Cloning of porcine neuron growth inhibitory factor (metallothionein III) cDNA and expression of the gene in Saccharomyces cerevisiae.
Growth inhibitory factor (GIF), a member of the metallothionein (MT) family, is also known as MTIII. This protein distinguishes itself from other MT isoforms by exerting an inhibitory effect on cortical neuron growth instead of metal ion buffering. In this work, we cloned MTIII genes from a porcine brain cDNA library. Two species of clones were isolated that vary with respect to one nt in the coding sequence. This discrepancy results in the translation of two MTIII primary structures having a different amino acid at residue 46. Herein, both MTIII cDNAs were constructed into an expression vector and transformed into yeast cells, respectively. The yeast carrying either MTIII gene displayed a similar metal tolerance when cultured in a medium containing metal. The resistance to metal toxicity was attributed to the expression of MTIII gene which was confirmed by RNA and protein analyses. The characteristics of the protein stability, metal binding affinity and ultraviolet absorption spectrum of the yeast produced MTIII are also compared with those of MTII. The comparison reveals that both MTs have similar physical characteristics. Moreover, circular dichroism spectrum of Cd saturated MTIII was analyzed as well. Typical Cys-Cd bands for MTII appear in the spectrum, indicating similar metal-thiol interactions for MTIII to those for other MT isoforms.
Wang
S H
SH
Institute of Radiation Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chang
C Y
CY
Chen
C F
CF
Tam
M F
MF
Shih
Y H
YH
Lin
L Y
LY
D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006131:Growth Inhibitors / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism; D000090023:Metallothionein 3; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry; D013552:Swine / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D006131:Growth Inhibitors; D000090023:Metallothionein 3; D008670:Metals; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein