Journal of Helminthology (1992) 66, 193-197
A hidden break in the 28.0S rRNA from Diphyllobothrium dendriticum K. A. KARLSTEDT, G. I. L. PAATERO, J.-H. MAKELA and B.-J. WIKGREN Department of Biology, Abo Akademi University, SF-20520 Abo, Finland
ABSTRACT Nondenatured and denatured total RNA from the tapeworm Diphyllobothrium dendriticum (Cestoda) was analysed by agarose gel electrophoresis. It was found that the large subunit ribosomal RNA (IrRNA) is 28.OS and the small subunit ribosomal RNA (srRNA) is 19.5S. Following denaturation the 28.OS rRNA was disrupted into a 19.5S subfragment and a 20.7S subfragment due to the presence of a centrally located hidden break. By hybridization of Northern blot membranes with oligonucleotide probes specific for the 5'- and 3'-ends of the IrRNA respectively, we have shown that the 19.5S subfragment is from the 5'-end (the a-subfragment) and the 20.7S subfragment from the 3'-end (the p"subfragment) of the 28.0S rRNA of D. dendriticum. KEY WORDS: Diphyllobothrium dendriticum, rRNA, hidden break. Northern blot, oligonuclcotide probes, Cestoda
INTRODUCTION
The presence of a hidden break in the central domain of the large subunit ribosomal RNA (IrRNA) has been demonstrated in protozoa, in higher plant chloroplasts, and in almost all the protostomes examined, but not in deuterostomes (for review see ISHIKAWA, 1977b; RAUE et al., 1988). If the IrRNA exhibits a hidden break, denaturation of IrRNA results in dissociation of hydrogen bonds causing disruption of IrRNA into an a-subfragment (5'-end) and a p-subfragment (3'-end). A hidden break in the IrRNA was first observed by APPLEBAUM et al. (1966) in insects. Later sequence studies of the central domain of insect IrRNA have revealed that there is not only a break in a phosphodiester bond but various amounts of nucleotides are eliminated during the nuclear processing of pre-lrRNA (WARE et al., 1985; FUJIWARA & ISHIKAWA, 1986; DE LANVERSIN & JACQ, 1989). Furthermore, it has recently been shown that the introduced gaps are located in specific expansion segments of the IrRNA (DE LANVERSIN & JACQ, 1989; ENGBERG et al., 1990; MERTZ et al., 1991; VAN KEULEN et al., 1991). In the present study we show, by using oligonucleotide probes specific for the 5'-end and the 3'-end of the IrRNA respectively, that due to a central hidden break the 28.OS rRNA from Diphyllobothrium dendriticum (Cestoda) upon heat treatment disrupts into a 19.5S a-subfragment and a 20.7S (3-subfragment.
MATERIALS AND METHODS
RNA isolation Extraction of total RNA from adult D. dendriticum was carried out essentially using the LiCI-urea method as described by SIMPSON et al. (1984). Gel electrophoresis Aliquots of 3 u.g of total RNA in 30 mM sodium acetate, pH 5-5, were electrophoresed in 1-5% agarose gels in TBE buffer (89 mM Tris-HCl, pH 8-3, 89 mM
194
K. A. K.ARLSTEDT el al.
boric acid, and 2 mM EDTA) containing 0-25 fig/ml ethidium bromide. The samples were incubated for 15 min either at 65°C, 37°C or 25°C before loading. For size determination nondenatured E. coli and rat RNA were run simultaneously in the gel. Northern blot analysis Prior to Northern blot analysis the gels (0-8% agarose) were incubated in 10% formaldehyde at 65°C for 30 min (KHANDJIAN & MERIC, 1986). Following this denaturing step the RNA was transferred to nylon membranes (Amersham, Hybond N) in 20XSSC (lxSSC is 015 M NaCI and 0015 M sodium citrate), and the RNA was then fixed to the membranes by UV irradiation for 2 min. Hybridization Prehybridization and hybridization were carried out in 6xSSC, 5xDenhardt's solution ( 0 1 % each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), 0-1% SDS, 10 (ig/ml of poly A and 50 u.g/ml of sonicated and denatured salmon sperm DNA at 55°C. The membranes were hybridized with 3'-end labelled (a-32P ddATP) oligonucleotide probes, the a-oligo and the p"-oligo, each representing a consensus sequence in the 5'-end (a-subfragment) and the 3'-end ((i-subfragment) of the lrRNA, respectively. The a-oligo has sequences complementary to bases 89104 and the (3-oligo to bases 4244-4259 in the rat 28.OS rRNA sequence (CHAN et al., 1983). The sequences are as follows: a-oligo: 5'-CTA AAC CCA GCT CAC-3' P-oligo: 5'-TCA CTC GCC GTT ACT-3' The oligonucleotide sequences were deduced, and their universal specificity was tested using the UWGCG software (DEVEREUX et al., 1984) to compare known rDNA sequences in the GenBank database. Following hybridization the membranes were washed in 6xSSC at 60°C for 15 min, and the exposures were done with intensifying screens at -7()°C. RESULTS AND DISCUSSION The electrophoretic separation of nondenatured total RNA from D. dendriticum in a 1-5% agarose gel revealed the lrRNA to be 28.OS and the small subunit ribosomal RNA (srRNA) to be 19.5S (Fig. 1 lane c). The size determination was based on the electrophoretic mobility of the E. coli 16.OS and 23.OS rRNA and that of the rat 18.0S and 28.0S rRNA (Fig. 1 lanes d and e, respectively). The corresponding sizes of rRNA from Echinococcus granulosus determined by ultracentrifugation are 29.4S and 19.6S (AGOSIN et al., 1971). When total RNA from D. dendriticum was electrophoretically separated following complete denaturation (pretreatment at 65°C) two close bands were observed at the positions of 19.5S and 20.7S (Fig. 1 lane a). Following partial denaturation (pretreatment at 37°C) the 28.OS rRNA was partly found in the 19.5S-20.7S region (Fig. 1 lane b). The electrophoretic pattern exhibiting successive disappearance of the 28.OS rRNA band in response to temperature increase suggests that there is a hidden break in the lrRNA of D. dendriticum. Previously, the presence of hidden breaks in some other parasitic flatworms, including Fasciola hepatica (ISHIKAWA, 1977a), Schistosoma mansoni (TENNISWOOD & SIMPSON, 1982; VAN KEULEN et al., 1991) and Echinococcus granulosus (MCMANUS et al., 1985), has been reported.
Hidden break in the 28.OS rRNA from D. dendriticum
195
In order to identify the a-subfragment (5'-end) and the |3-subfragment (3'-end) formed upon denaturation of the IrRNA from D. dendriticum, a- and |3-oligo probes were used for hybridization. The hybridization with the a-oligo, specific for the 5'-end of the IrRNA, showed that this probe specifically binds to nondenatured 28.OS rRNA (Fig. 2A lane a). The fact that no hybridization in this lane occurs to 19.5S rRNA excludes crosshybridization to the srRNA. However, upon heat treatment (65°C) of the sample the a-oligo binds only to 19.5S rRNA (Fig. 2A lane c). We thus conclude that this 19.5S rRNA is the a-subfragment. Hybridization with the (3-oligo, specific for the 3'-end of the IrRNA, also occurs specifically to nondenatured 28.OS rRNA (Fig. 2B lane a) but upon denaturation only to the 20.7S rRNA (Fig. 2B lane c). We therefore deduce this to be the (3-subfragment.
28.0S-*
23.0S-* 20.7S-* 19.5S-*
a
b
c
d
e
FIG. 1. Electrophoretic separation of total RNA from D. dendriticum in a 1-5% agarose gel stained with ethidium bromide. Samples of 3 ng of total RNA were treated for 15 min at a)65°C (denatured), b) 37°C (partially denatured), and c) 25°C (nondenatured). As size markers nondenatured RNA from E. coli d) and from Ratlus e) were run in parallel.
B Probe for 5'-end
a 25°C
b 37°C
c 65°C
Probe for 3'-end
a 25°C
b c 37°C 65°C
FIG. 2. Autoradiograms of Northern blot membranes hybridized with oligonucleotide probes specific for the 5'-end (A) and 3'-end (B) of the 28.OS rRNA, respectively. The lanes a. b, and c contain 3 u.g of total RNA from D. dendrilicum prepared as described in the legend of Fig. 1.
196
K. A. KARLSTEDT el al.
Furthermore, pretreatment at 37°C results in a partial disruption of the lrRNA, which leads to the binding of both oligos to the 28.OS rRNA, and to the binding of each oligo to the 19.5S and 20.7S rRNA, respectively (Figs. 2A lane b and 2B lane b). Our results show that a 19.5S a-subfragment and a 20.7S (3-subfragment are released due to the dissociation of hydrogen bonds within the 28.OS rRNA of D. dendriticum upon denaturation, and the smaller subfragment thus displays the same electrophoretic mobility as the srRNA. Previous size determinations of the subfragments formed owing to a centrally located hidden break in the lrRNA of several protostomes revealed a trend that the size of the larger subfragment equals the size of the srRNA. (CAMMARANO et al., 1975). CAMMARANO et al. (1975) found, however, as an exception, that the smaller subfragment of Artemia salina is of the same size as the srRNA which is consistent with our present results. Furthermore, it has been reported that the lrRNA of S. mansoni (TENNISWOOD & SIMPSON, 1982), Planarian sp. (CAMMARANO et al., 1975), and E. granulosus (MCMANUS et al. 1985) can be disrupted into two subfragments of equal size. Recently, MERTZ et al. (1991) and VAN KEULEN et al. (1991) have characterized the central region of the lrRNA of different schistosomes, and they have localized the hidden break to a gap region consisting of 54-67 bases. The central domain of the lrRNA is of special interest for our knowledge about the rRNA evolution as these domains contain variable regions, the expansion segments, which have increased in size during evolution compared to the E. coli lrRNA (see RAUE et al., 1988). The origin of the expansion segments is, however, not known. It has been suggested that they are insertions into the ancestral rRNA, which do not disturb the ribosomal function and are therefore tolerated (WARE et al., 1985). An alternative explanation is based on the assumption that the original rRNA was discontinuous, i.e. the functional domains were separated by nonfunctional sequences (CLARK, 1987). Recently, DE LANVERSIN & JACQ (1989) have demonstrated, by comparing the sequences of the central domain of lrRNA from a variety of organisms, that the lrRNA from prokaryotes and eukaryotes show a common structural core. As the ancestral flatworms are usually regarded as the ancestors of all metazoans (FIELD et al., 1988) it would be, from an evolutionary point of view, of particular interest to further analyse the central domain of the lrRNA of flatworms.
ACKNOWLEDGEMENTS The authors would like to thank The Finnish Academy of Science as well as The Finnish Society of Sciences and Letters for financial support.
REFERENCES AGOSIN. M., REPETTO, Y. & DICOWSKY, L. (1971) Ribonucleic acid ol' Echinococcus granutosus protoscolices. Experimental Parasilology, 30, 233-243. APPLEBAUM, S. W., EBSTEIN. R. P. & WYATT. G. R. (1966) Dissociation of ribosomal ribonucleic acid from silkmoth pupae by heat and dimethylsulfoxide; evidence for specific cleavage points. Journal of Molecular Biology, 21, 29-41. CAMMARANO. P., PONS. S. & LONDEI. P. (1975) Discontinuity of the large ribosomal subunit RNA and rRNA molecular weights in eukaryote evolution. Ada Biologica el Medica Germanica, 34, 1123-1135. CHAN, Y . - L , OLVERA, J. & WOOL. I. G. (1983) The structure of rat 2SS ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene. Nucleic Aciils Research. 11, 7819-7831. CLARK, C. G. (1987) On the evolution of ribosomal RNA. Journal oj Molecular Evolution, 25, 343350.
Hidden break in the 28.0S rRNA from D. dendriticum
197
DE LANVERSIN, G. & JACQ, B. (1989) Sequence and secondary structure of the central domain of Drosophila 26S rRNA: A universal model for the central domain of the large rRNA containing the region in which the central break may happen. Journal of Molecular Evolution, 28, 403417. DEVEREUX, J., HAEBERLI, P. & SMITHIES, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research, 12, 387-395. ENGBERG, J., NIELSEN, H., LENAERS, G., MURAYAMA. O., FUJITANI. H. & HIGASHINAKAGAWA, T. (1990) Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments. Journal of Molecular Evolution, 30, 514-521. FIELD, K. G., OLSEN, G. J., LANE, .D. J., GIOVANNONI, S. J., GHISELIN, M. T., RAFF, E. C , PACE, N. R. & RAFF, R. A. (1988) Molecular phylogeny of the animal kingdom. Science, 239, 748-753. FUJIWARA, H. & ISHIKAWA, H. (1986) Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: implication based on structural studies. Nucleic Acids Research, 14, 6393-6401. ISHIKAWA, H. (1977a) Comparative studies on the thermal stability of animal ribosomal RNA's-IV. Thermal stability and molecular integrity of ribosomal RNA's from several Protostomes (rotifers, round-worms, liver-flukes, and brine-shrimps). Comparative Biochemistry and Physiology, 56B, 229-234. ISHIKAWA, H. (1977b) Evolution of ribosomal RNA. Comparative Biochemistry and Physiology, 58B, 1-7. KHANDJIAN, E. W. & MERIC, C. (1986) A procedure for Northern blot analysis of native RNA. Analytical Biochemistry, 159, 227-232. McMANUS, D. P., KNIGHT, M. & SIMPSON, A. J. G. (1985) Isolation and characterisation of nucleic acids from the hydatid organisms, Echinococcus spp. (Cestoda). Molecular and Biochemical Parasitology, 16, 251-266. MERTZ, P. M., BOBEK. L. A., REKOSH, D. M. & LoVERDE, P. T. (1991) Schistosoma haematobium and S. japonicum: Analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences. Experimental Parasitology, 73, 137-149. RAUE, H. A., KLOOTWIJK, J. & MUSTERS, W. (1988) Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Progress in Biophysics and Molecular Biology, 51, 77-129. SIMPSON, A. J . G . , DAME, J. B., LEWIS, F. A. & McCUTCHAN.T. F. (1984) The arrangement of ribosomal RNA genes in Schistosoma mansoni. Identification of polymorphic structural variants. European Journal of Biochemistry, 139, 41-45. TENNISWOOD, M. P. R. & SIMPSON, A. J. G. (1982) The extraction, characterization and in vitro translation of RNA from adult Schistosoma mansoni. Parasitologv, 84, 253-261. VAN KEULEN, H., MERTZ, P. M., LoVERDE. P. T., SHI, H. & REKOSH, D. M. (1991) Characterization of a 54-nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni. Molecular and Biochemical Parasitology, 45, 205-214. WARE, V. C , RENKAWITZ, R. & GERBI, S. A. (1985) rRNA processing: removal of only nineteen bases at the gap between 28Sot and 28Sp rRNAs in Sciara coprophila. Nucleic Acids Research, 13, 3581-3597. Accepted llth March, 1992.
A hidden break in the 28.0S rRNA from Diphyllobothrium dendriticum K. A. KARLSTEDT, G. I. L. PAATERO, J.-H. MAKELA and B.-J. WIKGREN Department of Biology, Abo Akademi University, SF-20520 Abo, Finland
ABSTRACT Nondenatured and denatured total RNA from the tapeworm Diphyllobothrium dendriticum (Cestoda) was analysed by agarose gel electrophoresis. It was found that the large subunit ribosomal RNA (IrRNA) is 28.OS and the small subunit ribosomal RNA (srRNA) is 19.5S. Following denaturation the 28.OS rRNA was disrupted into a 19.5S subfragment and a 20.7S subfragment due to the presence of a centrally located hidden break. By hybridization of Northern blot membranes with oligonucleotide probes specific for the 5'- and 3'-ends of the IrRNA respectively, we have shown that the 19.5S subfragment is from the 5'-end (the a-subfragment) and the 20.7S subfragment from the 3'-end (the p"subfragment) of the 28.0S rRNA of D. dendriticum. KEY WORDS: Diphyllobothrium dendriticum, rRNA, hidden break. Northern blot, oligonuclcotide probes, Cestoda
INTRODUCTION
The presence of a hidden break in the central domain of the large subunit ribosomal RNA (IrRNA) has been demonstrated in protozoa, in higher plant chloroplasts, and in almost all the protostomes examined, but not in deuterostomes (for review see ISHIKAWA, 1977b; RAUE et al., 1988). If the IrRNA exhibits a hidden break, denaturation of IrRNA results in dissociation of hydrogen bonds causing disruption of IrRNA into an a-subfragment (5'-end) and a p-subfragment (3'-end). A hidden break in the IrRNA was first observed by APPLEBAUM et al. (1966) in insects. Later sequence studies of the central domain of insect IrRNA have revealed that there is not only a break in a phosphodiester bond but various amounts of nucleotides are eliminated during the nuclear processing of pre-lrRNA (WARE et al., 1985; FUJIWARA & ISHIKAWA, 1986; DE LANVERSIN & JACQ, 1989). Furthermore, it has recently been shown that the introduced gaps are located in specific expansion segments of the IrRNA (DE LANVERSIN & JACQ, 1989; ENGBERG et al., 1990; MERTZ et al., 1991; VAN KEULEN et al., 1991). In the present study we show, by using oligonucleotide probes specific for the 5'-end and the 3'-end of the IrRNA respectively, that due to a central hidden break the 28.OS rRNA from Diphyllobothrium dendriticum (Cestoda) upon heat treatment disrupts into a 19.5S a-subfragment and a 20.7S (3-subfragment.
MATERIALS AND METHODS
RNA isolation Extraction of total RNA from adult D. dendriticum was carried out essentially using the LiCI-urea method as described by SIMPSON et al. (1984). Gel electrophoresis Aliquots of 3 u.g of total RNA in 30 mM sodium acetate, pH 5-5, were electrophoresed in 1-5% agarose gels in TBE buffer (89 mM Tris-HCl, pH 8-3, 89 mM
194
K. A. K.ARLSTEDT el al.
boric acid, and 2 mM EDTA) containing 0-25 fig/ml ethidium bromide. The samples were incubated for 15 min either at 65°C, 37°C or 25°C before loading. For size determination nondenatured E. coli and rat RNA were run simultaneously in the gel. Northern blot analysis Prior to Northern blot analysis the gels (0-8% agarose) were incubated in 10% formaldehyde at 65°C for 30 min (KHANDJIAN & MERIC, 1986). Following this denaturing step the RNA was transferred to nylon membranes (Amersham, Hybond N) in 20XSSC (lxSSC is 015 M NaCI and 0015 M sodium citrate), and the RNA was then fixed to the membranes by UV irradiation for 2 min. Hybridization Prehybridization and hybridization were carried out in 6xSSC, 5xDenhardt's solution ( 0 1 % each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), 0-1% SDS, 10 (ig/ml of poly A and 50 u.g/ml of sonicated and denatured salmon sperm DNA at 55°C. The membranes were hybridized with 3'-end labelled (a-32P ddATP) oligonucleotide probes, the a-oligo and the p"-oligo, each representing a consensus sequence in the 5'-end (a-subfragment) and the 3'-end ((i-subfragment) of the lrRNA, respectively. The a-oligo has sequences complementary to bases 89104 and the (3-oligo to bases 4244-4259 in the rat 28.OS rRNA sequence (CHAN et al., 1983). The sequences are as follows: a-oligo: 5'-CTA AAC CCA GCT CAC-3' P-oligo: 5'-TCA CTC GCC GTT ACT-3' The oligonucleotide sequences were deduced, and their universal specificity was tested using the UWGCG software (DEVEREUX et al., 1984) to compare known rDNA sequences in the GenBank database. Following hybridization the membranes were washed in 6xSSC at 60°C for 15 min, and the exposures were done with intensifying screens at -7()°C. RESULTS AND DISCUSSION The electrophoretic separation of nondenatured total RNA from D. dendriticum in a 1-5% agarose gel revealed the lrRNA to be 28.OS and the small subunit ribosomal RNA (srRNA) to be 19.5S (Fig. 1 lane c). The size determination was based on the electrophoretic mobility of the E. coli 16.OS and 23.OS rRNA and that of the rat 18.0S and 28.0S rRNA (Fig. 1 lanes d and e, respectively). The corresponding sizes of rRNA from Echinococcus granulosus determined by ultracentrifugation are 29.4S and 19.6S (AGOSIN et al., 1971). When total RNA from D. dendriticum was electrophoretically separated following complete denaturation (pretreatment at 65°C) two close bands were observed at the positions of 19.5S and 20.7S (Fig. 1 lane a). Following partial denaturation (pretreatment at 37°C) the 28.OS rRNA was partly found in the 19.5S-20.7S region (Fig. 1 lane b). The electrophoretic pattern exhibiting successive disappearance of the 28.OS rRNA band in response to temperature increase suggests that there is a hidden break in the lrRNA of D. dendriticum. Previously, the presence of hidden breaks in some other parasitic flatworms, including Fasciola hepatica (ISHIKAWA, 1977a), Schistosoma mansoni (TENNISWOOD & SIMPSON, 1982; VAN KEULEN et al., 1991) and Echinococcus granulosus (MCMANUS et al., 1985), has been reported.
Hidden break in the 28.OS rRNA from D. dendriticum
195
In order to identify the a-subfragment (5'-end) and the |3-subfragment (3'-end) formed upon denaturation of the IrRNA from D. dendriticum, a- and |3-oligo probes were used for hybridization. The hybridization with the a-oligo, specific for the 5'-end of the IrRNA, showed that this probe specifically binds to nondenatured 28.OS rRNA (Fig. 2A lane a). The fact that no hybridization in this lane occurs to 19.5S rRNA excludes crosshybridization to the srRNA. However, upon heat treatment (65°C) of the sample the a-oligo binds only to 19.5S rRNA (Fig. 2A lane c). We thus conclude that this 19.5S rRNA is the a-subfragment. Hybridization with the (3-oligo, specific for the 3'-end of the IrRNA, also occurs specifically to nondenatured 28.OS rRNA (Fig. 2B lane a) but upon denaturation only to the 20.7S rRNA (Fig. 2B lane c). We therefore deduce this to be the (3-subfragment.
28.0S-*
23.0S-* 20.7S-* 19.5S-*
a
b
c
d
e
FIG. 1. Electrophoretic separation of total RNA from D. dendriticum in a 1-5% agarose gel stained with ethidium bromide. Samples of 3 ng of total RNA were treated for 15 min at a)65°C (denatured), b) 37°C (partially denatured), and c) 25°C (nondenatured). As size markers nondenatured RNA from E. coli d) and from Ratlus e) were run in parallel.
B Probe for 5'-end
a 25°C
b 37°C
c 65°C
Probe for 3'-end
a 25°C
b c 37°C 65°C
FIG. 2. Autoradiograms of Northern blot membranes hybridized with oligonucleotide probes specific for the 5'-end (A) and 3'-end (B) of the 28.OS rRNA, respectively. The lanes a. b, and c contain 3 u.g of total RNA from D. dendrilicum prepared as described in the legend of Fig. 1.
196
K. A. KARLSTEDT el al.
Furthermore, pretreatment at 37°C results in a partial disruption of the lrRNA, which leads to the binding of both oligos to the 28.OS rRNA, and to the binding of each oligo to the 19.5S and 20.7S rRNA, respectively (Figs. 2A lane b and 2B lane b). Our results show that a 19.5S a-subfragment and a 20.7S (3-subfragment are released due to the dissociation of hydrogen bonds within the 28.OS rRNA of D. dendriticum upon denaturation, and the smaller subfragment thus displays the same electrophoretic mobility as the srRNA. Previous size determinations of the subfragments formed owing to a centrally located hidden break in the lrRNA of several protostomes revealed a trend that the size of the larger subfragment equals the size of the srRNA. (CAMMARANO et al., 1975). CAMMARANO et al. (1975) found, however, as an exception, that the smaller subfragment of Artemia salina is of the same size as the srRNA which is consistent with our present results. Furthermore, it has been reported that the lrRNA of S. mansoni (TENNISWOOD & SIMPSON, 1982), Planarian sp. (CAMMARANO et al., 1975), and E. granulosus (MCMANUS et al. 1985) can be disrupted into two subfragments of equal size. Recently, MERTZ et al. (1991) and VAN KEULEN et al. (1991) have characterized the central region of the lrRNA of different schistosomes, and they have localized the hidden break to a gap region consisting of 54-67 bases. The central domain of the lrRNA is of special interest for our knowledge about the rRNA evolution as these domains contain variable regions, the expansion segments, which have increased in size during evolution compared to the E. coli lrRNA (see RAUE et al., 1988). The origin of the expansion segments is, however, not known. It has been suggested that they are insertions into the ancestral rRNA, which do not disturb the ribosomal function and are therefore tolerated (WARE et al., 1985). An alternative explanation is based on the assumption that the original rRNA was discontinuous, i.e. the functional domains were separated by nonfunctional sequences (CLARK, 1987). Recently, DE LANVERSIN & JACQ (1989) have demonstrated, by comparing the sequences of the central domain of lrRNA from a variety of organisms, that the lrRNA from prokaryotes and eukaryotes show a common structural core. As the ancestral flatworms are usually regarded as the ancestors of all metazoans (FIELD et al., 1988) it would be, from an evolutionary point of view, of particular interest to further analyse the central domain of the lrRNA of flatworms.
ACKNOWLEDGEMENTS The authors would like to thank The Finnish Academy of Science as well as The Finnish Society of Sciences and Letters for financial support.
REFERENCES AGOSIN. M., REPETTO, Y. & DICOWSKY, L. (1971) Ribonucleic acid ol' Echinococcus granutosus protoscolices. Experimental Parasilology, 30, 233-243. APPLEBAUM, S. W., EBSTEIN. R. P. & WYATT. G. R. (1966) Dissociation of ribosomal ribonucleic acid from silkmoth pupae by heat and dimethylsulfoxide; evidence for specific cleavage points. Journal of Molecular Biology, 21, 29-41. CAMMARANO. P., PONS. S. & LONDEI. P. (1975) Discontinuity of the large ribosomal subunit RNA and rRNA molecular weights in eukaryote evolution. Ada Biologica el Medica Germanica, 34, 1123-1135. CHAN, Y . - L , OLVERA, J. & WOOL. I. G. (1983) The structure of rat 2SS ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene. Nucleic Aciils Research. 11, 7819-7831. CLARK, C. G. (1987) On the evolution of ribosomal RNA. Journal oj Molecular Evolution, 25, 343350.
Hidden break in the 28.0S rRNA from D. dendriticum
197
DE LANVERSIN, G. & JACQ, B. (1989) Sequence and secondary structure of the central domain of Drosophila 26S rRNA: A universal model for the central domain of the large rRNA containing the region in which the central break may happen. Journal of Molecular Evolution, 28, 403417. DEVEREUX, J., HAEBERLI, P. & SMITHIES, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research, 12, 387-395. ENGBERG, J., NIELSEN, H., LENAERS, G., MURAYAMA. O., FUJITANI. H. & HIGASHINAKAGAWA, T. (1990) Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments. Journal of Molecular Evolution, 30, 514-521. FIELD, K. G., OLSEN, G. J., LANE, .D. J., GIOVANNONI, S. J., GHISELIN, M. T., RAFF, E. C , PACE, N. R. & RAFF, R. A. (1988) Molecular phylogeny of the animal kingdom. Science, 239, 748-753. FUJIWARA, H. & ISHIKAWA, H. (1986) Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: implication based on structural studies. Nucleic Acids Research, 14, 6393-6401. ISHIKAWA, H. (1977a) Comparative studies on the thermal stability of animal ribosomal RNA's-IV. Thermal stability and molecular integrity of ribosomal RNA's from several Protostomes (rotifers, round-worms, liver-flukes, and brine-shrimps). Comparative Biochemistry and Physiology, 56B, 229-234. ISHIKAWA, H. (1977b) Evolution of ribosomal RNA. Comparative Biochemistry and Physiology, 58B, 1-7. KHANDJIAN, E. W. & MERIC, C. (1986) A procedure for Northern blot analysis of native RNA. Analytical Biochemistry, 159, 227-232. McMANUS, D. P., KNIGHT, M. & SIMPSON, A. J. G. (1985) Isolation and characterisation of nucleic acids from the hydatid organisms, Echinococcus spp. (Cestoda). Molecular and Biochemical Parasitology, 16, 251-266. MERTZ, P. M., BOBEK. L. A., REKOSH, D. M. & LoVERDE, P. T. (1991) Schistosoma haematobium and S. japonicum: Analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences. Experimental Parasitology, 73, 137-149. RAUE, H. A., KLOOTWIJK, J. & MUSTERS, W. (1988) Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Progress in Biophysics and Molecular Biology, 51, 77-129. SIMPSON, A. J . G . , DAME, J. B., LEWIS, F. A. & McCUTCHAN.T. F. (1984) The arrangement of ribosomal RNA genes in Schistosoma mansoni. Identification of polymorphic structural variants. European Journal of Biochemistry, 139, 41-45. TENNISWOOD, M. P. R. & SIMPSON, A. J. G. (1982) The extraction, characterization and in vitro translation of RNA from adult Schistosoma mansoni. Parasitologv, 84, 253-261. VAN KEULEN, H., MERTZ, P. M., LoVERDE. P. T., SHI, H. & REKOSH, D. M. (1991) Characterization of a 54-nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni. Molecular and Biochemical Parasitology, 45, 205-214. WARE, V. C , RENKAWITZ, R. & GERBI, S. A. (1985) rRNA processing: removal of only nineteen bases at the gap between 28Sot and 28Sp rRNAs in Sciara coprophila. Nucleic Acids Research, 13, 3581-3597. Accepted llth March, 1992.
