Nucleotide sequences of three distinct cDNA clones coding for the rat class I heavy chain RT1n antigen.

We isolated and sequenced three different RT1n cDNA clones that each encode the entire major histocompatibility complex (MHC) class I heavy chain protein, including the 39 untranslated region (UTR) and the poly-A tail. The mRNA was isolated from Concanavalin A-stimulated (Pharmacia, Piscataway, NJ) splenocytes of BN (RT1n) rats (Harlan Sprague-Dawley, Indianapolis, IN). The cDNA library was synthesized from BN mRNA using an reverse transcription (RT)-polymerase chain reaction (PCR)-based cloning method (Wang et al. 1996). Using previously described upstream and downstream primers in a PCR-based method (Wang et al. 1996) we isolated three RT1n clones. To assure the accuracy of each RT1n clone, we individually sequenced the cDNA samples from triplicate PCR reactions as well as from triplicate TA-cloning reactions. Multiple RT1n clones for each cDNA were then sequenced in both directions to eliminate any possibility of random PCR mutations. Although we performed sequencing of multiple clones, we cannot completely exclude the possibility of PCR artifact. As previously suggested (Joly et al. 1995), undesirable PCR reactions such as polymerase “jumping” between different sequences are possible, especially at high concentrations of templates. We found that our three RT1n sequences are similar to rat class I MHC sequences available in the GenBank, including two RT1n sequences (Joly et al. 1995; X90375, X90376; Fig. 1). Interestingly, our clones 4 and 12 are identical in the 39 UTR and at the 39 half of the coding region. Furthermore, the sequence of our clone 12 (U50448) is similar to the partial cDNA sequence of RT1n (X90375) with the exception of two nucleotides (GC) at positions 564 and 565, which differ from those (CG) presented in their sequence. This difference in our amino acid sequence produced Trp and Leu amino acids at positions 191 and 192, instead of Cys and Val amino acids as in the RT1n (X90375) sequence. Our three cDNAs were subcloned into pMAMneo (CloneTech, Palo Alto, CA), an inducible eukaryotic expression vector, and transfected into rat BUF (RT1b) hepatoma cells. Neomycin (Life Technologies; Grand Island, NY)-resistant transfectants (0.5 × 106) stimulated with 1 μg dexamethasome (DEX; Sigma, St. Louis, MO) for 6 h were tested for cell-membrane expression of class I MHC molecules by FACS analysis with an FITC-conjugated mouse anti-rat class I MHC-specific monoclonal antibody (OX18; Pharmingen, San Diago, CA). Both non-induced and DEXinduced normal BUF cells expressed almost identical baseline amounts of class I RT1.Ab molecules (Fig. 2 A, B). Similarly, DEX-stimulated control BUF cells that had been transfected with pMAMneo alone (without RT1n cDNA) expressed a similar number of class I MHC molecules as non-transfected controls (Fig. 2 C). In contrast, following DEX induction, BUF hepatoma cells transfected with any of three RT1n clones displayed increased expression of membrane-bound class I MHC molecules (Fig. 2 D, E, F). Thus, our results document that all three transfected RT1n genes may be expressed on the cell surface, when under control of a DEX-inducible promoter.