Neutrophil and macrophage cells express several proteins which defend the host from invading bacteria. These proteins are the primary host defense mechanisms. Among them, Defensins are a family of antibiotic proteins whose primary and secondary structures have been elucidated. The primary focus of this project is to understand the Defensin molecules' essential elements that elicit antibacterial activity. A human Defensin gene was chosen as subject. Because artificial genes provide clear advantages for altering genes, the defensin gene was assembled and cloned from synthetic oligonucleotides. During this period, several methods of gene synthesis were evaluated and developed a new method to accurately assemble synthetic genes. The technique involved the synthesis of only one of the DNA strands. The method was optimized by synthesizing the lacZ gene of Escherichia coli. Another aspect of the project involved identification of newer antibacterial genes in mice. For this purpose a cDNA library from mouse macrophage messenger RNA was prepared. This cDNA library is being used to isolate a large number of genes which might produce antibacterial proteins.
