MOLECULAR CLONING OF A NOVEL BASEMENT MEMBRANE COMPONENT

点击翻译按钮获取中文摘要

The candidate, Lawrence S. Chan, is an Assistant Professor of Dermatology at the Northwestern University Medical School. After graduating form the University of Pennsylvania School of Medicine in 1985, Dr. Chen obtained his Dermatology residency training at the University of Michigan Medical School under the leadership of Dr. John J. Voorhees. He was also trained as an Immunodermatology fellow at the same institution under the mentorship of Dr. Kevin D. Coper, a cellular immunologist, and Dr. James T Eler, a molecular biologist. The candidate's career interest is in the area of human autoimmunity against skin basement membrane zone (BMZ) components. During his residency, Dr. Chan discovered a new immunemediated blistering skin disease characterized by autoantibodies against a 105-kDa BMZ component at the lower lamina lucida. The candidate's short-term goals are to purify this new BMZ component and clone the human gene encoding for this new component. The candidate's long-term goals are to understand the functions of this new BMZ component: its relationship to other BMZ components, its influence on epidermal cell biology, its antigenic domains, and its pathophysiologic role in inducing autoimmune reaction. The research specific aims for the next five years, are to purify this 105- kD component, to isolate the human cDNA that encodes for this component, and to determine its functional domains. To achieve these specific aims, fibroblast protein will be subjected to multiple chromatographic columns, including Mono Q anion-exchange and reverse phase columns, for purification. The purified fractions will be analyzed by 2-dimensional gel electrophoresis and immunoblotting. The internal amino acid sequences will be obtained after proteolytic digestion of th purified protein. Monoclonal antibodies raised against the 105-kDa component and polyclonal antibodies raised against the amino-terminal peptide will be utilized to screen a human fibroblast cDNA expression library. Positive plaques will be subjected to cloning and DNA sequencing. Once the entire cDNA is delineated, multiple fusion proteins will be generated to examine the antigenic domains, the cellular and extracellular attachment domains, and the domains that influence keratinocyte biology. Understanding the structure and function of this new lamina lucida component will shed light on our understanding of the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering diseases, gestational development, and wound healing. The research environment provided for Dr. Chan at the northwestern University include; a Biotechnology Core Facility for protein microsequencing, peptide and oligonucleotide synthesis, monoclonal antibody production, two-dimensional gel electrophoresis, flow cytometry, molecular biology supplies, and an Animal Care Facility, all in the same research building as Dr. Chan's laboratory. The Department of Dermatology has several faculty members who can assist Dr. Nageswararao, another collaborator, is a protein biochemist, Dr. Woodley, the candidate's primary sponsor, is a protein biochemist and is well acquainted with epithelial cell biology. The Department of Dermatology has a departmental library which contains many major scientific journals and a state-of-the-art Fast Protein Liquid Chromatography system that can facilitate Dr. Chan's protein purification. Dr. Chan has been provided a fully equipped 660 square-feet laboratory space, a 200 square-feet office space adjacent to his laboratory, a culture room, and an electron microscope. In addition, the candidate has 80% projective time devoted to research. Together, this environment provides sufficient support for the candidate to succeed in his research effort.