Abstract
Heat shock proteins/cognates 70 are chaperones essential for proper protein folding. This protein family comprises inducible members (Hsp70s) with expression triggered by the increased concentration of misfolded proteins due to protein-destabilizing conditions, as well as constitutively expressed cognate members (Hsc70s). Previous works on non-model amphipod species Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, both endemic to Lake Baikal in Eastern Siberia, have only revealed a constitutively expressed form, expression of which was moderately further induced by protein-destabilizing conditions. Here we describe heat-inducible hsp70s in these species. Contrary to the common approach of using sequence similarity with hsp/hsc70 of a wide spectrum of organisms and some characteristic features, such as absence of introns within genes and presence of heat shock elements in their promoter areas, the present study is based on next-generation sequencing for the studied or related species followed by differential expression analysis, quantitative PCR validation and detailed investigation of the predicted polypeptide sequences. This approach allowed us to describe a novel type of hsp70 transcripts that overexpress in response to heat shock. Moreover, we propose diagnostic sequence features of this Hsp70 type for amphipods. Phylogenetic comparisons with different types of Hsp/Hsc70s allowed us to suggest that the hsp/hsc70 gene family in Amphipoda diversified into cognate and heat-inducible paralogs independently from other crustaceans. Thus, the cognate and inducible hsp70 types in distant taxa may not be recognized by sequence similarity.
Highlights
Www.nature.com/scientificreports conditions and up-regulated in response to stress[6]
To further characterize the transcript, a gene model containing the complete open reading frame (ORF) was generated by mapping transcriptome reads to the corresponding sequence of Ommatogammarus flavus (Dybowsky, 1874) ORF, as it was the closest relative to the Eulimnogammarus genus among all species in which we found the putative heat-inducible hsp[70], according to Naumenko et al.[29]
In the heat-inducible forms of H. azteca, we found an heat shock elements (HSEs)-like sequence AATCGAGAAATTCTGGAAG located 1187 bp upstream of the ORF start, and one HSE TTTCATGAACTTTCC located in 1565 bp upstream of the start codon in the inverted non-cytoplasmic hsp[70] (JQDR01033640.1); and CTTCTGGAACATCCA located 1159 bp upstream the start codon (JQDR01033640.1)
Summary
Www.nature.com/scientificreports conditions and up-regulated in response to stress[6]. Using changes in the hsp[70] expression level as a comprehensive biomarker in environmental monitoring requires fast and reliable detection of the strong increase in the mRNA concentration (two to three orders of magnitude increase compared to the control level). This pattern was observed in many model objects, as well as in some non-model organisms and in vitro systems (Drosophila melanogaster Meigen, 1830, Saccharomyces cerevisiae Meyen ex E.C. Hansen, Xenopus laevis Daudin, 1802, Caenorhabditis elegans (Maupas, 1900), mammalian cell cultures). Without deeper consideration of this functional variability, erroneous interpretations are often possible[3]
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