Construction and Characterization of a Library of Metallothionein Coding Sequence Mutants

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Abstract
New possibilities for experimental investigation of the metallothionein system at the DNA, RNA, and protein levels are now available via a library of metallothionein coding sequence mutants. Appropriate treatment in vitro with sodium bisulfite of the sense or antisense DNA strands in M13 phage vectors and propagation in Escherichia coli BD1528 (ung–) produced two unique collections in the library containing either C to T or G to A transition mutations in the codons at low and high frequencies. The strategy for mutagenesis takes advantage of degeneracy in the genetic code so that no cysteine codons are replaced in the CT mutants while all are subject to change in the GA collection. Two hundred and sixty-four clones from the library have been sequenced and G to A transitions affecting each of the 20 cysteine codons in metallothionein have been detected. Other mutations in codons for amino acids proposed to be important for metallothionein function have also been identified.