Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.
Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.
Hickey
D R
DR
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, NY 14642.
Jayaraman
K
K
Goodhue
C T
CT
Shah
J
J
Fingar
S A
SA
Clements
J M
JM
Hosokawa
Y
Y
Tsunasawa
S
S
Sherman
F
F
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D010856:Columbidae / Q000235:genetics; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D005813:Genes, Synthetic / Q000235:genetics; D006736:Horses / Q000235:genetics; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013053:Spectrophotometry; D014413:Tuna / Q000235:genetics
D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008715:Methionine