PhD

dissertationThe molecular events controlling the expression of the mouse Crry and Cr2 complement receptor genes were investigated. The Crry gene is ubiquitously expressed and encodes a protein product of 65 kilodaltons (kDa). A 1.9 kilobase genomic fragment containing the Crry promoter and other 5' regulatory sequences was fused to a reporter gene. Deletional analysis of 5' sequences revealed an enhancer site. Gel shift and methylation interference assays localized a sequence that was capable of forming a DNA-protein complex. The sequence identified by these assays was able to enhance transcription from a heterologous promoter in a position and orientation independent manner. These experiments identified a Crry gene transcriptional enhancer. The murine Cr2 gene encodes two protein products of 145 kDa and 190 kDa that are restricted in expression to a subset of hematopoietic lineage cells. Sequencing of a genomic fragment of the Cr2 promoter-enhancer region revealed a 49 base pair region that was 70% homologous to an analogous site in the human CR2 promoter-enhancer. Gel shift and methylation interference assays revealed that a single sequence outside the homology area was responsible for the majority of the gel shift complexes. The sequence was similar to the octamer consensus sequence. The size and tissue specificity of the Cr2 gene octamer site complexes is consistent with the size and expression pattern of the Oct-1 and Oct-2 transcriptional control proteins. It was found that bacterial infection decreased both Oct-2 and Cr2 transcription. These experiments indicate that Oct-2 may play a role in B-cell specific transcription of the Cr2 gene. An early report stated that the products of the Cr2 gene were expressed on murine macrophages. It had also been suggested that these cells phagocytized complement coated particles via the 190 kDa Cr2 protein. The transcriptional activity of the Cr2 gene in mouse macrophages was investigated using reverse transcription-rapid polymerase chain reaction and indicated a lack of Cr2 gene expression in macrophages of three different derivations. Protein products of the Cr2 gene were not found on the surface of macrophages. These experiments indicate that the CR1-like activity on mouse macrophages is not due to the presence of Cr2 proteins