SELECTION OF NEW BIOLOGICALLY-ACTIVE MOLECULES FROM RANDOM NUCLEOTIDE-SEQUENCE

Genetic diversity can be achieved in vitro by inserting random nucleot ide (nt) sequences into cloned genes. In the case of enzymes, subseque nt genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activitie s, or altered temperature sensitivities. Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of an enzyme. Selecting biologically active DN A sequences from large random populations provides a new method for id entifying nt sequences with unique functions. Analogous random sequenc e selection techniques have been applied to determine the consensus se quence of the Escherichia coli promoters, DNA and RNA sequences that b ind specific protein(s), DNA regulatory sequences, ribozyme(s) and lig and-specific RNA(s). In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences i nto the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have al tered substrate specificity and/or catalytic rates when compared to th ose of the wild-type enzyme.