Centrifugation on 5-50~ gradients resolved the RNA into 24.9_ 0.2, 17.8 + 0.1 and 5.2 + 0.2S (mean + SE for 5 determinations). The molecular size estimated by polyacrylamide gel electrophoresis was 1.24 and 0.84 M, for the heavier ribosomal components; there were two lighter bands migrating with the rat liver 5S RNA. The 1.24 M RNA is unstable giving 0.73 and 0.57 M bands (Fig. 1). For further details see Morales & Roberts (1977). L. tropica R N A RNA isolated by the DEP method and centrifuged on 5-50~ sucrose gradients showed two large peaks of 5S and 17S, with a shoulder of approximately 26.9S on the 17S peak; presence of EDTA in the gradient or pronase treatment of the RNA prior to centrifugation did not improve the resolution. RNA prepared by the SDS method, in either the presence or absence of 0.59/o bentonite resolved into 27.4 + 0.2, 19.0 + 0.3 and 4.9 + 0.2S components (means _ SE for 3 determinations), when centrifuged on 5-50% sucrose gradients (Fig. 2). Polyacrylamide gel electrophoresis resulted in separation of the RNA obtained with the DEP method, molecular sizes thus estimated were 1.28 M, 0.89 M and 0.62 M for the three heavier bands (mean for 5 b
a
c
d
e
f
g
h
i
1.75M
0.7
M
~ m
_,~m' ,,
m--
m m ~
m
mm
m m
~
m
m
m
---~-
~
m~
mmmm
m m m
m
m
| m - -
m
m
Fig. 1. Electrophoretic analysis of trypanosomatid and rat liver RNAs in a 2.5~ polyacrylamide slab gel. a--Rat liver RNA. b--C. fasciculata RNA. c--C. fasciculata RNA from the 25S region of a sucrose gradient, d--C. fasciculata RNA from the 18S region of a sucrose gradient, e--C. fasciculata RNA from the 5S region of a sucrose gradient. f~C. fasciculata RNA incubated for 3 rain at 52°C. g--C. fasciculata 25S RNA incubated for 10 rain at 52°C. h--T. brucei RNA. i--L. tropica RNA.
0.2
\
,,,/. I
"4 I
I
I0
t
20 50 Fraction
L
)
4O
Fig. 2. Sucrose gradient analysis of L. tropica rRNA. RNA prepared by the SDS method was placed on a 40 ml 5-50% sucrose gradient and centrifuged for 17 hr at 4°C and 75,465 g,v in the Beckman SW 25.2 rotor. determinations The SE varied between 0.003 and 0.004). As in the case of C. fasciculata, there were also two light bands migrating with the rat liver 5S RNA (Fig. 1l Although 27S RNA was not seen in the samples prepared by the DEP method and analyzed by sucrose gradient centrifugation, the intensity of the 1.28 M band obtained from these preparations with electrophoresis is greater than the intensity of either the 0.89 M or 0.62 M bands, suggesting the absence of extense degradation of the 27S RNA and indicating a possible interaction of the 27 and 19S RNAs, that is not negated by the presence of the chelating agent, EDTA. T. brucei R N A RNA isolated by the SDS-bentonite method and centrifuged on sucrose gradients resolved into 25.4 + 0.1, 19.6 + 0.3 and 4.5 + 0.09S (means + SE for 4 determinations) peaks (Fig. 3). Polyacrylamide gel electrophoresis of RNA prepared by phenol extraction separated into 1.28, 0.85 and 0.58 M components (means for 6 determinations), plus two lighter bands, migrating close to the 5S RNA (Fig. 1). Occasionally a 0.72 M band was also present. All bands were resistant to DNase and pronase, but sensitive to RNase. Degradation of the 1.28 M RNA occurred easily either during the extraction or during the preparation of the samples for electrophoresis with resulting enhancement of the 0.72 and 0.58 M bands. DISCUSSION High molecular size rRNA isolated from different species of Trypanosomatids, by different methods, showed a tendency to interact, forming a complex that was very difficult to be resolved with sucrose gradient centrifugation. C. fasciculata RNA could be separated only through the use of 5-509/0 gradients.
Study of the ribonucleic acids serve size classes, The three species of trypanosomarids studied showed a 25-27S RNA and an 18-20S 1.5 RNA, which compare with values obtained with other protozoa (Barker & Swales, 1972; Chi & Suyama, 20S 1970; Krawiec & Eisenstadt, 19701 Gel electrophoresis revealed three high molecular size bands E ranging from 1.24-1.28, 0.844).89, and 0.57-0.62 M. In E 0 some occasions, a band of 0.72 M could also be 1.0 oJ detected. Electrophoretic results obtained with RNA <~ from different regions of a sucrose gradient (see Morales & Roberts, 1977, and Fig. l) and with RNA samples previously incubated at elevated temperatures, indicate that the 1.24-1.28 M RNA corresponds to the 25-27S RNA and that it easily undergoes 0.5 degradation to give the 0.72 and 0.57-0.62 M bands. ! The fact that the 0.844).89 M RNA was only found in the region of the sucrose gradient corresponding / to 18S RNA and that none of the degradation products of the 25S RNA has this molecular size indiI I I I cates that this RNA corresponds to the 18S RNA. l 2O 30 40 T. brucei 1.28 M RNA seemed to be more unstable I0 than the corresponding RNA of the other two species Fraction studied. No 0.72 M component was detected in L. troFig. 3. Sucrose gradient analysis of T. brucei rRNA. Cen- pica RNA; in the case of C. fasciculata and T. brucei trifugation conditions as described in Fig. 2. the 0.72 M band was absent in some preparations and was more evident after heat treatment of the samples. This suggests that this band is a product of degradation and does not correspond to the 0.7 M rRNA L. tropica RNA isolated by the DEP method, and of other eukaryotes. The instability of heavy rRNA T. rangeli (results not shown) isolated by the phenol has been reported for several organisms such as: method, could not be resolved even in 5-50~ gra- Tetrahymena (Bastock et al., 1971), Euglena (Loening, dients. The fact that the same preparation could be 1968), Amoeba (Loening, 1968; Stevens & Pachler, separated by electrophoresis, and that with the SDS 1972) and insects (Shine & Dalgarno, 1973). It has method three peaks were obtained even in the been suggested that the instability is due to the presabsence of bentonite, rules out the possibility of ence of "hidden nicks" in the RNA molecule, the degradation of the heavier peak and banding of the resulting strands being held together by hydrogen products with the lighter peak. The interaction is bonding. The presence of such nicks probably indiprobably not an artifact introduced by the method cates some spatial configuration of the RNA within of isolation alone (since rat liver RNA isolated by the ribosomes, that leaves certain nucleotides exposed the same methods could be resolved with typical to the action of nucleases. Lava-Sanchez & Puppo sucrose gradients) but a property inherent to the (1975) have done experiments that support this idea. RNA of the trypanosomatids studied. The interaction In Musca carnaria the 26S RNA (1.42 M) breaks into is probably not due to cation bridges, since EDTA 0.74 M and 0.68 M components upon incubation for in the gradients failed to improve the resolution. Pro- 3 min at 60°C. Newly formed 26S RNA (first 3 hr nase treatment of the RNA before ultracentifugation labeling) is heat stable, suggesting that the "nicking" did not improve the resolution either, suggesting that occurs in the cytoplasm. Incubation of 26S RNA and the aggregation is not due to the presence of proteins. ribosomes obtained during the first three hours of RNA prepared by the SDS-bentonite method from labeling, in the presence of 0.01-1 ng/ml RNase any species could always be separated into 3 com- resulted in random breakage of the 26S RNA; ponents by sucrose gradient centrifugation. The basic whereas the 26S RNA extracted from the ribosomes difference between this method and the other methods had only 3 nicks. employed was that instead of precipitating the RNA The 1.24-1.28 M RNAs compare to the heavier with ethanol overnight, the RNA extract was immedi- component of plants and some protozoa (Loening, ately placed on a gradient and separated, probably 1968), but the 0.82-0.9 M RNA has a considerably not allowing enough time for the aggregation to higher molecular size than the 0.7 M reported for the occur. The importance of time in the formation of second heavy component of rRNA of most eukarthe complex is also suggested by the fact that a C. yotes. It compares, however, with values obtained for fasciculata RNA sample prepared by the DEP this second heavy component in Amoeba and Euglena method that had been previously analyzed by sucrose (Loening, 1968). The information now available gradient centrifugation, could no longer be separated reveals the presence of this 0.8-0.9 M rRNA species by ultracentrifugation after several months of storage in Protozoa of the Classes Mastigophora and Sarcoin the freezer, although gel electrophoresis still dina; the members of the Class Cilliata (Paramecium, showed the usual bands, indicating absence of major Tetrahymena) having a 0.7M RNA comparable to degradation. that of other eukaryotes (Attardi & Amaldi, 1970; As already mentioned, rRNA seems to have in- Loening, 1968). It is intriguing that although most creased in size with evolution, with a tendency to pre- biological evidence suggests the emergence of higher
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NYDIA M. MORALESAND JOHN F. ROBERTS
animals from flagellates, it is the cilliates that have R N A that compares to the R N A of higher animals. The study of the R N A from more species of protozoa might clarify the evolutionary significance of the sizes of rRNA. REFERENCES
ATTARDI G. & AMALDI F. (1970) Structure and synthesis of ribosomal RNA. A. Rev. Biocher~ 39, 183-226. BARKER D. C. & SWALESL. S. (1972) Characteristics of ribosomes during differentation from trophozoite to cyst in axenic Entamoeba sp. Cell Differentiation 1, 297-306. BASTO~I~C. J., PRESCOTTD. M. ~--LAUTH M. 0971) Lability of 26S rRNA in Tetrahymena pyriformis. Expl Cell Res. 66, 260-262. Cm J. C. H. & SUVAI~L,~Y. (1970) Comparative studies on mitochondrial and cytoplasmic ribosomes of Tetrahymena pyriformis. J. molec. Biol. 53, 531-556. GROOT P. H. E., A~aJ C. & BORSTP. (1970) Variation with temperature of the apparent molecular weight of rat liver mitochondrial RNA, determined by gel electrophoresis. Biochent biophys. Res. Commun. 41, 1321-1327. KRAWIEC S. & EISENSTADTJ. M. (1970) Ribonucleic acids from the mitochondria of bleached Euglena gracilis. Biochim. biophys. Acta 217, 132-141. LANHAM S. M. (1968) Separation of trypanosomes from the blood of infested rats and mice by anion exchangers. Nature, Lond. 218, 1273-1274.
LAVA-SANCHEZP. A. & PuvvO S. (1975) Occurrence in vivo of "hidden breaks" at specific sites of the 26S ribosomal RNA of Musca carnaria. J. molec. Biol. 95, 9-20. LOENING U. E. (1968) Molecular weights of ribosomal RNA in relation to evolution. J. molec. Biol. 38, 355-365. MARTIN R. G. d?¢.AMESB. N. (1961) A method for determining sedimentation behaviour of enzymes: application to protein mixtures. J. molec. Biol. 236, 1372- 1379. MORALES N. M. & ROBERTSJ. F. (1977) The ribonucleic acids of Crithidia fasciculata. Submitted to J. Protozool. PEACOCKA. C. & D1NGrCtANW. (1967) Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827. POULSONR. 0973) Isolation, purification and fractionation of RNA. In The Ribonucleic Acids. (Edited by STEWART P. R. & LETHAN D. S.), pp. 243 259. Springer Verlag, New York. SCHERRERK. (1969) Isolation and sucrose gradient analysis of RNA. In Fundamental Techniques in Virology. (Edited by HABEL K. & SALZMON. P.), pp. 413-432. Academic Press, New York. SHINE J. & DALGARNOL. (1973) Occurrence of heat dissociable rRNA in insects: the presence of 3 polynucleotide chains in 26S RNA from cultured Aedes aegypti cells. J. molec. Biol. 75, 57-72. STEVENS A. R. & PACHLER P. F. (1972) Discontinuity of 26S rRNA in Acanthamoeba castellani. J. molec. Biol. 66, 225-237.