Objectives: To determine whether annular rDNA is complexed with the nuclear envelope proteins.
Methods: From a batch of lupus sera with anti-nDNA, we selected a lupus serum containing annular anti-nDNA autoantibodies resistant to DNase digestion and used it to isolate several cDNA clones from a lambda gt11 HeLa cell library.
Results: The cloned fusion protein immunoadsorbed the annular anti-nDNA autoantibodies, and the immunoaffinity autoantibodies eluted from the recombinant filters produced an annular pattern around the nucleus in fluorescent assays on HEp-2 cells; by Western blot, they also recognized a 70 kDa protein from HEp-2 cell extracts. Annular-lambda gt11 lysogens generated in E. coli Y1089 produced a fusion protein that recognized annular anti-nDNA autoantibody-containing lupus sera by Western blot. The recombinant filters and annular fusion protein were also recognized by a prototype anti-lamin serum. To determine whether the annular recombinant protein bound DNA, an interaction assay was performed in vitro using DNA minicircles and DNA from HEp-2 cells; this assay resulted in a slowing of the electrophoretic mobility of the DNA.
Conclusions: 1) The annular DNA in eukaryotic cells is complexed with nuclear envelope proteins. 2) Annular anti-nDNA autoantibodies from lupus patients cross-react with perinuclear proteins. 3) Perinuclear proteins recognized by anti-nDNA are lamins. 4) An interaction between DNA and the 70 kDa protein is inducible in vitro. Whether this interaction affects cell function is still unknown.