Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3(1)

A cotton Ltp3 gene and its 5' and 3' flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli beta-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5' flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.