MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H(+)-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923-17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H(+)/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, alpha cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H(+)/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.
In order to develop simple sequence repeat (SSR) markers in Italian ryegrass, we constructed a genomic library enriched for (CA)n-containing SSR repeats. A total of 1,544 clones were sequenced, of which 1,044 (67.6%) contained SSR motifs, and 395 unique clones were chosen for primer design. Three hundred and fifty-seven of these clones amplified products of the expected size in both parents of a two-way pseudo-testcross F(1) mapping population, and 260 primer pairs detected genetic polymorphism in the F(1) population. Genetic loci detected by a total of 218 primer pairs were assigned to locations on seven linkage groups, representing the seven chromosomes of the haploid Italian ryegrass karyotype. The SSR markers covered 887.8 cM of the female map and 795.8 cM of the male map. The average distance between two flanking SSR markers was 3.2 cM. The SSR markers developed in this study will be useful in cultivar discrimination, linkage analysis, and marker-assisted selection of Italian ryegrass and closely related species.
Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear β(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy.
We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription-PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.
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