Comparison of Secondary Structure of the Ribosomal Internal Transcribed Spacer 2 of Eight Lepidopteran species from Diverse Geographical Locations

INTRODUCTION

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Lepidopteran insects are basically phytophagous in nature. The larvae of these lepidopteran insects are voracious. Hence, they have become major pests of various crops (Metcalf and Flint, 1962; Nair, 1986; David, 1991; Srivastava, 2000). Intra-specific conservation and variations have been reported using ITS2 region collected from diverse geographic locations that are used for phylogenetic studies (Van der Sande et al., 1992; Michot et al., 1984; Shinohara et al., 1999; Crabtree et al., 1995). The internal transcribed spacers (ITS) are located between the repeating array of nuclear 18S, 5.8S and 28S ribosomal RNA genes, a locus that has 100-200 copies per genome. The ITS spacers are versatile genetic markers and have been used for phylogenetic analysis, evaluation of the evolutionary process, as well as for determination of taxonomic identities (Atanas and Nazar, 1999). The lepidopteran insects selected for the present study were: Helicoverpa punctigera, Luehdorfia longicaudata, Luehdorfia chinensis, Luehdorfia puziloi puziloi, Luehdorfia puziloi yessoensis, Heliconius charithonia, Heliconius hecale and Heliconius melpomene.

Mature rRNAs are produced by the processing of a large precursor from which different transcribed spacer regions are sequentially removed through an elaborate pathway of cleavage steps (Perry, 1976). In eukaryotes, transcribed spacer regions may represent a very substantial fraction of the length of the primary transcript. Although these transcribed spacer regions are obvious candidates for important roles in the control of ribosome biogenesis, elucidation of their biological function and of the molecular mechanisms involved in their accurate excision still remain a major challenge. Recent functional analyses performed on yeast ribosomal RNA genes clearly show that the structural integrity of the transcribed spacer regions is an essential prerequisite for correct processing of mature rRNA and biogenesis of active ribosomal subunits (Michot et al., 1984; Musters et al., 1990). The derivation of reliable secondary structure models for each transcribed spacer region would undoubtedly represent a major step towards a detailed understanding of their biological role. The comparative sequence analysis provides the most powerful tool for identifying the biologically relevant folding pattern of an RNA molecule, i.e., its native structure within the cellular context (Michot et al., 1984). However, an essential prerequisite for its effective utilization is the availability of a collection of sequences exhibiting a substantial number of nucleotide differences while remaining similar enough for unequivocal sequence alignments. Due to the high rate of sequence variation of transcribed spacers, this may exhibit dramatic size variation and extensive sequence divergence even among moderately distant species (Michot et al., 1983; Furlong and Maden, 1983). Nevertheless, the presence of phylogenetically conserved secondary structure elements in the 5’ externally transcribed spacer was recently revealed by the comparative analysis of a limited set of vertebrate sequences (Michot and Bachellerie, 1991).

The present study is focused on internal transcribed spacer, ITS2, which interrupts the eukaryotic large subunit rRNA molecule and has no prokaryotic equivalent (Michot et al., 1984). The sequence and secondary structures of ITS2 of selected geographically variant Lepidopteran insect species were comprehensively investigated. Such case studies are relevant in broader phylogenetic contexts and for analyzing the function in ribosome biogenesis. Since the secondary structures of ITS region are more conserved than the nucleotide sequences their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus the structure models developed in this study can be used for future phylogenetic analysis.