Here, we present a simple modular extendable vector system for introducing the T7
RNA polymerase and tetracycline repressor genes into Trypanosoma
brucei. This novel system exploits developments in our
understanding of gene expression and genome organization to produce a
streamlined plasmid optimized for high levels of expression of the introduced
transgenes. We demonstrate the utility of this novel system in bloodstream and
procyclic forms of Trypanosoma brucei, including the genome
strain TREU927/4. We validate these cell lines using a variety of inducible
experiments that recapture previously published lethal and non-lethal
phenotypes. We further demonstrate the utility of the single marker (SmOx)
TREU927/4 cell line for in vivo experiments in the tsetse fly
and provide a set of plasmids that enable both whole-fly and salivary
gland-specific inducible expression of transgenes.
We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was approximately 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassium-dependent sodium/calcium exchange activity were observed for each spliced variant.
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