Studies on the murine alpha(1)-1 protease inhibitor gene family

Abstract

Murine {dollar}\alpha\sb1{dollar}-protease inhibitor ({dollar}\alpha\sb1{dollar}-PI) is encoded by a small multigene family. Humans have a single homologous gene, {dollar}\alpha\sb1{dollar}-antitrypsin ({dollar}\alpha\sb1{dollar}-AT), whose primary physiologic substrate is neutrophil elastase. Interest in these protease inhibitors is founded on two main observations. First, humans homozygous for the Z allele develop an early onset form of pulmonary and liver disease. These diseases have focused attention on the molecular structure-function relationships determining the interaction of these proteases with their cognate substrates and how abberrations in these interactions can result in pathogenesis. Second, these genes are tissue specifically expressed in a developmentally regulated fashion with expression limited predominantly to hepatocytes and some lymphocytes. This complex pattern of regulated expression has focused attention on the cis and trans acting genetic elements responsible.;Towards understanding these issues, several independent but complementary experimental approaches to characterize the {dollar}\alpha\sb1{dollar}-PI gene family in C57BL/6 mice were pursued. Chapter One describes a new polymerase chain reaction methodology (PCR + 1) which was developed as a tool to study individual members of any highly related multigene family. This technique was applied to the {dollar}\alpha\sb1{dollar}-PI gene family in order to delineate five distinct genes and develop gene specific oligonucleotide probes for each. These probes were used under stringent conditions to demonstrate that each gene is abundantly expressed in the liver. Full length liver cDNA clones were obtained for each gene and a comparison of the nucleotide sequences demonstrated marked hypervariability in the region of the gene encoding the reactive site. The deduced amino acid sequences reveal regions of polymorphism among the five cDNAs which can be mapped to distinct structural motifs in the crystal structure of the single homologous human {dollar}\alpha\sb1{dollar}-AT protein. Chapter Two describes an in vitro transcription assay that was employed to map the promoter elements of a Balb/c {dollar}\alpha\sb1{dollar}-PI gene. This assay has proven useful in identifying and partially characterizing a heat stable {dollar}\alpha\sb1{dollar}-PI promoter transcription factor which appears to be ubiquitous. It is involved in establishing the transcription initiation site, as measured by the "G" free cassette in vitro transcription assay. This promoter factor bears homology to sequences in other liver specifically expressed genes which also bind heat stable transcription factors.;This work has established the number and diversity of the genes which comprise the C57BL/6 {dollar}\alpha\sb1{dollar}-protease inhibitor gene family. It provides the groundwork required to delineate the genomic organization of these genes, their evolutionary relationships and functional differences at the protein level. In addition, preliminary data is presented on a previously unrecognized promoter element in the Balb/c {dollar}\alpha\sb1{dollar}-PI gene promoter which may also be present in other liver specifically expressed genes.