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Abstract Aided by a host of bioinformatics tools, primary and secondary structural analyses of the internal transcribed spacer 2 (ITS2) from the eukaryotic ribosomal RNA repeat have a long and enviable record of service to diversity studies of fungi, plants and protists. Automation of annotation, secondary structure estimation and sequence alignment have become routine for the vast majority of ITS2 sequences. Challenges to the bioinformatics pipeline for ITS2 analysis generally arise in cases where the sequence length lies well outside the norm. These sequences generally defy protocols for annotation and secondary structure prediction. The long ITS2 sequences (ca. 600 nucleotides) from the green alga, Jenufa , offered an opportunity to explore this problem. Custom BLAST parameters revealed the presence of 4-helix structures (200–250 nucleotides) embedded in the 5′ portion of several long ITS2 sequences of Jenufa. Of special note is the ITS2 sequence of J. lobulosa where a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence. Phylogenetic analysis of these typically-sized sequences resolved Golenkinia longispicula as the sister to Jenufa. Our observations indicate that other long ITS2 sequences should be examined for evidence of expansion or duplication. In addition, if the embedded ITS2 sequences are functional, then ribogenesis is almost certainly more diverse than is already apparent from studies of humans and yeast. Highlights • Custom BLAST parameters revealed the presence of 4-helix structures embedded in the long ITS2 sequences of Jenufa • For J. lobulosa a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence • ITS2 sequence-structure phylogenetics affirmed an alliance between Jenufa and Golenkinia (Chlorophyta, Chlorophyceae) [ABSTRACT FROM AUTHOR]

Abstract: Studies of alpha-2-macroglobulin (α2M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition – through physical entrapment of the pathogen-derived protease – makes α2M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of α2M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of α2M and α2M-like isoforms exist and (2) at least some codons in the functional domains of α2M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of α2M. The presence of two, non-monophyletic α2M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of α2M is consistent with similar observations of other immune system components. [Copyright &y& Elsevier]