Structural Characterization of the Internal Transcribed Spacer 2 (ITS2) of the Ribosomal DNA (rDNA) Cluster in Calyptratae (Diptera: Schizophora) and its Implications for Molecular Phylogenetic Analyses

Abstract

The internal transcribed spacer 2 (ITS2) of the eukaryotic ribosomal DNA (rDNA) cluster plays an essential role in processing of the ribosomal RNA, which is primarily accomplished by the secondary structures acquired by the molecule after transcription. Two possible structural conformation models have been proposed for the ITS2 region, the "ring model" and the "hairpin model," and the former has been widely used in many molecular phylogenetic analyses incorporating structural information available to date. To evaluate the validity of this model, in vitro transcribed ITS2 molecules from species representing the three superfamilies of the Calyptratae clade (Diptera: Schizophora), namely Cochliomyia hominivorax, Musca domestica, and Glossina morsitans, were submitted to enzymatic digestion with single- and double-stranded specific nucleases (RNases I, A, T1, and V1). The resulting fragments were analyzed by capillary electrophoresis and digestion sites were mapped in the secondary structure models which were obtained by in silico prediction with further refinement by homology comparisons. The pattern of RNA fragments generated by these RNases show a high degree of correlation to most of the predicted helix-loop regions and structural motifs. Discrepancies to the models can be explained by alternative structural conformation dynamics (in M. domestica and G. morsitans) and by higher-order factors (such as tertiary interactions) that may stabilize thermodynamically unfavored structures (in C. hominivorax).

The first phylogenetic study of Mesembrinellidae (Diptera: Oestroidea) based on molecular data: clades and congruence with morphological characters

“…For the region ITS2 , the nomenclature adopted is based on the works of Young and Coleman () and Marinho et al. (, , ).…”

“…For the ITS2 region, we used a combined approach of de novo and homology‐based modelling, comprising: (1) in silico prediction based on thermodynamic parameters with the mfold software (Zuker, ); (2) comparison with previously published structures for D. melanogaster (Young and Coleman, ) and some species of the Calliphoridae and Mesembrinellidae families (Marinho et al., , , ); and (3) comparison among the structures predicted for all species and the establishment of a common folding pattern.…”

“…This nomenclature differs from the one used in a previous analysis (Marinho et al, 2012), which was based on an older publication by Buckley et al (2000). For the region ITS2, the nomenclature adopted is based on the works of Young and Coleman (2004) and Marinho et al (2011Marinho et al ( , 2012Marinho et al ( , 2013.…”

Molecular phylogeny of the diverse parasitoid wasp genus Ophion Fabricius (Hymenoptera: Ichneumonidae: Ophioninae)

“…As the ITS2 sequences were folded in silico , further studies are needed to confirm that these structures are accurate (Eddy, ; Marinho et al , ). Overall, the ITS2 structures were consistent with the conserved patterns observed by Coleman (), loosely corresponding to the four‐domain model predicted for ITS2 (Schultz et al , ).…”

Research Article ITS2 as a molecular marker for the identification of Diatraea saccharalis and D. flavipennella and possible infection with Cotesia spp.

“…ITS 2 plays an essential role in the maturation of the pre-ribosomal RNA as its secondary structure contained the cleavage sites for the pre-rRNA processing (Marinho et al, 2013). Previous studies showed that ITS 2 is a good molecular marker to phylogenetic inferences among families, subfamilies, and especially as a molecular marker in lower taxonomic groups as genera and species.…”