Human skin fibroblasts in culture have provided an important and useful tool for studies designed to detect aberrations of connective tissue in diseases. In continuation of this project, we propose extensive studies on the structure and metabolism of collagen and its biosynthetic precursor, procollagen, utilizing human skin fibroblast cultures. The overall goal of these studies is to define the molecular defects which may occur in collagen in acquired and heritable diseases. The main emphasis of these studies will be on two distinct entities, the fibrotic skin diseases and the Ehlers-Danlos syndrome. Cells from these patients will be subjected to in-depth analyses using a variety of techniques, with emphasis on recombinant DNA technology. In case of fibrotic skin diseases, we will examine alterations in collagen gene expression at translational and transcriptional level. Specifically, we will a) determine the steady-state levels of genetically distinct procollagen mRNAs; b) elucidate the transcriptional mechanisms leading to altered mRNA abundance; c) examine the relationship between the gene expression and the state of the gene by assay of DNase hypersensitivity and the methylation of the gene; d) determine the procollagen gene copy number as an indication of gene amplification. In case of the Ehlers-Danlos syndrome we attempt to define structural mutations in collagen by a) examination of type I and type III procollagen mRNAs by Northern blot and dot blot hybridizations; b) detection of mutated sequences by S1-nuclease digestion of mRNA-cDNA hybrids; c) examination of genomic DNA by restriction enzyme mapping; d) detection of single-base substitutions in total genomic DNA by denaturing gradient gel electrophoresis; e) linkage analyses utilizing restriction fragment length polymorphism of the procollagen genes. These studies are expected to yield precise information on the underlying molecular mechanisms of diseases affecting connective tissues. Such data will help us to define the normal structure-function relationships of genetically distinct collagens in skin and other tissues, as it pertains to specific domains and amino acid sequences in the protein. The results will also provide us with knowledge highly useful for the development of rationale treatment modalities for these conditions.