Robert M. Horton

[10] Specific amplification of complementary DNA from targeted members of multigene families

Publisher Summary The specific amplification of complementary DNA (cDNA) from targeted members of a multigene family was achieved by designing oligonucleotide PCR primers that are complementary to localized segments of diversity within the 5’ and 3’ untranslated regions of the encoded mRNAs. In cases where the primers differentiated among all the different species of cDNA, only a single amplification product was recovered. However, in cases where more than one homologous mRNA species was amplified in the same PCR, multiple products were recovered. Among these products were the targeted cDNA as well as related recombinant DNA species. The presence of the chimeric amplification products highlights a major limitation of using regions of homology to design PCR primers for the analysis of multigene families. Even under conditions where there are only two or three homologous species of cDNA amplified by a set of primers, the presence of recombinants among the amplification products significantly complicated the analysis.

Novel MHC Variants Spliced by Overlap Extension

We have developed a new technique, based on the polymerase chain reaction (Ho 1989), called “splicing by overlap extension” (SOE), which simplifies site-directed mutagenesis and allows the introduction of larger systematic changes in gene structure in a manner independent of the distribution of restriction endonuclease sites in the targeted area of the gene (Ho 1989). An added feature of SOE is that it provides a straight-forward strategy for the generation of chimeric genes from unrelated sources, such that our ability to make genetic constructs is limited by knowledge of how protein domains will interact and not by the structure of the genes that encode them (Horton 1989). We have used the SOE strategy to generate novel MHC class I variants designed to investigate the antigen presenting properties of the encoded glycoproteins. Here we present examples of how SOE has been applied in the generation of these constructs and the characterization of their gene products using mAbs.

Contributions of Interlocus Exchange to the Structural Diversity of the H-2K, D, and L Alleles

The nature of diversity that distinguishes alleles form each other and from sequences of other class I loci was analyzed using 24 H-2K, D and L sequences, providing statistical evidence that alleles of the K and D/L loci are evolving independently throughout most of their coding regions. However, sequences determining the structure of the antigen binding domains of both allelic series appear to be co-evolving. Sequence motifs that distinguish alleles in these series are present at both the K and D/L loci, a pattern of diversity that is reminiscent of the class I mutants that have been described in the mouse.

The Functional Significance of Amino Acid Polymorphisms in Class I MHC Molecules

The x-ray crystal model of the HLA-A2 molecule depicts an antigen presenting domain consisting of two α-helices under which are found eight antiparallel β-strands. Furthermore, the position, orientation, and functional contribution of many amino acid side chains has been proposed (Bjorkman 1987a,b). In general, amino acid residues that point inward/upward from the α-helices or point upward from the β-strand floor are thought to be involved with either peptide binding or T-cell receptor (TcR) interaction. Amino acid side chains that point outward from the α-helices, project downward from the β-strand floor, or are located on loops outside of the antigen recognition site (ARS) are thought to be silent with regard to TcR interaction or peptide binding (Bjorkman 1987b).