Heat Shock-regulated Production of Escherichia Coli Beta-galactosidase in Saccharomyces Cerevisiae

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1983 Sep 1

PMID 6415404

Citations 19

Authors

D B Finkelstein

S Strausberg

Affiliations

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Abstract

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

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References

Yamamori T, Ito K, Nakamura Y, Yura T . Transient regulation of protein synthesis in Escherichia coli upon shift-up of growth temperature. J Bacteriol. 1978; 134(3):1133-40. PMC: 222364. DOI: 10.1128/jb.134.3.1133-1140.1978. View

Finkelstein D, Butow R . DNA-binding proteins in yeast. Effect of growth phase and mitochondrial function. Arch Biochem Biophys. 1976; 174(1):52-65. DOI: 10.1016/0003-9861(76)90323-4. View

Ashburner M, Bonner J . The induction of gene activity in drosophilia by heat shock. Cell. 1979; 17(2):241-54. DOI: 10.1016/0092-8674(79)90150-8. View

Stinchcomb D, Struhl K, Davis R . Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979; 282(5734):39-43. DOI: 10.1038/282039a0. View

Tschumper G, Carbon J . Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980; 10(2):157-66. DOI: 10.1016/0378-1119(80)90133-x. View

Broach J, Hicks J . Replication and recombination functions associated with the yeast plasmid, 2 mu circle. Cell. 1980; 21(2):501-8. DOI: 10.1016/0092-8674(80)90487-0. View

Casadaban M, Chou J, Cohen S . In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980; 143(2):971-80. PMC: 294402. DOI: 10.1128/jb.143.2.971-980.1980. View

Guarente L, Ptashne M . Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981; 78(4):2199-203. PMC: 319311. DOI: 10.1073/pnas.78.4.2199. View

Rose M, Casadaban M, Botstein D . Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci U S A. 1981; 78(4):2460-4. PMC: 319366. DOI: 10.1073/pnas.78.4.2460. View

10.

Burnette W . "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; 112(2):195-203. DOI: 10.1016/0003-2697(81)90281-5. View

11.

Corces V, Pellicer A, Axel R, Meselson M . Integration, transcription, and control of a Drosophila heat shock gene in mouse cells. Proc Natl Acad Sci U S A. 1981; 78(11):7038-42. PMC: 349189. DOI: 10.1073/pnas.78.11.7038. View

12.

Bennetzen J, Hall B . Codon selection in yeast. J Biol Chem. 1982; 257(6):3026-31. View

13.

Zaret K, Sherman F . DNA sequence required for efficient transcription termination in yeast. Cell. 1982; 28(3):563-73. DOI: 10.1016/0092-8674(82)90211-2. View

14.

Finkelstein D, Strausberg S, McAlister L . Alterations of transcription during heat shock of Saccharomyces cerevisiae. J Biol Chem. 1982; 257(14):8405-11. View

15.

Finkelstein D, Strausberg S . Identification and expression of a cloned yeast heat shock gene. J Biol Chem. 1983; 258(3):1908-13. View

16.

Beggs J . Transformation of yeast by a replicating hybrid plasmid. Nature. 1978; 275(5676):104-9. DOI: 10.1038/275104a0. View