Summary
Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
Similar content being viewed by others
References
Baim SB, Sherman F (1988) Mol Cell Biol 8:1591–1602
Bennetzen JL, Hall BD (1982) J Biol Chem 257:3026–3031
Boulnois GJ (ed) (1987) Gene cloning and analysis: a laboratory guide. Blackwell Scientific Publications, Oxford, UK
Bradford MM (1976) Anal Biochem 147:248–254
Casey GP, Xiao W, Rank GH (1988) J Inst Brew 94:93–97
Cashmore AM, albury MS, Hadfield C, Meacock PA (1986) Mol Gen Genet 203:154–162
chen CY, Oppermann H, Hitzeman RA (1984) Nucleic Acid Res 12:8951–8970
Cigan AM, Donahue TF (1987) Gene 59:1–18
Fogel S, Welch J (1982) Proc Natl Acad Sci USA 79:5342–5346
Gale EF, Cundliffe E, Reynolds PE, Richmond MH, Waring MJ (1981) The Molecular Basis of Antibiotic Action, 2nd, edn. Wiley, London, pp 503–506
Haas MJ, Dowding JE (1975) Methods Enzymol 43:611–628
Hadfield C, Cashmore AM, Meacock PA (1986) Gene 45:149–158
Hadfield C, Cashmore AM, Meacock PA (1987) Gene 52:59–70
Henderson RCA, Cox BS, Tubb R (1985) Curr Genet 9:133–138
Hames BD (1981) An introduction to polyacrylamide gel electrophoresis. In: Hames BD, Rickwood D (eds) Gel electrophoresis of proteins. IRL Press, Oxford, pp 1–91
Hoekema A, Kastelein RA, Vasser M, deBoer HA (1987) Mol Cell Biol 7:2914–2924
Hollenberg CP (1982) Curr Topics Microbiol Immunol 96:119–144
Ito H, Fukuda Y, Murata K, Kimra A (1983) J Bacteriol 153:163–168
Jimenez A, Davies J (1980) Nature 287:869–871
Kozak M (1986) Cell 44:283–292
Kozak M (1989) Mol Cell Biol 9:5134–5142
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Mellor J, Dobson MJ, Roberts NA, Kingsman AJ, Kingsman SM (1985) Gene 33:215–226
Mellor J, Dobson MJ, Kingsman AJ, Kingsman SM (1987) Nucleic Acids Res 15:6243–6260
Norrander J, Kempe T, Messing J (1983) Gene 26:101–106
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Proc Natl Acad Sci USA 78:6354–6358
Parent SA, Fenimoe CM, Bostian KA (1985) Yeast 1:83–138
Reiss B, Sprengel R, Will H, Schaller H (1984) Gene 30:211–218
Sharp PM, Tuohy TMF, Mosurski KR (1986) Nucleic Acids Res 14:5125–5143
Sherman F, Stewart JW (1982) Mutations altering initiation of translation of yeast iso-1-cytochrome c; contrasts between the eukaryotic and prokaryotic initiation process. In: Strathern NJ, Jones EW, Broach JR (ed) The molecular biology of the yeast Saccharomyces. Metabolism and gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 301–333
Shine J, Dalgarno L (1974) Proc Natl Acad Sci USA 71:1342–1346
Stark MJR (1987) Gene 51:255–267
Vieira J, Messing J (1982) Gene 19:259–268
Webster TD, Dickson RC (1983) Gene 26:243–252
Zhu J, Contreras R, Gheysen D, Erst J, Fiers W (1985) Biotechnology 3:451–456
Zitomer RS, Hall D (1976) J Biol Chem 251:6320–6326
Zitomer RS, Walthall DA, Rymond BC, Hollenberg CP (1984) Mol Cell Biol 4:1191–1197
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hadfield, C., Jordan, B.E., Mount, R.C. et al. G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae . Curr Genet 18, 303–313 (1990). https://doi.org/10.1007/BF00318211
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00318211