Comp. Biochem. Physiol. Vol. 103B,No. 3, pp. 651-655, 1992 Printed in Great Britain

0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd

NUCLEOTIDE SEQUENCE AND PRESUMED SECONDARY STRUCTURE OF THE INTERNAL TRANSCRIBED SPACERS OF rDNA OF THE PEA APHID, ACYRTHOSIPHON PISUM O-Yu KWON and HAJIMEISHIKAWA* Zoological Institute, Faculty of Science, University of Tokyo, Japan (Tel: 81-3-3812-2111 ext. 4446; Fax: 81-3-3816-1965) (Received 3 April 1992; accepted 5 May 1992)

Abstract--1. Internal transcribed spacer (ITS) 1 and ITS 2 of rDNA of the pea aphid, Acyrthosiphonpisum consisted of 229 and 280 nucleotides, whose G + C contents were 70 and 74%, respectively. 2. Secondary structure models constructed for the ITS 1 and ITS 2 suggested that certain structural motifs have been conserved in these regions despite extensive divergence in nucleotide sequence due to species.

INTRODUCTION The formation of mature 18S rRNA, 5.8S r R N A and 28S r R N A in eukaryotes involves a series of posttranscriptional processing of a long precursor r R N A (Perry, 1976). Within the precursor rRNA, mature r R N A sequences are separated by two special segments, which are called internal transcribed spacers, ITS 1 between 18S r R N A and 5.8S rRNA, and ITS 2 between 5.8S r R N A and 28S rRNA. While the nucleotide sequence of mature rRNAs has been strongly conserved throughout t h e evolution of prokaryotes and eukaryotes, both ITS 1 and ITS 2 sequences of distant organisms show dramatic differences in size and G + C content (Michot et al., 1983). While eukaryotic ribosomal ITS 1 and ITS 2 have been relatively well characterized in yeast (Rubin, 1973; Veldman et al., 1981) and several vertebrates (Furlong and Maden, 1981; Michot et al., 1983; Subramanyam et al., 1982), no invertebrate, including insects, ribosomal ITS 1 and ITS 2 have been so far characterized. In this paper we report the complete primary sequences of ITS 1 and ITS 2 of the pea aphid rDNA. In addition, their presumed secondary structure models are constructed based on calculation of free energy. MATERIALS

AND METHODS

Isolation of the genomic DNA of pea aphids, construction of the genomic library, screening of the rDNA and mapping with restriction endonueleases were performed as described previously (Fujiwara and Ishikawa, 1986; Herrmann et al., 1987; Maniatis et al., 1982; Ogino et al., 1990). The 1.4 kb EcoRI-ECORI fragment was derived from 2 ApR8 which encodes the full length of the pea aphid rDNA (Kwon eta/., 1991), and subeloned into the same sites of the plasmid Bluescript SK +, which was named pApR815 (see Fig. 1). The plasmid subelone pApR815 contained the coding regions for the 3'-terminal of 18S *To whom correspondence should be addressed. 651

rRNA, 5.8S rRNA and the Y-terminal of 28S rRNA, as well as the internal transcribed spacers. Plasmid subclones of deletion series were constructed and sequenced by the dideoxy chain termination method (Sanger et aL, 1977) using the Sequenase version 2.0 Kit (U.S. Bioehem. Co.). The secondary structures of the ITS 1 and ITS 2 region were constructed using the DNASIS V 6.00 program (Hitachi Software Engineering Co., Japan), which found the best stable and fit secondary structures with minimum free energy for those regions. RESULTS AND DISCUSSION The sequencing strategy of the insect pApR815 is outlined in Fig. 1, and the nucleotide sequence determined shown in Fig. 2. Since the nucleotide sequences of the aphid 18S r R N A (Kwon et al., 1991) and 5.8S r R N A (Ogino et al., 1990) was known, ITS 1 was automatically identified in Fig. 2. The start point of the aphid 28S r R N A was deduced by comparison with other insect 28S rRNAs (Tautz et aL, 1988), and confirmed by the S I mapping analysis (data not shown), which facilitated to identify ITS 2. It was shown that ITS 1 and ITS 2 consist of 229 and 280 nucleotides, respectively. The two ITS sequences shared a very high G + C content, 70 and 7 4 0 for ITS 1 and ITS 2, respectively. It is generally known that in a given species the two ITS sequences are very similar to each other in both length and G + C content (Schnare et al., 1990). The G + C content of ITS sequences of higher eukaryotes is higher than that of the corresponding sequences of insects and lower eukaryotes (Fig. 3) (Schnare et al., 1990). The aphid ITS sequences were quite exceptional in that their G + C content was very high and comparable to that of the ITSs of vertebrates. While the rRNAs of aphids and vertebrates have high G + C contents, the G + C content of the ITSs of these organisms were still higher (Schnare et aL, 1990). In contrast, insects and lower eukaryotes generally share rRNAs of a low G + C content, and the G + C content of their ITSs is still lower. This

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Fig. 1. A unit structure of rDNA and sequencing strategy for the internal transcribed spacers. Horizontal arrows indicate the direction and extent of the sequences which were read. 10

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AAAGCCCATCGCCGAATCTCGCGCGCACTTTTGTGTCCGCCGTATGAGAGTTGTGGCGTACGGGCCGGCCTCGTT 1135 1145 1155 1165 1175 1185 1195 GCCGGGCGCACGTGACGGTCCAAGTCTCCCTTGAACGGGCCATGGCCCGCAGATGGTGCCAGGCCAGTAGAGGCC 1210 1220 1230 1240 1250 1260 1270 GTCACAGCGTTCCGGGATCGGACCGCGCCTTCGAGTCGGGTTGCTTGAGAGTGCAGCCCGAAGCGGGTGGTAAAC 1285 1295 1305 1315 1325 1335 1345 TCCATCTAACCGTAAATACGGCCACGAGACCGATAGTTTACAAGTACCGTGAGGGAAAGTTGAAACAATTTGAA

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Fig. 2. Nucleotide sequence of the pea aphid internal transcribed spacers. Boxed sequences represent regions coding for the 18S, 5.8S and 28S rRNA, respectively. ITS 1 extends from position 236 to 464 (between the 18S and 5.8S rRNA sequence), ITS 2 being from 625 to 904 (between 5.8S and 28S rRNA sequence).

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Nucleotide sequence of aphid rDNA may suggest that higher and lower eukaryotes are under different evolutionary pressures. In this context, it is intriguing that the aphid seems to be under pressure similar to that affecting vertebrates. Like in other ITSs so far examined, there was no common tract shared by both ITS 1 and ITS 2 of the aphid rDNA. Additionally, neither tRNA-like sequence nor open reading frame was detected in the two aphid ITS sequences. The proposed secondary structures of the ITS 1 and ITS 2 region of the pea aphid precursor rRNA are shown in Fig. 4. The model for ITS 1 contains four stem-loop structures (I-IV). The proposed stems occur at nucleotide positions 4-68 (I), 73-100 and 185-210 (II), 104-114 (III) and 115-185 (IV) (with residue 1 being the first nucleotide of the ITS 1 region). While in general these stems are very stable and rich in G/C pairs, stem I has a large loop containing 17 residues. The last part of this structure contains a U-rich pyrimidine tract. Free energy for the proposed secondary structure of the ITS 1 is - 123.3 kcal/mol. The model for ITS 2 contains six stem-loop structures (I-VI). The proposed stems occurred at nucleotide positions 3-43 and 236-276 (I), 217-234 (II), 47-77 and 187-216 (III), 78-133 (IV), 138-153 (V) and 154-186 (VI) (with residue 1 being the first nucleotide of the ITS 2 region). All of these stems, except stem V, are stable and rich in G/C pairs. Free energy for the proposed secondary structure of the ITS 2 is -207.7 kcal/mol. The models of the secondary structure for the pea aphid ITS 1 and ITS 2 are surprisingly similar to general structures (Yeh et al., 1990) that have been reported for ITSs of other organisms, even though the aphid ITSs were very different from those of otber organisms (Schnare et al., 1990) in nucleotide sequence and G + C content. Recently, Yeh and Lee (1990) reported secondary structures for ITS 1 and ITS 2 of several organisms, which also showed that certain common folding features appear to be conserved in spite of extensive sequence divergence. Whether those common helical structures play any role in the recognition mechanism of ribosomal RNA processing must await further experimentation. research was supported by Grants-in Aid for General (No. 01440004) and Developmental Research (No. 01840028)and for ScientificResearch on Priority Areas, "Symbiotic Biosphere: Ecological Interaction Network Promoting the Coexistence of Many Species" (No. 03269102) from the Ministry of Education, Science and Culture of Japan. The research was also supported by a grant from Kirin Brewery Co. Ltd. Acknowledgements--This

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