Metallothioneins with interdomain hinges expanded by insertion mutagenesis.
Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.
Rhee
I K
IK
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Lee
K S
KS
Huang
P C
PC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004361:Drug Tolerance; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D003062:Codon; D008670:Metals; D012333:RNA, Messenger; D004247:DNA; D008668:Metallothionein