Abstract
DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.
Full text
PDF



















Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abraham J., Nasmyth K. A., Strathern J. N., Klar A. J., Hicks J. B. Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region. J Mol Biol. 1984 Jul 5;176(3):307–331. doi: 10.1016/0022-2836(84)90492-3. [DOI] [PubMed] [Google Scholar]
- Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
- Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Derynck R., Singh A., Goeddel D. V. Expression of the human interferon-gamma cDNA in yeast. Nucleic Acids Res. 1983 Mar 25;11(6):1819–1837. doi: 10.1093/nar/11.6.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edmonds M., Vaughan M. H., Jr, Nakazato H. Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1336–1340. doi: 10.1073/pnas.68.6.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Faye G., Leung D. W., Tatchell K., Hall B. D., Smith M. Deletion mapping of sequences essential for in vivo transcription of the iso-1-cytochrome c gene. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2258–2262. doi: 10.1073/pnas.78.4.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goeddel D. V., Leung D. W., Dull T. J., Gross M., Lawn R. M., McCandliss R., Seeburg P. H., Ullrich A., Yelverton E., Gray P. W. The structure of eight distinct cloned human leukocyte interferon cDNAs. Nature. 1981 Mar 5;290(5801):20–26. doi: 10.1038/290020a0. [DOI] [PubMed] [Google Scholar]
- Hitzeman R. A., Chen C. Y., Hagie F. E., Patzer E. J., Liu C. C., Estell D. A., Miller J. V., Yaffe A., Kleid D. G., Levinson A. D. Expression of hepatitis B virus surface antigen in yeast. Nucleic Acids Res. 1983 May 11;11(9):2745–2763. doi: 10.1093/nar/11.9.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hitzeman R. A., Clarke L., Carbon J. Isolation and characterization of the yeast 3-phosphoglycerokinase gene (PGK) by an immunological screening technique. J Biol Chem. 1980 Dec 25;255(24):12073–12080. [PubMed] [Google Scholar]
- Hitzeman R. A., Hagie F. E., Hayflick J. S., Chen C. Y., Seeburg P. H., Derynck R. The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. Nucleic Acids Res. 1982 Dec 11;10(23):7791–7808. doi: 10.1093/nar/10.23.7791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hitzeman R. A., Hagie F. E., Levine H. L., Goeddel D. V., Ammerer G., Hall B. D. Expression of a human gene for interferon in yeast. Nature. 1981 Oct 29;293(5835):717–722. doi: 10.1038/293717a0. [DOI] [PubMed] [Google Scholar]
- Hitzeman R. A., Leung D. W., Perry L. J., Kohr W. J., Levine H. L., Goeddel D. V. Secretion of human interferons by yeast. Science. 1983 Feb 11;219(4585):620–625. doi: 10.1126/science.6186023. [DOI] [PubMed] [Google Scholar]
- Jayaram M., Li Y. Y., Broach J. R. The yeast plasmid 2mu circle encodes components required for its high copy propagation. Cell. 1983 Aug;34(1):95–104. doi: 10.1016/0092-8674(83)90139-3. [DOI] [PubMed] [Google Scholar]
- Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Losson R., Lacroute F. Interference of nonsense mutations with eukaryotic messenger RNA stability. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5134–5137. doi: 10.1073/pnas.76.10.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McLaughlin C. S., Warner J. R., Edmonds M., Nakazato H., Vaughan M. H. Polyadenylic acid sequences in yeast messenger ribonucleic acid. J Biol Chem. 1973 Feb 25;248(4):1466–1471. [PubMed] [Google Scholar]
- Mellor J., Dobson M. J., Roberts N. A., Tuite M. F., Emtage J. S., White S., Lowe P. A., Patel T., Kingsman A. J., Kingsman S. M. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene. 1983 Sep;24(1):1–14. doi: 10.1016/0378-1119(83)90126-9. [DOI] [PubMed] [Google Scholar]
- Moir D., Mao J., Schumm J. W., Vovis G. F., Alford B. L., Taunton-Rigby A. Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin. Gene. 1982 Jul-Aug;19(1):127–138. doi: 10.1016/0378-1119(82)90197-4. [DOI] [PubMed] [Google Scholar]
- Ratzkin B., Carbon J. Functional expression of cloned yeast DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):487–491. doi: 10.1073/pnas.74.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stinchcomb D. T., Struhl K., Davis R. W. Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979 Nov 1;282(5734):39–43. doi: 10.1038/282039a0. [DOI] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tschumper G., Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. doi: 10.1016/0378-1119(80)90133-x. [DOI] [PubMed] [Google Scholar]
- Tuite M. F., Dobson M. J., Roberts N. A., King R. M., Burke D. C., Kingsman S. M., Kingsman A. J. Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae. EMBO J. 1982;1(5):603–608. doi: 10.1002/j.1460-2075.1982.tb01215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valenzuela P., Medina A., Rutter W. J., Ammerer G., Hall B. D. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature. 1982 Jul 22;298(5872):347–350. doi: 10.1038/298347a0. [DOI] [PubMed] [Google Scholar]
- Zitomer R. S., Montgomery D. L., Nichols D. L., Hall B. D. Transcriptional regulation of the yeast cytochrome c gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3627–3631. doi: 10.1073/pnas.76.8.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]