This thesis can be divided into three parts: Chapter Two and Chapter Three contain study on the class I genes of the major histocompatibility complex (MHC), Chapter Four and Chapter Five contain study on the T-cell receptor genes, and the appendices deal with my initial attempt to clone the eDNA encoding the T-cell receptor α chain and various other research projects that I have been involved in to some extent (mouse MHC class II genes, PDGF genes and rat class I and class II genes).
The first part of my thesis describes the study of the organization of the genes encoding the mouse class I MHC molecule. 54 cosmid clones containing 36 class I genes were isolated and, by restriction enzyme mapping, the 54 clones could be divided into 13 clusters. Using low-copy probes isolated from each cosmid cluster and the restriction enzyme site polymorphism of those probes, I was able to map each of the class I gene clusters into the precise location of the mouse MHC. Surprisingly, most of the class I genes map into the Tla region, only five class I genes (three cosmid clusters) map into the classical H-2 region. The functions of these class I genes in the Tla region are still largely unknown.
The remainder of my thesis contains the study on the T-cell receptor genes. In an effort to isolate the cDNA clone encoding the T-cell receptor α chain, I have isolated 64 T–cell specific cDNA clones, using a T-cell minus B-cell subtractive cDNA probe. The T-cell receptor α and β chain cDNA clones were among these 64 clones. Using the T-cell receptor α chain cDNA as a probe, I subsequently isolated clones encoding a germline variable(V) gene segment and cosmid clones spanning 120 kb of DNA encoding the joining(J) and constant(C) gene segments of the T-cell receptor α chain. Analysis of these clones, including sequencing of one germline Vα and six germline Jα gene segments, showed that the DNA recognition sequence for the α chain DNA rearrangment is similar to that of the β chain counterpart. In contrast to the general J gene segment organization in the β chain, γ chain and the immunoglobulin gene families, I showed that the 18 Jα gene segments I analyzed were spread over 60 kb of DNA and lay as far as 63 kb 5' to the Cα gene.
In a step to dissect the structure function relationship of the T-cell receptor molecules, I have cloned and determined the nucleotide sequences of seven functional α chains and six β chains of the T-cell receptor genes from nine T-helper hybridomas specific for the C-terminal peptide of pigeon cytochrome c and the E class II molecule. Northern blot analyses using the isolated Vα and Vβ gene segments were performed on the RNAs isolated from a total of 15 T–helper hybridomas specific for the C-terminal peptide of cytochrome c. A single Vα gene segment is predominantly used in these 15 T-helper hybridomas, whereas at least five different Vβ gene segments are utilized. I conclude that the Vα gene segment mis important for the cytochrome c response and might provide most of the contact residues with the C-terminal region of cytochrome c. I also found that the junctional sequences of the β chain may alter the antigen fine specificity of the T-cell clones. Finally, somatic hypermutation does not appear to play a crucial role in generating diversity for the T-cell receptor α or β chains.
