Summary
Derivatives of Drosophila 70,000 dalton heat shock protein (hsp70) genes were constructed in which all of the hsp70 coding sequence but for the first seven codons had been substituted by a DNA segment coding for E. coli β-galactosidase. The constructs were capable of directing the synthesis of active β-galactosidase in COS1 (SV40 transformed African Green Monkey Kidney) cells. The hybrid genes were then used to develop a procedure permitting the introduction of genes and their transient expression in cultured cells of Drosophila melanogaster. Introduction of hybrid genes was achieved by DEAE-dextran-mediated transfection. Substantial gene activity was observed in heat-treated cells only 4 h, maximal activity 24 h after transfection. Various parameters of the transfection/transient expression system including the effects of different 3'nontranslated sequences on hybrid gene expression were investigated in an attempt to provide a useful procedure for studies of the expression of other genes in D. melanogaster cells. To show that promoters which are weaker than that of the hsp70 gene direct the synthesis of easily measurable amounts of β-galactosidase in D. melanogaster cells, the expression of a hsp84-β-galactosidase hybrid gene was also examined. Expression of the hsp70 hybrid gene occurs during heat shock, at temperatures at which other proteins are not made, and decreases sharply after heat treatment. The expression of the transfected gene therefore closely follows that of the endogenous hsp70 genes. This result suggests that a short hsp70 gene segment consisting of 195 base pairs of upstream sequence and a complete RNA leader region contain all the information required for the induced synthesis of proteins during heat shock.
Similar content being viewed by others
References
Ashburner M, Bonner JJ (1979) The induction of gene activity in Drosophila by heat shock. Cell 17:241–254
Bienz M, Pelham HRB (1982) Expression of a Drosophila heat shock protein in Xenopus oocytes: conserved and divergent regulatory signals. EMBO J 1:1583–1588
Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980
Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J (1983) β-galactosidase gene fusions for analyzing gene expression in E. coli and yeast. In: Wu R (ed) Methods in enzymology, vol 100 B. Academic Press Inc, New York
Corces V, Pellicer A, Axel R, Meselson M (1981) Integration, Transcription, and control of a Drosophila heat shock gene in mouse cells. Proc Natl Acad Sci USA 78:7038–7042
DiDomenico BJ, Bugaisky GE, Lindquist S (1982 a) The heat shock response is self-regulated at both the transcriptional and post-transcriptional levels. Cell 31:593–603
DiDomenico BJ, Bugaisky GE, Lindquist S (1982 b) Heat shock and recovery are mediated by different translational mechanisms. Proc Natl Sci USA 79:6181–6185
DiNocera PP, Dawid IB (1983) Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci USA 80:7095–7098
Holmgren R, Corces V, Morimoto R, Blackman R, Meselson M (1981) Sequence homologies in the 5′ regions of four Drosophila heat shock genes. Proc Natl Acad Sci USA 78:3775–3778
Ingolia TD, Craig EA (1981) Primary sequence of the 5′ flanking regions of the Drosophila heat shock genes in chromosome subdivision 67B. Nucl Acids Res 9:1627–1642
Ireland RC, Berger E, Sirotkin K, Yund MA, Osterbur D, Fristrom D (1982) Ecdysterone induces the transcription of four heat shock genes in Drosophila S3 cells and imaginal discs. Dev Biol 93:498–507
Karch F, Torok I, Tissieres A (1981) Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster. J Mol Biol 148:219–230
Kruger C, Benecke BJ (1981) In vitro translation of Drosophila heat shock and non-heat-shock mRNAs in heterologous and homologous cell-free systems. Cell 23:595–603
Lis J, Costlow N, de Banzie J, Knipple D, O'Connor D, Sinclair L (1982) Transcription and Chromatin Structure of Drosophila heat-shock genes in yeast. In: Schlesinger MJ (ed) Heat shock from bacteria to man. Cold Spring Harbor Laboratory, New York, pp 57–62
Luthman H, Magnusson G (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucl Acids Res 11:1295–1308
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning. Cold Spring Harbor Laboratory, New York
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564
Mellon P, Parker V, Gluzman Y, Maniatis T (1981) Identification of DNA sequences required for transcription of the human α1-globin gene in a new SV40 host-vector system. Cell 27:279–288
Mirault ME, Goldschmidt-Clermont M, Moran L, Arrigo P, Tissieres A (1978) The effect of heat shock on gene expression in Drosophila melanogaster. Cold Spring Harbor Symp Quant Biol 42:819–827
Mirault ME, Southgate R, Delwart E (1982) Regulation of heat-shock genes: a DNA sequence upstream of Drosophila hsp70 genes is essential for their induction in monkey cells. EMBO J 1:1279–1285
Moran L, Mirault ME, Tissieres A, Schedl P, Artavanis-Tsakonas S, Gehring W (1979) Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein. Cell 17:1–8
O'Connor D, Lis J (1981) Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation. Nucl Acids Res 9:5075–5092
Pelham HRB (1982) A regulatory upstream promoter element in the Drosophila hsp70 heat-shock gene. Cell 30:517–528
Rubin GM, Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353
Savakis C, Demetri G, Cherbas P (1980) Ecdysteroid-inducible polypeptides in a Drosophila cell line. Cell 22:665–674
Schlesinger MJ, Ashburner M, Tissieres A, eds (1982) Heat shock from bacteria to man. Cold Spring Harbor Laboratory, New York
Southgate R, Ayme A, Voellmy R (1983) Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B. J Mol Biol 165:35–57
Storti RV, Scott MP, Rich A, Pardue ML (1980) Translational control of protein synthesis in response to heat shock in D. melanogaster cells. Cell 22:825–834
Subramani S, Mulligan R, Berg P (1981) Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol 1:854–864
Voellmy R, Rungger D (1982) Transcription of a Drosophila heat shock gene is heat-induced in Xenopus oocytes. Proc Natl Acad Sci USA 79:1776–1780
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Lawson, R., Mestril, R., Schiller, P. et al. Expression of heat shock-β-galactosidase hybrid genes in cultured Drosophila cells. Molec Gen Genet 198, 116–124 (1984). https://doi.org/10.1007/BF00328710
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00328710