The major goal of the work proposed is to determine the biological significance of the large metaphase chromosome anomalies termed homogeneously staining regions (HSRs) and the small, paired chromatin bodies termed double-minute chromosomes (DMs) that are prevalent features of human neuroblastoma cells. Since there is direct evidence that the HSRs and indirect evidence that the DMs of antifolate-resistant Chinese hamster and mouse cells which overproduce target enzyme dihydrofolate reductase contain selectively amplified genes coding for the enzyme, we plan to determine: 1) whether the cytologically similar anomalies characterizing most human neuroblastoma cell lines likewise represent disproportionate increases in specific DNA sequences, and 2) whether HSRs, in particular, contain protein-encoding genes. Approaches include: karyotype studies of our own collection of human neuroblastoma and of new lines in an ongoing evaluation of HSRs and DMs (Aim #1), two-dimensional (2D) gel electrophoretic analysis of proteins in selected near-diploid HSR-containing and HSR-lacking human neuroblastoma and in control (nonneuroblastoma) cells and of in vitro translated products from some of these lines to detect increased synthesis of polypeptides in HSR-bearing cells (Aim #2), and cloning of HSR-specific DNA sequences from BE(2)-C cells, the principal HSR-bearing neuroblastoma line, to be used as probes for these sequences in other HSR- and DM-containing human neuroblastoma lines (Aim #3). In addition to gaining specific information about malignant neuronal cell development, we anticipate that ultimately we will generate new information about chromosomal manifestations of gene expression and mechanisms of DNA sequence amplification.