RESUMEN
Four complete mitochondrial DNA genomes for rainbow trout, Oncorhynchus mykiss, were sequenced and compared to the previously reported complete mitochondrial sequence. Analyses revealed 13 additional bp and 31 insertion/deletion (indels) sites between the four new sequences and the previously published sequence. Twenty-eight indels were single base pairs while the remaining were multiple base pair insertions, two in the ATPase6 gene, one of three bases and one of nine bases, and a two base insert in the control region. Indels in the new genomes were compared to a wide range of salmonid species and corresponded with sequences in closely related species, indicating that errors were likely incorporated into the previously published sequence. Although variable nucleotide positions were observed throughout the newly sequenced genomes, non-synonymous amino acid substitutions were observed in only six of seven ND genes. While there were only 14 amino acid substitutions between genomes, K(a)/K(s) ratios ranged between 0 and 1. A single amino acid deletion in the ND1 coding region unique to rainbow trout was also detected. Using complete mitochondrial genomes for all sequenced Salmonidae species, a phylogenetic analysis was performed to establish a salmonid mitochondrial phylogeny revealing support for Salvelinus, rather than Salmo, as the sister taxa to Oncorhynchus.
RESUMEN
A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.
