Comparison Of Secondary Structure Of The Ribosomal Internal Transcribed Spacer 2 (ITS2) Of Eight Lepidopteran Species From Diverse Geographical Locations
M Kuracha, B Rayavarapu, S Kumar, P Rao
diverse geographical locations, ribosomal internal transcribed spacer 2 its2, secondary structure
M Kuracha, B Rayavarapu, S Kumar, P Rao. Comparison Of Secondary Structure Of The Ribosomal Internal Transcribed Spacer 2 (ITS2) Of Eight Lepidopteran Species From Diverse Geographical Locations. The Internet Journal of Genomics and Proteomics. 2005 Volume 2 Number 1.
A comparative study of common core secondary structure in the ribosomal internal transcriber 2 (ITS2) of 8 Lepidopteron species selected from different geographical locations was carried out. Among the selected insects some are serious pests of agricultural crops. Ex:
Lepidopteran insects are basically phytophagous in nature. The larvae of these lepidopteran insects are voracious. Hence, they have become major pests of various crops [1,2,3,4]. Intra-specific conservation and variations have been reported using ITS2 region collected from diverse geographic locations that are used for phylogenetic studies [5, 6, 7, 8]. 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 . The lepidopteran insects selected for the present study were:
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 . 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
The present study is focused on internal transcribed spacer, ITS2, which interrupts the eukaryotic large subunit rRNA molecule and has no prokaryotic equivalent . The sequence and secondary structures of ITS2 of selected geographically variant
Materials and Methods
ITS 2 sequences of eight
Multiple sequence alignments were performed using CLUSTALW with a gap opening penalty of 15 and gap extension penalty of 6.66.
Secondary structure prediction
The RNA secondary structures for ITS2 were predicted using RNADRAW . RNADRAW predicts RNA structures by identifying suboptimal structures using the free energy optimization methodology at a default temperature of 370C. In the current study, ITS2 and 5.8S regions (the first 170 nucleotides) were used for RNA structure prediction. The minimum energy structure prediction algorithm in RNADRAW was ported from the RNAFOLD program included in the Vienna RNA package . The dynamic programming algorithm employed in RNADRAW was based on the work of Zuker and Stiegler  and uses energy parameters taken from Freier  and Jaeger .
The phylogenetic Genebee service was used for phylogenetic tree construction .
The length of ITS2 elements of eight selected lepidopteran species ranged in size between 644 and 699 bp. eight dispersed but unambiguously conserved sequence segments encompassing about a third of the ITS2 length have been identified. They were interspersed with variable regions and gaps where size variations accumulate. The characteristics of the sequences for each species are shown in Table 1. The length variations were observed with maximum length being 699 bp and minimum of 644 bp for
Secondary structural features of ITS2 regions were given in the Table: 1. The secondary structures of the mentioned
The selected 8
However accumulated substitutions in the ITS sequence leading to length variation also had a profound effect on the conservedness among the structures. The length variation observed was may be due to insertions effected by many factors including genetic drift, the relative number and size of repeats, rates of unequal crossover, gene conversion, immigration and the number of the loci . But still high level of sequence conservation was found between some species like
These results suggest that the differences and conserved ness observed between ITS-2 of different species are not “neutral” and are not simple accumulated random nucleotide changes, but bear a significant functional load. In the previous study of three related mosquito genera (
The present study shows two contrasting aspects of ITS2 regions i.e. the general trend of variability among the species as well as the conservedness between few species. Surprisingly, the species displaying the conservedness belong to different geographical locations with diverse climatic and ecological conditions. Our study implies that the ITS2 regions though have less selective pressure than the ribosomal regions but still evolve slower than the intergenic spacers, indicating that some selective pressure do exists on them, probably from the constraint to maintain the RNA secondary structure required for post-transcriptional processing and are more species specific than geographically influenced. Several common structural folds were shared among the selected lepidopteran insects for maintaining functional equivalents. Identifying the homologous regions and reconstructing their evolution increases the traits available for the phylogenetic analysis. Construction of an evolutionary tree using more isolates of
Prof. Poduri Nagaraja Rao, Department of Zoology, Osmania University College for Women, Koti, Hyderabad, Andhra Pradesh, India. e-mail: email@example.com