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3543020553
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note
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The α wild-type, β wild-type, αIII, and βIII constructs have been previously described (8). The α wild-type amino acid sequence from the interchain Cys to the COOH-terminus is CDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (30), with the α-CPM indicated in bold. The β wild-type amino acid sequence from the interchain Cys to the COOH-terminus is CGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAM VKRKNS. Similarly, the corresponding αIII amino acid sequence is CDATLTEKSFETVTVHTEKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF. The underlined sequences are derived from murine Cδ and consequently, the distal five amino acids (DMNLN) of the α-CPM have been replaced. To permit surface expression of this chimeric α-chain, the αIII construct was paired with the βIII cDNA (8), which encodes the corresponding amino acid sequence CGITSASYQQGVLSATILYLLLLLKSVIYLAIISFSLLRRTSVCGNEKKS. The underlined sequences are derived from murine Cγ1. Although this TCR is encoded by chimeric α and β chains, the functional defects associated with the αIII/βIII TCR are due to the absence of an intact α-CPM (8). cDNAs were excised with Eco RI and Bam and individually cloned into the Sal I and Bam sites of the expression vector pHSE3′ (31).
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13
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3543016388
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note
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In contrast to irradiated B6 mice, irradiated B6 athymic nude mice reconstituted with T cell-depleted bone marrow from α-CPM mutant animals failed to generate significant numbers of transgenic T cells. Therefore, the appearance of mutant T cells in the periphery is dependent on the presence of a thymus (12).
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30
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3543022947
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S. Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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31
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0024438903
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34
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3543021757
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note
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bm12 mAb 3JP (33) were purified from culture supernatants using protein G Sepharose beads (Pharmacia). Cells were analyzed on a FACScan or a FAC-Star Plus (Becton Dickinson) using the CellQuest software (Becton Dickinson).
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35
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1842411319
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36
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3543024766
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note
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-/- mice expressing the wild-type or the α-CPM mutant TCR. Cells were then harvested and stained with Hoechst 33342 (1 μg/ml) followed by staining with CD4 mAb, CD8 mAb, and propidium iodide (2.5 μg/ml) and analyzed by flow cytometry.
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37
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3542992314
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note
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-/- (A. Rolink) mice and the pHSE3′ (H. Pircher) vector are gratefully acknowledged. Care of animals was carried out in accordance with the cantonal and federal laws of Switzerland. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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