MUTAGENESIS OF BOVINE BASIC FIBROBLAST GROWTH-FACTOR THROUGH HYBRID CLONEMID CONSTRUCTION - A NEW APPROACH TO DNA MANIPULATION

Basic fibroblast growth factor (bFGF) is a heparin-binding protein whi ch is mitogenic for a wide variety of cells derived from the mesoderm and neuroectoderm. It is also known as an angiogenic and neurotrophic molecule. The mutagenesis of bovine bFGF gene into the human bFGF gene and the expression of human bFGF have been carried out in this labora tory In this study, efficient linkage (ligation) of single strands of DNA at their terminal complementary areas was found to be obtained eas ily at low temperature. Based upon this discovery, we have advanced a general method of DNA manipulation. The major principle is to make use of complementary sequences in a DNA linker to circularise a cut vecto r or use the single-strand DNA fragments to rebuild the plasmid throug h heteroduplex plasmid (hybrid clonemid) construction. The use of sing le DNA strands in DNA manipulation produces high flexibility in the co urse of hybrid clonemid construction. It enables DNA cloning and mutag enesis to be carried out concurrently The primer-induced and polycatio n-reliant unidirectional deletion (PPUD) phenomenon is also observed i n this study. The factors that affect the efficiency of mutagenesis ar e discussed. This work offers a new understanding of DNA manipulation.