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Secundary structure of the large ribosomal subunit RNA of the moss Funaria hygrometrica
••••••AL.F.
Short Communication
PIIM PII,.~.I.., © 1997 by Gustav Fischer Verlag, Jena
Secundary Structure of the Large Ribosomal Subunit RNA of the Moss Funaria hygrometrica 1 INGRID CAPESIUS ,
* and YVES VAN DE PEER2
1
Botanisches Institut der Universitiit Heidelberg, 1m Neuenheimer Feld 360, D-69120 Heidelberg, Germany
2
Departement Biochemie, Universiteit Antwerpen (VIA), Universiteitsplein 1, B-261O Antwerpen, Belgium
Received November 22, 1996· Accepted December 10, 1996
Summary
In this study, the complete nucleotide sequence of the large ribosomal subunit RNA of the bryophyte Funaria hygrometrica was determined. The RNA sequence, which is the first one reported for bryophytes, was used to infer a secondary structure model. It delivers the base for further evolutionary studies in this group.
Key words: Large ribosomal subunit (ISU) RNA, Funaria hygrometrica, moss, secondary structure, bryo-
phyte. Introduction
The small ribosomal subunit (SSU) RNA gene is probably the molecule most frequendy used at present for the inference of evolutionary relationships among species, and its merits as a tool in molecular evolution have been discussed frequendy (Woese, 1987; Sogin, 1989; Doolitde and Brown, 1994). The main advantages of the SSU rRNAs are undoubtedly their structural and functional constancy and universal occurrence. Furthermore, SSU rRNA genes show an alternation of conserved and variable areas (Van de Peer et al., 1996), which makes them suitable for the study of a wide range of evolutionary divergences. The large ribosomal subunit (LSU) RNA shares these advantages with the SSU rRNA and has the added bonus of a longer chain-length. This implies a higher information content and as a result the LSU rRNA might be more reliable for the inference of phylogenies (De Rijk et al., 1995). At the moment, about 50 complete SSU rRNA gene sequences of bryophytes have been determined (Capesius, 1995; Kranz et al., 1995; Hedderson et al., 1995; Bopp and Capesius, 1996). However, until now, no complete sequence of the LSU rRNA was available from this group. In this * The nucleotide sequences reported in this paper have been submitted to the GenBanklAMBL Data Bank with accession numbers X99331 for the 25S rRNA and X74114 for 5.8S rRNA. j. Plant PhysioL WJL 151. pp. 239-241 (1997)
study, we report the secondary structure model of the first complete LSU rRNA sequence from the moss Funaria hygrometrica.
Materials and Methods
Plant material Funaria hygrometrica was grown on sterile solid nutrient medium at 20·C with 16 h light according to Bopp and Knoop (1984) in a Sanyo cooled incubator. Isolation ofribosomal DNA and sequencing Total genomic DNA was isolated by the method of Murray and Thompson (1980). The ribosomal DNA was separated from genomic DNA by centrifugation in a Hoechst 33258-CsCI density gradient. The band containing ribosomal-DNA was digested with EcoRI and the fragments giving hybridization signals to the 25S rRNA from Sinapis alba (Capesius, 1997) were ligated in pUC 18 vector and used to transform DH-5 cells. The specific 25S primer and the commercial universal primers were used together with the Sequenase 2.0 kit (USB) to determine the contiguous sequence of both strands. Sequencing was performed by the dideoxy method of Sanger et al. (1977) with a 35S-dATP. Sequences were determined manually from autoradiograms.
240
INGRID CAPES IUS
and YVES VAN
DE PEER
Fig. 1: Secondary structure model of the 5.8S and the 25S (LSU) rRNA genes of the bryophyte Funaria hygromarica. Helix numbering is as in De Rijk et al., 1996. The position of the inuon is indicated by an i.
LSU rRNA structure of Funaria Results and Discussion
The LSU rRNA sequence of Funaria hygrometrica was included in a large sequence alignment maintained in the research group of Dr. De Wachter (De Rijk et al., 1996). The LSU rRNA alignment comprises about 50 complete or nearly complete nuclear eukaryotic sequences, 9 of which are from green plants. The sequence alignment was carried out with the DCSE program (De Rijk and De Wachter, 1993) on the basis of similarity in primary and secondary structures. The complete LSU rRNA from Funaria hygrometrica is 3581 bases long. This is longer than the hitherto reported complete LSU rRNA sequences from other plants such as Oryza sativa (Takaiwa et al., 1985), Lycopersicon esculentum (Kiss et al., 1988), Arabidopsis thaliana (Unfried and Gruendler, 1990), and Sinapis alba (Capesius, 1991). The LSU rONA sequence of Funaria hygrometrica contains an intron, and this is the first report of an intervening sequence in an LSU rONA of a land plant. In the alga Chlorella eUipsoidea an intron is also found (Aimi et al., 1994). An intervening sequence is also present at the same position in the partly determined gene of moss physcomitrella patens (Capesius, 1997). The secondary structure of the 25S rRNA sequence of Funaria hygrometrica is given in Fig. 1. The intron sequence is situated between positions 11 and 179 (not included in Fig. 1), and has a length of 169 nucleotides. The similarity of the LSU of Funaria hygrometrica to Saccharomyces cerevisiae is 79%, to Sinapis alba or Oryza sativa 85.2%-85.6%. Previous studies showed that the 18S rRNA is not very well suited to resolve all branching patterns within the different orders and families of bryophytes (Bopp and Capesius, 1996). In the Jungermanniopsida, and the pleurocarpous mosses of the Hypnales, the gene lengths (1810 and 1819 bp resp.) as well as the sequences are similar. Therefore, it is very difficult to resolve the exact branching order within both groups. So far, only partial LSU rRNA sequences, and from single strand analyses, have been published from bryophytes (Waters et al., 1992). We expect that if more LSU rRNA sequences of mosses become available in the future, a phylogeny on the basis of the LSU will help to resolve these and other problems concerning the phylogeny of bryophytes.
241
Acknowledgements
The work was supponOO by a grant of the Deutsche Forschungsgemeinschaft Ca 5616-2. We thank Sabine Hdm for excellent technical assistance, Peter De Rijk for aligning the sequence, and Sabine Chapelle for drawing the secondary structure. References
AIMI, T., T. 'YAMADA, M. YAMAsHITA, and Y. MUROOKA: Gene 145, 139-144 (1994). Bopp, M. and I. WESIUS: Bot. Acta 109,368-372 (1996). Bopp, M. and B. KNoop: In: VASIL, I. (00.): Cell culture and somatic cell genetics of plants, pp. 96-106. Academic Press, New York, London (1984). CAPESIUS, I.: Plant Mol. BioI. 16, 1093-1094 (1991). - ]. Plant Physiol. 146, 59-63 (1995). - Plant Mol. BioI. 33, 559-564 (1997). DE RI]K, P. and R. DE WACHTER: Comput. Applic. Biosci. 9,735740 (1993). DE RI]K, P., Y. VAN DE PEER, I. VAN DE BROECK, and R. DE WACHTER:]. Mol. Evol. 41,366-375 (1995). DE RI]K, P., Y. VAN DE PEER, and R. DE WACHTER: Nucleic Acids Res. 24, 92-97 (1996). DOOUTTLE, w. E and]. R. BROWN: Proc. Nat!. Acad. Sci. USA 91, 6721-6728 (1994). HEDDERSON, T. A., R. L. CHAPMAN, and W. L. ROOTES: Plant. Syst. Evol. 200,213-224 (1995). KIss, T., M. KIss, and E SOLYMOSY: Nucl. Acids Res. 17, 796 (1988). KR.ANz, H. D., D. MIKS, M.-L. SIEGLER, I. CAPESIUS, CH. W. SENSEN, and V. A. R. Huss: J. Mol. Evol. 41,74-84 (1995). MURRAY, M. G. and W. E THOMPSON: Nucleic Acids Res. 8/19, 4321-4325 (1980). SANGER, E, S. NICKLEN, and A. R. COULSON: Proc. Nat!. Acad. Sci. USA 74, 5463-5467 (1997). SOGIN, M. L.: Amer. Zool. 29, 487-499 (1989). TAKAIWA, E, K OONO, Y. bDA, and M. SUGIURA: Gene 37, 255259 (1985). UNFRIED, I. and P. GRUENDLER: Nucleic Acids. Res. 18, 4011 (1990). VAN DE PEER, Y., S. CHAPELLE, and R. DE WACHTER: Nucleic Acids Res. 24, 3381-3391 (1996). WOESE, C. R.: Microbiol. Rev. 51,221-271 (1987). WATERS, D. A., M. A. BUCHHEIM, R. A. DEWEY, and R. L. CHAPMAN: Amer. ]. Bot. 79 (4), 459-466 (1992).
Short Communication
PIIM PII,.~.I.., © 1997 by Gustav Fischer Verlag, Jena
Secundary Structure of the Large Ribosomal Subunit RNA of the Moss Funaria hygrometrica 1 INGRID CAPESIUS ,
* and YVES VAN DE PEER2
1
Botanisches Institut der Universitiit Heidelberg, 1m Neuenheimer Feld 360, D-69120 Heidelberg, Germany
2
Departement Biochemie, Universiteit Antwerpen (VIA), Universiteitsplein 1, B-261O Antwerpen, Belgium
Received November 22, 1996· Accepted December 10, 1996
Summary
In this study, the complete nucleotide sequence of the large ribosomal subunit RNA of the bryophyte Funaria hygrometrica was determined. The RNA sequence, which is the first one reported for bryophytes, was used to infer a secondary structure model. It delivers the base for further evolutionary studies in this group.
Key words: Large ribosomal subunit (ISU) RNA, Funaria hygrometrica, moss, secondary structure, bryo-
phyte. Introduction
The small ribosomal subunit (SSU) RNA gene is probably the molecule most frequendy used at present for the inference of evolutionary relationships among species, and its merits as a tool in molecular evolution have been discussed frequendy (Woese, 1987; Sogin, 1989; Doolitde and Brown, 1994). The main advantages of the SSU rRNAs are undoubtedly their structural and functional constancy and universal occurrence. Furthermore, SSU rRNA genes show an alternation of conserved and variable areas (Van de Peer et al., 1996), which makes them suitable for the study of a wide range of evolutionary divergences. The large ribosomal subunit (LSU) RNA shares these advantages with the SSU rRNA and has the added bonus of a longer chain-length. This implies a higher information content and as a result the LSU rRNA might be more reliable for the inference of phylogenies (De Rijk et al., 1995). At the moment, about 50 complete SSU rRNA gene sequences of bryophytes have been determined (Capesius, 1995; Kranz et al., 1995; Hedderson et al., 1995; Bopp and Capesius, 1996). However, until now, no complete sequence of the LSU rRNA was available from this group. In this * The nucleotide sequences reported in this paper have been submitted to the GenBanklAMBL Data Bank with accession numbers X99331 for the 25S rRNA and X74114 for 5.8S rRNA. j. Plant PhysioL WJL 151. pp. 239-241 (1997)
study, we report the secondary structure model of the first complete LSU rRNA sequence from the moss Funaria hygrometrica.
Materials and Methods
Plant material Funaria hygrometrica was grown on sterile solid nutrient medium at 20·C with 16 h light according to Bopp and Knoop (1984) in a Sanyo cooled incubator. Isolation ofribosomal DNA and sequencing Total genomic DNA was isolated by the method of Murray and Thompson (1980). The ribosomal DNA was separated from genomic DNA by centrifugation in a Hoechst 33258-CsCI density gradient. The band containing ribosomal-DNA was digested with EcoRI and the fragments giving hybridization signals to the 25S rRNA from Sinapis alba (Capesius, 1997) were ligated in pUC 18 vector and used to transform DH-5 cells. The specific 25S primer and the commercial universal primers were used together with the Sequenase 2.0 kit (USB) to determine the contiguous sequence of both strands. Sequencing was performed by the dideoxy method of Sanger et al. (1977) with a 35S-dATP. Sequences were determined manually from autoradiograms.
240
INGRID CAPES IUS
and YVES VAN
DE PEER
Fig. 1: Secondary structure model of the 5.8S and the 25S (LSU) rRNA genes of the bryophyte Funaria hygromarica. Helix numbering is as in De Rijk et al., 1996. The position of the inuon is indicated by an i.
LSU rRNA structure of Funaria Results and Discussion
The LSU rRNA sequence of Funaria hygrometrica was included in a large sequence alignment maintained in the research group of Dr. De Wachter (De Rijk et al., 1996). The LSU rRNA alignment comprises about 50 complete or nearly complete nuclear eukaryotic sequences, 9 of which are from green plants. The sequence alignment was carried out with the DCSE program (De Rijk and De Wachter, 1993) on the basis of similarity in primary and secondary structures. The complete LSU rRNA from Funaria hygrometrica is 3581 bases long. This is longer than the hitherto reported complete LSU rRNA sequences from other plants such as Oryza sativa (Takaiwa et al., 1985), Lycopersicon esculentum (Kiss et al., 1988), Arabidopsis thaliana (Unfried and Gruendler, 1990), and Sinapis alba (Capesius, 1991). The LSU rONA sequence of Funaria hygrometrica contains an intron, and this is the first report of an intervening sequence in an LSU rONA of a land plant. In the alga Chlorella eUipsoidea an intron is also found (Aimi et al., 1994). An intervening sequence is also present at the same position in the partly determined gene of moss physcomitrella patens (Capesius, 1997). The secondary structure of the 25S rRNA sequence of Funaria hygrometrica is given in Fig. 1. The intron sequence is situated between positions 11 and 179 (not included in Fig. 1), and has a length of 169 nucleotides. The similarity of the LSU of Funaria hygrometrica to Saccharomyces cerevisiae is 79%, to Sinapis alba or Oryza sativa 85.2%-85.6%. Previous studies showed that the 18S rRNA is not very well suited to resolve all branching patterns within the different orders and families of bryophytes (Bopp and Capesius, 1996). In the Jungermanniopsida, and the pleurocarpous mosses of the Hypnales, the gene lengths (1810 and 1819 bp resp.) as well as the sequences are similar. Therefore, it is very difficult to resolve the exact branching order within both groups. So far, only partial LSU rRNA sequences, and from single strand analyses, have been published from bryophytes (Waters et al., 1992). We expect that if more LSU rRNA sequences of mosses become available in the future, a phylogeny on the basis of the LSU will help to resolve these and other problems concerning the phylogeny of bryophytes.
241
Acknowledgements
The work was supponOO by a grant of the Deutsche Forschungsgemeinschaft Ca 5616-2. We thank Sabine Hdm for excellent technical assistance, Peter De Rijk for aligning the sequence, and Sabine Chapelle for drawing the secondary structure. References
AIMI, T., T. 'YAMADA, M. YAMAsHITA, and Y. MUROOKA: Gene 145, 139-144 (1994). Bopp, M. and I. WESIUS: Bot. Acta 109,368-372 (1996). Bopp, M. and B. KNoop: In: VASIL, I. (00.): Cell culture and somatic cell genetics of plants, pp. 96-106. Academic Press, New York, London (1984). CAPESIUS, I.: Plant Mol. BioI. 16, 1093-1094 (1991). - ]. Plant Physiol. 146, 59-63 (1995). - Plant Mol. BioI. 33, 559-564 (1997). DE RI]K, P. and R. DE WACHTER: Comput. Applic. Biosci. 9,735740 (1993). DE RI]K, P., Y. VAN DE PEER, I. VAN DE BROECK, and R. DE WACHTER:]. Mol. Evol. 41,366-375 (1995). DE RI]K, P., Y. VAN DE PEER, and R. DE WACHTER: Nucleic Acids Res. 24, 92-97 (1996). DOOUTTLE, w. E and]. R. BROWN: Proc. Nat!. Acad. Sci. USA 91, 6721-6728 (1994). HEDDERSON, T. A., R. L. CHAPMAN, and W. L. ROOTES: Plant. Syst. Evol. 200,213-224 (1995). KIss, T., M. KIss, and E SOLYMOSY: Nucl. Acids Res. 17, 796 (1988). KR.ANz, H. D., D. MIKS, M.-L. SIEGLER, I. CAPESIUS, CH. W. SENSEN, and V. A. R. Huss: J. Mol. Evol. 41,74-84 (1995). MURRAY, M. G. and W. E THOMPSON: Nucleic Acids Res. 8/19, 4321-4325 (1980). SANGER, E, S. NICKLEN, and A. R. COULSON: Proc. Nat!. Acad. Sci. USA 74, 5463-5467 (1997). SOGIN, M. L.: Amer. Zool. 29, 487-499 (1989). TAKAIWA, E, K OONO, Y. bDA, and M. SUGIURA: Gene 37, 255259 (1985). UNFRIED, I. and P. GRUENDLER: Nucleic Acids. Res. 18, 4011 (1990). VAN DE PEER, Y., S. CHAPELLE, and R. DE WACHTER: Nucleic Acids Res. 24, 3381-3391 (1996). WOESE, C. R.: Microbiol. Rev. 51,221-271 (1987). WATERS, D. A., M. A. BUCHHEIM, R. A. DEWEY, and R. L. CHAPMAN: Amer. ]. Bot. 79 (4), 459-466 (1992).