The biosynthesis, structure and function of the principal differentiation products of human and mouse epidermis are being studied. Keratin subunits polymerize in vitro into native-type intermediate filaments. Details of their structure are being investigated by use of: solid state NMR on isotopically-labeled filaments; transmission and scanning transmission electron microscopy of intact filaments or subfilamentous forms; optical diffraction and image analysis procedures; and limited proteolysis experiments to ascertain alignment of constituent subunits. Model structures generated by these methods are being computationally tested for compatibility with other physico- chemical data and amino acid sequence information on individual subunits. cDNA clones to keratins 1 and 10 are being used to characterize the number, organization and complexity of their genes isolated from cosmid libraries. The expression of the human genes is being studied by the production of transgenic mice produced from various constructs of these genes. cDNA clones are being used to isolate and characterize the genes for mouse and human filaggrin, which appear to be initially expressed as a very large polyprotein precursor. cDNA clones encoding a major cell envelop protein have been isolated and are being sequenced. Constructs of keratins, filaggrin and the cell envelop protein clones are being assembled with pGEM vectors for use in situ hybridization experiments in order to study the expression of these proteins in epidermal keratinizing disorders.
