Characterization of Coptis japonica CjABCB2, an ATP-binding cassette protein involved in alkaloid transport.

Higher plants produce a large number of secondary metabolites. Among these are the alkaloids, a group of small nitrogen-containing molecules. Alkaloids often have strong biological activity that protects alkaloid-producing plants from herbivores, and often accumulate to high concentrations in a specific organelle of a particular organ in the producing plant. However, knowledge of the membrane transport mechanism of alkaloids is still limited. Coptis japonica, a perennial Ranunculaceous plant, produces the benzylisoquinoline alkaloid berberine. This alkaloid, though biosynthesized in root tissues, accumulates in the rhizome, suggesting translocation of the molecule via xylem. In this study, a gene encoding a ATP-binding cassette (ABC) protein of B-type, Cjabcb2, was isolated from C. japonica. Northern analysis showed that Cjabcb2 was preferentially expressed in the rhizome, which is the sink organ of berberine. Functional analysis of CjABCB2 using yeast suggested that CjABCB2 transports berberine in an inward direction. Membrane separation and in situ hybridization data indicated that CjABCB2 might be involved in translocation of berberine from the root to the rhizome by transporting berberine at the plasma membrane of cells around the xylem of the rhizome.

Transient gene expression mediated by integrase-defective retroviral vectors.

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica.

Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome.