Abstract
GPD1 (encoding glyceraldehyde-3-phosphate dehydrogenase) is a constitutively expressed gene in Cochliobolus heterostrophus that produces a single transcript. The steady state level of GPD1 mRNA is 14-fold greater than that of the constitutively-expressed TRP1 gene (encoding a tryptophan biosynthesis enzyme) indicating that GPD1 has a stronger promoter and/or a more stable mRNA. A set of lacZ translational fusion vectors was constructed to compare the gene expression signals of GPD1, TRP1 and PRO1 (a C. heterostrophus genomic fragment selected for promoter activity) in C. heterostrophus as single copies at the same site in the chromosome. Under conditions that repressed endogenous β-galactosidase expression, β-galactosidase activity in transformants was constitutive and required the GPD1, TRP1 or PRO1 expression signals. In-frame GPD1::lacZ activities were 6-fold greater than in-frame TRP1::lacZ and PRO1::lacZ activities, indicating that GPD1 has more efficient expression signals.
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References
Bernatzky R, Tanksley SD (1986) Methods for detection of single-or low-copy sequences in tomato on Southern blots. Plant Mol Biol Rep 4:37–41
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 72:248–354
Casadaban MJ, Martinez-Arias M, Shapira SK, Chou J (1983) β-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol 100:293–308
Davis MA, Cobbett CS, Hynes MJ (1988) An amdS-lacZ fusion for studying gene regulation in Aspergillus. Genet 63:199–213
Froshauer S, Beckwith J (1984) The DNA sequence to the malF gene and of lacZ fusions to it. J Biol Chem 259:10896–10903
Gorcom RFM van, Pouwels PH, Goosen T, Visser J, van den Broek AWJ, Hamer JE, Timberlake WE, van den Hondel CAMJJ (1985) Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans. Gene 40:99–106
Gorcom REM van, Punt PJ, Pouwels PH, van den Hondel CAMJJ (1986) A system for the analysis of expression signals in Aspergillus. Gene 48:211–217
Guarente L, Ptashne M (1981) Fusion of Escherchia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae Proc Natl Acad Sci USA 78:2199–2203
Hall CV, Jacob PE, Ringold GM, Lee F (1983) Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet 2:101–109
Hamer JE, Timberlake WE (1987) Functional organization of the Aspergillus nidulans trpC promoter. Mol Cell Biol 7:2352–2359
Holland M J, Holland JP (1978) Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry 17:4900–4907
Kaster KR, Burgett SG, Rao RN, Ingolia TD (1983) Analysis of a bacterial hygromycin B gene by transcriptional and translational funsions and DNA sequencing. Nucleic Acids Res 11:6895–6911
Leach J, Lang BR, Yoder OC (1982) Methods for selection of mutants and in vitro culture of Cochliobolus heterostrophus. J Gen Micro 128:1719–1729
Lee SB, Milgroom MG, Taylor JW (1988) A rapid, high-yield miniprep method for isolation of total genomic DNA from fungi. Fungal Genet Newslett 21:23–23
Lis JT, Simon JA, Sutton CA (1983) New heat-shock puffs and β-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid fusion. Cell 35:403–410
Luttrell ES (1958) The perfect stage of Helminthosporium turcicum. Phytopathology 48:281–287
Maniatis T, Fritsch EF, Sambrook (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Piechaczyk M, Blanchard JM, Marty L, Dani Ch, Panabieres F, Sabouty S El, Fort Ph, Jeanteur Ph (1984) Post-transcriptional regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression in rat tissues. Nucleic Acids Res 12:6951–6962
Punt PJ, van den Hondel CAMJJ (1992) Analysis of transcription control sequences in filamentous fungi. In: Tudzynski P, Stahl U (Eds) Proceedings of the EMBO Workshop on Molecular Biology of Filamentous Fungi. CCH Verlag, Berlin, pp 177–180
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CAMJJ (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124
Punt PJ, Dingemanse MA, Jacobs-Meijsing BJM, Pouwels PH, van den Hondel CAMJJ (1988) Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. Gene 69:49–57
Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede RDM, Pouwels PH, van den Hondel CAMJJ (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene coding for glyceraldehyde-3-phosphate dehydrogenase. Gene 93:101–109
Punt PJ, Kramer C, Kuyvenhoven A, Pouwels PH, van den Hondel CAMJJ (1992) An upstream activating sequence from the Aspergillus nidulans gpdA gene. Gene 120:67–73
Rose M, Botstein D (1983) Construction and use of gene fusions to lacZ (β-galactosidase) that are expressed in yeast. Methods Enzymol 101:167–180
Schåfer W, Straney D, Ciuffetti L, Van Etten HD, Yoder OC (1989) One enzyme makes a fungal pathogen, but not a saprophyte, virulent on a new host plant. Science 246:247–249
Silhavy TJ, Beckwith JR (1985) Uses of laxZ fusions for the study of biological systems. Microbiol Rev 49:398–418
Silver PA, Keegan LP, Ptashne M (1984) Amino-terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci USA 81:5951–5955
Simon JA, Lis JT (1987) A germline transformation analysis reveals flexibility in the organization of heat-shock consensus elements. Nucleic Acids Res 15:2971–2988
Streatfield SJ, Toews S, Roberts CF (1992) Functional analysis of the expression of the 3′-phosphoglycerate kinase pkg gene in Aspergillus nidulans. Mol Gen Genet 233:231–240
Sun X-H, Lis JT, Wu R (1988) The positive and negative transcriptional regulation of the Drosophila Gapdh-2 gene. Genes Dev 2:743–753
Turgeon BG, MacRae WD, Garber RC, Fink GR, Yoder OC (1986) A cloned tryptophan-synthesis gene from the ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast, and Aspergillus nidulands Gene 42:79–88
Turgeon BG, Garber RC, Yoder OC (1987) Development of a fungal transformation system based on selection of sequences with promoter activity. Mol Cell Biol 7:3297–3305
Van Wert SL (1990) Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene in Cochliobolus heterostrophus. PhD Dissertation, Cornell University, Ithaca, New York
Van Wert SL, Yoder OC (1992) Structure of the Cochliobolus heterostrophus glyceraldehyde-3-phosphate dehydrogenase gene. Curr Genet 22:29–35
Weinstock GM, Berman ML, Silhavy TJ (1983) In: Papas TS, Rosenberg M, Chirikjian JC (eds) Gene amplification and analysis, vol 3. Expression of cloned genes in procaryotic and eucaryotic cells. Elsevier Scientific Publishing Co. Inc. pp 27–64
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Communicated by H. Bertrand
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Van Wert, S.L., Yoder, O.C. Relative strengths of promoters from Cochliobolus heterostrophus . Curr Genet 25, 217–222 (1994). https://doi.org/10.1007/BF00357165
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DOI: https://doi.org/10.1007/BF00357165