Identification of the immunodominant regions of the melanoma antigen tyrosinase by anti-tyrosinase monoclonal antibodies.

Tyrosinase, the critical enzyme in melanin synthesis, is also found to be expressed in most malignant melanomas and can serve as a target for the immune response by both CD4+ and CD8+ T-cells. Therefore it could be used as a potential target for therapeutic intervention in tyrosinase-positive melanomas. In order to develop serological reagents for the immunodetection of human tyrosinase and to find the most immunogenic region of the protein, we have raised a panel of monoclonal antibodies (MAbs) against recombinant tyrosinase expressed and purified from bacteria. Epitope mapping revealed the 79 amino acid long stretch between 163 and 241 residues to be the most immunodominant region of the tyrosinase. This region could be further divided into three parts by binding different MAbs. These MAbs were very useful tools for the detection of tyrosinase expression from different constructs in tissue culture cells by immunocytochemistry and in melanocytes by immunohistochemistry. Some of the MAbs that recognized epitopes between 163 and 204 amino acids also recognized an additional distinct protein of about 70 kDa seen on Western blot analysis of transfected and non-transfected COS-7 cells. One of these, the MAb 4B1, was used in immunohistochemistry, and cross reaction with the basement membrane of the human tissue was observed. The analysis of the 4B1 MAb epitope showed that the C-terminal part of that region almost entirely overlaps with the sequence of the recently reported basement membrane protein beta-netrin.

Induction of the bovine papillomavirus origin "onion skin"-type DNA replication at high E1 protein concentrations in vivo.

We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially "onion skin"-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products.