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 physicochemical data and amino acid sequence information on individual subunits. PCR-generated probes to type I and type II keratins are being used to characterize the number, organization and complexity of their genes. The expression of the human keratin genes 1 and 10 are being studied by the production of transgenic mice produced from various constructs of these genes. Both cDNA and alphaDNA clones have been used to describe the properties of the human filaggrin gene. It encodes a large polyprotein precursor that displays considerable variation in sequence and size. Clones encoding a major cell envelope protein, loricrin, have also been isolated and are being sequenced. Clones encoding an epidermal transglutaminase have been isolated and will be characterized. Constructs of keratins, filaggrin and the cell envelop protein clones have been assembled with pGEM vectors for use in insitu hybridization experiments in order to study the expression of these proteins in epidermal keratinizing disorders.
