FIG contributed to NMR analysis, MA performed the phylogenetic analysis. MRB performed some growth experiments and trehalose

determination, JJN participated in bioinformatic analysis and figure preparation. MEA and CV conceived the study, participated in the design, coordination, bioinformatic analysis, and writing of the manuscript. All authors have read and approved the final manuscript.”
“Background Epitope tagging has been widely used for the analysis of protein localization, interaction, and function (reviewed in [1]). It is extremely useful in studying the proteins of the ciliated protozoan Tetrahymena thermophila because epitope tags can be introduced efficiently into endogenous chromosomal loci by homologous recombination Alpelisib research buy in this organism [2]. In many cases, a protein of interest is tagged by introducing a tag at its C-terminus [3–5]

because a drug-resistance marker, which must be introduced in proximity to the tag see more for the establishment of transgenic strains, rarely disturbs the gene promoter if it is inserted downstream of a target gene; thus, the tagged protein can be expressed at its endogenous levels. We previously established a set of convenient modules designed for PCR- and plasmid-based C-terminal tagging (Kataoka et al. submitted). However, sometimes a C-terminal tag renders the protein dysfunctional, disturbs the localization of the protein, or interferes with the protein’s interaction with other molecules. In these cases, tagging the protein at its N-terminus might be advised. There

is a drawback to the N-terminal epitope tagging strategy in general: an insertion of a drug-resistance marker into the upstream region of a gene could BIIB057 purchase disturb its promoter activity. This possibility is especially an issue in the Tetrahymena system because intergenic sequences are relatively short in this organism [6]. To avoid this problem, in previous experiments, N-terminally tagged proteins were expressed from ectopic genome locations, such as rDNA or β-tubulin 1 (BTU1) loci, and/or by ectopic promoters at their endogenous loci [7–10]. However, expression levels and patterns of these ectopically expressed N-terminally tagged proteins could differ from those of their endogenous counterparts and thus might cause mislocalization of proteins or artificial interaction with other molecules. Alternatively, Adenosine a drug-resistance marker can be inserted into the downstream region of a gene for N-terminal tagging. However, in this case, the entire coding sequence and both the upstream and the downstream flanking sequences of the gene have to be cloned as a single construct, which is sometimes not easy for large genes. In addition, if homologous recombination occurs within the coding sequence, an epitope tag at the N-terminus in the construct would be lost. Moreover, the inserted selectable marker could disturb the expression of the downstream gene.