Specificity of Anti-Dna Antibodies in Systemic Lupus Erythematosus

188 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.In an effort to clarify the involvement of anti-DNA antibodies in the disease systemic lupus erythematosus (SLE), the specificity of naturally occuring anti-DNA antibody in plasma of human patients with SLE was studied in detail.Colicin E1 plasmid DNA was employed as a test antigen in these studies utilizing an ammonium sulfate precipitation assay. Intact colicin E1 plasmid was shown to be specific for anti-double-stranded DNA (anti-dsDNA) antibodies and did not react with single-stranded DNA (ssDNA) specific antisera. ssDNA test antigen was similarly shown to be specific for anti-ssDNA antibodies and was bound by dsDNA-specific antibodies.Studies employing ss and dsDNA from calf thymus and mononucleotides as inhibitors of DNA binding demonstrated the presence of two anti-DNA populations in IgG fractions of the plasma tested: Type I--specific for ssDNA, and type II--cross-reactive, binding both ssDNA and dsDNA with apparently equal affinity. The specificity of the cross-reactive population was investigated in greater detail.ssDNA binding by type II antibodies was less effective at higher temperatures when compared to dsDNA binding, indicating structural and possibly nucleotide specificity. Structural specificity of type II antibodies was confirmed when synthetic double-stranded deoxynucleotide polymers proved to be more effective inhibitors of dsDNA binding than were the substituent single-stranded polymers.Type II anti-DNA antibodies were shown to interact electrostatically with the deoxyribose-phosphate backbone of DNA as dsDNA binding was sensitive to increasing sodium chloride concentrations.Recognition of nucleotide bases in dsDNA by type II antibodies was demonstrated by several lines of evidence: (1) Mononucleotides inhibited dsDNA binding while deoxyribose-phosphate did not. (2) One plasma tested (PS) showed specificity for GC rich DNA. (3) Poly(dG)(.)poly(dC) inhibited a different population of anti-DNA antibodies than poly(dA)(.)poly(dT) or poly(dA-dT)(.)poly(dA-dT).It was concluded that the linear deoxyribose-phosphate backbone, secondary structure, and nucleotide bases of DNA were all important to dsDNA recognition by type II anti-DNA antibodies in SLE plasma, although the relative contribution of each varied from patient to patient.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Characterization of BCE-1, a Transcriptional Enhancer Regulated by Prolactin and Extracellular Matrix and Modulated by the State of Histone Acetylation

We have previously described a 160-bp enhancer (BCE-1) in the bovine β-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-β and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-β but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones