Expression of a wheat alpha-gliadin gene in Saccharomyces cerevisiae.
A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae. This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S. cerevisiae. The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene. A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site. A CYC1::alpha-gliadin transcript of approx. 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region. The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence. The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx. 0.1% of the total cellular protein. The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin. This is consistent with recognition and cleavage of the signal peptide by yeast. Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.
Neill
J D
JD
Department of Plant Molecular Physiology, Hawaii Institute of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu 96822.
Litts
J C
JC
Anderson
O D
OD
Greene
F C
FC
Stiles
J I
JI
D003062:Codon; D005822:Genetic Vectors; D005903:Gliadin / Q000096:biosynthesis / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012346:RNA, Transfer, Amino Acyl / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic; D014908:Triticum / Q000235:genetics*
D003062:Codon; D010940:Plant Proteins; D012346:RNA, Transfer, Amino Acyl; D011993:Recombinant Fusion Proteins; D005903:Gliadin