Skip to main content

Advertisement

SpringerLink
Log in

Construction of the complete rat fatty acid synthase cDNA and its expression inSaccharomyces cerevisiae

  • Original Paper
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

The 272 647-dalton polypeptide of fatty acid synthase (FAS) fromRattus norvegicus has been expressed in a proteinase-deficient strain ofSaccharomyces cerevisiae. The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking. In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeastADH1 promoter. Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA. However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon. This was obtained when the 86 by of the rat FAS cDNA immediately 5′ to the start codon were replaced by a nonamer corresponding to the immediate 5′-vicinity of the translational start codon of the yeastADH1 gene. Nevertheless, the translation product could be detected only by Western blotting. The FAS proteins ofS. cerevisiae and rat are not functionally interchangeable. Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Achstetter T, Wolf DH (1985) Proteinases, proteolysis and biological control in the yeastSaccharomyces cerevisiae. Yeast 1:139–157

    PubMed  Google Scholar 

  • Andersson SGE, Kurland CG (1990) Codon preferences in freeliving microorganism. Microbiol Rev 54:198–210

    PubMed  Google Scholar 

  • Beck K-F, Schreglmann R, Stathopulos I, Klein H, Hoch J, Schweizer M (1992) The fatty acid synthase (FAS) gene and its promoter inRattus norvegicus. J DNA Sequencing and Mapping 2:359–386

    Google Scholar 

  • Bennetzen JL, Hall BD (1982 a) The primary structure of theSaccharomyces cerevisiae gene for alcohol dehydrogenase 1. J Biol Chem 257:3018–3025

    PubMed  Google Scholar 

  • Bennetzen JL, Hall BD (1982 b) Codon selection in yeast. J Biol Chem 257:3026–3031

    PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    PubMed  Google Scholar 

  • Carter AT, Narbad A, Pearson BM, Beck K-F, Logghe B, Contreras R, Schweizer M (1994) Phosphoribosylpyrophosphate synthetase (PRS): a new gene family inSaccharomyces cerevisiae. Yeast 10:1031–1044

    PubMed  Google Scholar 

  • Chen Y, Pioli D, Piper PW (1994) Overexpression of the gene for polyubiquitin in yeast confers increased secretion of a human leucocyte protease inhibitor. Bio/Technol 12:819–823

    Google Scholar 

  • Cigan AM, Donahue TF (1987) Sequence and structural features associated with translation intiation regions in yeast. Gene 59:1–18

    PubMed  Google Scholar 

  • Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

    PubMed  Google Scholar 

  • De Blas A, Cherwinsky H (1983) Detection of antigens on nitrocellulose paper immunoblots with monoclonal antobodies. Anal Biochem. 133:214–219

    PubMed  Google Scholar 

  • Emr SD (1991) Heterologous gene expression in yeast. Methods Enzymol 185:231–233

    Google Scholar 

  • Frankel A, Schlossman D, Welsh P, Hertler A, Withers D, Johnston S (1989) Selection and characterization of ricin toxin A-chain mutations inSaccharomyces cerevisiae. Mol Cell Biol 9:415–420

    PubMed  Google Scholar 

  • Hamilton R, Watanabe CK, de Boer HA (1987) Compilation and comparison of the sequence context around the ATG start codons inSaccharomyces cerevisiae mRNAs. Nucleic Acids Res 15:3581–3593

    PubMed  Google Scholar 

  • Hinnen A, Meyhack B, Heim J (1989) Heterologous gene expression in Yeast. In: Barr PJ, Brake AJ, Valenzuela P (eds) Yeast genetic engineering. Butterworths, pp 193–213

  • Holland JP, Holland MJ (1980) Structural comparison of two nontandemly repeated yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 255:2596–2605

    PubMed  Google Scholar 

  • Ikemura T (1982) Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. J Mol Biol 158:573–597

    PubMed  Google Scholar 

  • Ito H, Fukada Y, Muraka K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    PubMed  Google Scholar 

  • Jones EW (1991) Vacuolar proteases in yeastSaccharomyces cerevisiae. Methods Enzymol 185:372–386

    Google Scholar 

  • Kaiser C, Michaelis S, Mitchell A (Eds) (1994) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  • Knudsen K (1979) Medium-chain fatty acid synthesis in lactating rabbit mammary gland. Biochem J 181:267–274

    PubMed  Google Scholar 

  • Kozak M (1992) Regulation of translation in eucaryotic systems. Annu Rev Cell Biol 8:197–225

    PubMed  Google Scholar 

  • Kühn L, Castorph H, Schweizer E (1972) Gene linkage and gene-enzyme relations in the fatty acid synthetase system ofSaccharomyces cerevisiae. Eur J Biochem 24:492–497

    PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Laux T, Schweizer M (1990) Dietary-induced pre-translational control of rat fatty acid synthase. Biochem J 266:793–797

    PubMed  Google Scholar 

  • Ludwig DL, Ugolini S, Bruschi CV (1993) High-level heterologous gene expression inSaccharomyces cerevisiae from a stable 2-micron plasmid system. Gene 132:33–40

    PubMed  Google Scholar 

  • Lynen F (1969) Yeast fatty acid synthetase. Methods Enzymol 14:17–33

    Google Scholar 

  • Martin-Eauclaire MF, Søgaad M, Ramos C, Cestèle S, Bougis PE, Svensson B (1994) Production of active, insect-specific scorpion neurotoxin in yeast. Eur J Biochem 223:637–645

    PubMed  Google Scholar 

  • McAleer WJ, Buynak EB, Maigetter RZ, Wampler E, Miller WJ, Hilleman MR (1984) Human hepatitis B vaccine from recombinant yeast. Nature 307:178–180

    PubMed  Google Scholar 

  • Mylin LM, Hofmann KJ, Schultz LD, Hopper JE (1991) RegulatedGAL4 expression cassette providing controllable and high-level output from high-copy galactose promoters in yeast. Methods Enzymol 185:297–308

    Google Scholar 

  • Nepokroeff CM, Lakshmanan MR, Porter JW (1975) Fatty acid synthase from rat liver. Methods Enzymol 35:37–44

    PubMed  Google Scholar 

  • Randez-Gil F, Sanz P (1993) Expression of Aspergillus oryzae α-amylase gene inSaccharomyces cerevisiae. FEMS Microbiol Lett 112:119–124

    PubMed  Google Scholar 

  • Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast. Yeast 8:423–488

    PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • Schneider JC, Guarente L (1991) Vectors of expression of cloned genes in yeast: regulation, overproduction and underproduction. Methods Enzymol. 194:373–388

    PubMed  Google Scholar 

  • Schüller HJ, Förtsch B, Ramenstrauss B, Wolf DH, Schweizer E (1992) Differential proteolysic sensitivity of yeast fatty acid synthetase subunits α and β contributing to a balanced ratio of both fatty acid synthetase components. Eur J Biochem 203:607–614

    PubMed  Google Scholar 

  • Schultz LD, Tanner J, Hofmann KJ, Emini EA, Condra JH, Jones RE, Kieff E, Ellis RW (1987) Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus. Gene 54:113–123

    PubMed  Google Scholar 

  • Schweizer M (1986) Yeast fatty acid synthase genes. In: Hardie DG, Coggins JR (eds) Multidomain proteins-structure and evolution. Elsevier Biomedical Press, Amsterdam, New York, pp 195–228

    Google Scholar 

  • Schweizer M, Takabayashi K, Geiger T, Laux T, Biermann G, Buhler J.-M, Roberts LM, Heinrich PC (1987) Identification and sequencing of cDNA clones for the rodent negative acute-phase protein α1-inhibitor 3. Eur J Biochem 164:375–381

    PubMed  Google Scholar 

  • Schweizer M, Takabayashi K, Laux T, Beck K-F, Schreglmann R (1989) Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA. Nucleic Acids Res 17:567–586

    PubMed  Google Scholar 

  • Southgate VJ, Steyn AJC, Pretorius IS, Van Vuuren HJJ (1993) Expression and secretion ofBacillus amyloliquefaciens α-amylase by using the yeast pheromone α-factor promoter and leader sequence inSaccharomyces cerevisiae. Appl Environ Microbiol 59:1253–1258

    PubMed  Google Scholar 

  • Tautz D, Renz M (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132:14–19

    PubMed  Google Scholar 

  • Vaessen RTMJ, Kreike J, Groot GSP (1981) Protein transfer to nitrocellulose filters. FEBS Lett 124:193–196

    PubMed  Google Scholar 

  • Vernet T, Dignard D, Thomas DY (1987) A family of yeast expression vectors containing the phage fl intergenic region. Gene 52:225–233

    PubMed  Google Scholar 

  • Yanish-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC 19 vectors. Gene 33:103–119

    PubMed  Google Scholar 

  • Witkowska HE, Green BN, Smith S (1990) The carboxyl-terminal region of thioesterase 11 participates in the interaction with fatty acid synthase. J Biol Chem 265:5662–5665

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Biochemie, Universität Erlangen-Nürnberg, Staudtstrasse. 5, D-91058, Erlangen, Germany

    Rosemarie Kupfer

  2. Genetics and Microbiology Department, Norwich Research Park, Institute of Food Research, NR4 7UA, Colney, Norwich, UK

    Flora Beiche & Michael Schweizer

Authors
  1. Rosemarie Kupfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Flora Beiche
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Schweizer
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by K. Wolf

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kupfer, R., Beiche, F. & Schweizer, M. Construction of the complete rat fatty acid synthase cDNA and its expression inSaccharomyces cerevisiae . Curr Genet 29, 219–226 (1996). https://doi.org/10.1007/BF02221551

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02221551

Key words

Search

Navigation