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 principal 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 are experimentally preferable to naturally occurring ones, the gene was assembled from synthetic oligonucleotides. During this study, a new method to rapidly assemble synthetic genes was developed. This technique required the synthesis of only one strand of DNA and facilitated the assembly and cloning of large stretches of DNA in a single step. The method was tested and standardized by synthesizing the lacZ gene of E. coli. Refinements of this method for synthesizing larger genes are in progress. Although Defensin genes have been reported in several mammalian species, they have never been identified in mice. In these studies, Defensin gene homologues from a mouse genomic library were isolated by oligonucleotide probes. Restriction endonuclease analyses indicate that three of these recombinants have originated from independent chromosomal regions. The primary structure analysis of these genes is continuing.
