caa a22 4500 465757774 CHVBK 20180323111852.0 cr unu---uuuuu 170327e19901101xx s 000 0 eng 10.1007/BF00318211 doi (NATIONALLICENCE)springer-10.1007/BF00318211 G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae [Elektronische Daten] [C. Hadfield, B. Jordan, R. Mount, G. Pretorius, E. Burak] 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. Springer-Verlag, 1990 Saccharomyces cerevisiae nationallicence G418 resistance nationallicence Gene cartridges nationallicence Heterologous Gene expression nationallicence Hadfield C. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut Jordan B. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut Mount R. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut Pretorius G. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut Burak E. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut Current Genetics Springer-Verlag 18/4(1990-11-01), 303-313 0172-8083 18:4<303 1990 18 294 https://doi.org/10.1007/BF00318211 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00318211 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hadfield C. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut NATIONALLICENCE 700 1- Jordan B. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut NATIONALLICENCE 700 1- Mount R. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut NATIONALLICENCE 700 1- Pretorius G. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut NATIONALLICENCE 700 1- Burak E. Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK aut NATIONALLICENCE 773 0- Current Genetics Springer-Verlag 18/4(1990-11-01), 303-313 0172-8083 18:4<303 1990 18 294 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer