The Structure of Mammalian Genes: (1) Antibody Heavy Chain Variable Region Genes: Organization, Diversity, and Somatic Mutation. (2) Structure and Transcription of the DNA Encompassing the Origin of Replication of Human Mitochondrial DNA

This thesis describes two experimental systems utilized to study mammalian gene structure and expression: (1) antibody heavy chain variable region genes and (2) mitochondrial DNA. In order to study the organization and structure of antibody genes and the relative germline and somatic contributions towards antibody diversity, we have analyzed the germline genes encoding the murine immune response to phosphorylcholine. Molecular cloning studies were undertaken and conclusively show that there is only one germline VH gene segment encoding the immune response to phosphorylcholine. Protein sequencing work on monoclonal antibodies that bind phosphorylcholine reveals many different protein sequences related to one predominant sequence. We are able to conclude that these variant sequences are the result of somatic diversification operating on one germline gene segment. We are further able to show that this diversification is mutational and not recombinational. Finally, somatic mutation is correlated with the class of the antibody; IgG and IgA antibodies undergo somatic mutation, IgM antibodies do not. We have isolated and sequenced a family of four closely related VH gene segments designated V1, V3, V11 and V13. Their function varies: V1 encodes the immune response to phosphorylcholine, V3 is a pseudogene, V11 encodes the immune response to influenza hemagglutinin, and V13 has an unknown function but is not obviously a pseudogene. Structural analysis of recombinant clones containing this family of related VH gene segments and other VH gene segments reveals several important points about the organization of VH gene segments. First, closely related VH gene segments can be clustered together within the VH gene locus. Second, the spacing distance between adjacent VH gene segments is variable; it may be as short as 5 kb and greater than 30 kb. Finally, the average spacing distance between VH gene segments is large, at least 23 kb. Assuming a minimum of 200 germline VH gene segments, the size of the VH gene locus may be greater than 5 million base pairs. The human mitochondrial genome is the second system that has been chosen to study gene structure and expression, and to accomplish this, we have applied both DNA and RNA sequencing technologies. We sequenced the DNA encompassing the origin of DNA replication and then localized the origin at the nucleotide level. The human mitochondrial origin of DNA replication shares structural characteristics with other known origins of DNA replication; in particular, the presence of extensive secondary structure in the form of a stem-loop structure. In order to precisely localize mitochondrial transcripts to the DNA, we developed techniques that allowed the isolation and sequencing of the 5'-ends of mitochondrial transcripts. This technology was utilized to precisely localize the 5'-end of the mitocohndrial 12S rRNA species 457 nucleotide pairs 5'-to the origin of DNA replication. Analysis of the DNA sequence in this region revealed a phenylalanine tRNA gene whose 3'-end was joined end-to-end with the 5'-end of the 12S rRNA. This analysis first demonstrated the extreme enconomy of genetic material in mammalian mitochondrial DNA.