Reactive site polymorphism in the murine protease inhibitor gene family is delineated using a modification of the PCR reaction (PCR + 1).
Murine protease inhibitor (alpha 1-PI) proteins are encoded by a multigene family which has undergone recent duplication. It has been suggested that the evolution of diversity within this gene family may be driven by unusual selection for novel function at the reactive site of the duplicated members (1,2,3). In an attempt to use polymerase chain reaction (PCR) to generate and sequence clones spanning the polymorphic reactive site region, a PCR artifact was identified and determined to result from heteroduplex formation during the co-amplification of the related sequences in this multigene system. This artifact results in sequences which are combinatorial mosaics of the template sequences. We present a simple and general method (PCR + 1) for overcoming this artifact and demonstrate its application in delineating five distinct alpha 1-PI reactive site sequences in C57BL/6 mice, thus providing sequence information to generate gene-specific probes. The significance of the reactive site diversity in this protease inhibitor gene family is discussed as well as the general applications and limitations of the PCR + 1 technique.
Borriello
F
F
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Krauter
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011480:Protease Inhibitors; D013698:Templates, Genetic
D011480:Protease Inhibitors