Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).
To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthetic oligonucleotides which were joined enzymatically in vitro into four fragments which were cloned. The synthetic transducin gene and cDNA encoding transducin were expressed to similar levels in monkey kidney cells (COS-1) using a vector in which transcription was under the control of the adenovirus major late promoter.
Sakmar
T P
TP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis; D005786:Gene Expression Regulation; D005796:Genes; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012374:Rod Cell Outer Segment; D015293:Transducin
D008565:Membrane Proteins; D011994:Recombinant Proteins; D004247:DNA; D015293:Transducin