Castillo G et al. (1995), A homologue of the mammalian multidrug resistan...

XB-ART-19610

Biochim Biophys Acta 1995 Jun 09;12622-3:113-23.

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A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis.

Castillo G , Shen HJ , Horwitz SB .

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P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels. A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus laevis (Xe-mdr). The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe. The Xe-mdr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl. The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters. Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine. Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine. In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium. Brush border membrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner. Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function. These studies indicate that the structure of Xe-mdr has been conserved and suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: abcb1 abcb6

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	Fig. I. Cloning strategy used to obtain the three overlapping fragments (A,B,C) for the full length Xenopus laeL'is mdr gene. Fragment B was isolated from a tadpole eDNA library. Fragmeats A and C were isolated by anchored PCR. The numbered arrows indicate the primers used. RT, reverse transcriptase; TdT, terminal deoxynucleotidyl transferase.
	Fig. 2. Nucleotide and predicted amino acid sequence of Xenopus laevis mdr gene product. DNA sequence is presented in the 5' to 3' orientation. Untranslated sequences are in lower case letters and translated sequences are in upper case letters. Numbering of the amino acids begins in the first in frame AT(]. Putative N-glycosylation sites are underlined and the polyadenylation signal is boxed.
	Fig. 3. Amino acid alignment of the mammalian multidrug resistance gene products and the Xenopus laet,is homologue. The amino acid sequences of the human (mdrl and mdr2) [9,55], the mouse (mdrla, mdrlb, and mdr2) [12,13,18] and the hamster (pgpl, pgp2 and pgp3) [56], were aligned with the deduced amino acid sequence of the Xenopus gene. The alignment was created using the PILEUP program of the Genetic Computer Group. Identical amino acids are replaced with a dashed line (-). The entire sequence for the human mdrl gene product is shown. Gaps are introduced to optimize the alignment and are indicated by a dot (.). The grey boxes represent the 12 predicted transmembrane domains. The white solid boxes represent the nucleotide binding domains and the white broken box represents the linker region.
	Fig. 4. Dendrogram representing a cluster analysis of members of the multidrug resistance gene family. A multiple analysis was created using the PILEUP program of the Genetic Computer Group. The dendrogram is a tree representation of clustering relationships among the deduced amino acid sequences of the genes used for the analysis. The protein sequences used for the analysis were the hamster pgpl, pgp2 and pgp3 (Ha-pgpl, Ha-pgp2 and Ha-pgp3) [56], the mouse mdrlb, mdrla and mdr2 (Mumdrlb, Mu-mdrl and Mu-mdr2) [12,13,18], the rat mdr (Ra-Mdr) [57], the human mdrl and mdr2 (Hu-mdrl and Hu-mdr2) [9,55], the Entamoeba histolytica pgpl and pgp2 (En-Pgpl and En-Pgp2) [58], the Drosophila mdr49 and mdr65 (Dr-Mdr49 and Dr-Mdr65) [59], the C. elegans cepgpA and cepgpC (Ce-PgpA and Ce-PgpC) [30], the Leishmania ldmdrl (Le-Mdrl) [60], the Plasmodium mdr (Pl-Mdrl) [61] and yeast ste6 (Ye-Ste6) [49]. The two numbers in parenthesis represent the percent homology/identity to the Xenopus laevis mdr gene, designated as Xe-mdr.
	Fig. 5. Northern blot analysis of total RNA from normal tissues of adult frog. Total RNA was isolated from normal tissue of Xenopus laevis, separated on a 1.5% agarose gel and transfened by capillary action to a Genescreen membrane that was hybridized with fragment B. As a control, the same blot was stripped and hybridized with a probe for the Xenopus histone H4. Lane 1, oocyte; 2, brain; 3, gall bladder; 4, heart; 5, large intestine; 6, liver; 7, lung; 8, muscle; 9, oviduct; 10, skin; 11. small intestine; 12, spleen; 13, stomach. (B) Northern blot analysis of total RNA from sequential segments of the small intestine, starting at the duodenum. Each lane represents approx. 1 cm of the small intestine.
	Fig. 6. Expression of endogenous P-glycoprotein in the small intestine of adult Xenopus. Membranes were purified by differential centrifugation and separated by SDS-PAGE on a 10% gel, electroblotted to nitrocellulose filters and immunoprobed with antibody 5. Lane 1, membranes from the drug resistance cell line J7-V1; lane 2, membranes from the drug sensitive cell line J7. Lane 3, membranes from the small intestine of Xenopus laevis. Approx. 1 /zg of membrane protein was loaded in lanes 1 and 2, 20 /zg in lane 3.
	Fig. 7. In situ hybridization of Xenopus intestine. Sequential sections of the small intestine were hybridized with single stranded 35S-labelled antisense (panels a,c and e), and sense (panels b,d and f), RNA probes. Panels a and b show brightfield view of hematoxylin and eosin stained small intestine. Panels c and d represent darkfield views of the same sections (magnification, X 65). Panels e and f represent a greater magnification of a brightfield view (magnification, X 265).
	Fig. 8. Vinblastine transport by brush border membrane vesicles. Vesicles isolated from the small intestine were visualized by electron microscopy (A) (magnification, X 50000). Vesicles were loaded with [3H]vinblastine in the presence or absence of ATP or ATP',/S. Efflux was measured (B) in the presence of 3 mM ATP (O), 3 mM ATPyS (zx), ATP+ 10 /xM verapamil (A) and in the absence of ATP (O). Differences in [3H]vinblastine efflux in the absence or presence of verapamil were significant at 5 min (P < 0.05) and at 10 min (P < 0.03).