Use of the Tn903 neomycin-resistance gene for promoter analysis in the fission yeast Schizosaccharomyces pombe

Abstract

The bacterial neo gene from transposon Tn903 (Tn601) was used for dominant transformation of the fission yeast Schizosaccharomyces pombe. It was found that high transformation efficiency was dependent on a high level of promoter activity, mediated by the strong promoter of the Schizosaccharomyces pombe alcohol dehydrogenase gene (adh1), as shown by comparing the efficiency of transformation to G418-resistance, the resistance levels of transformed cells, and the in vitro amino-glycoside phosphotransferase activity. On the other hand, the heterologous promoter of the Saccharomyces cerevisiae alcohol dehydrogenase I gene (adc1) is shown to be a weak promoter in Schizosaccharomyces pombe, though its activity is significantly enhanced in cells grown on glycerol as a carbon source. This system for selection and detection of promoter-active sequences may provide a useful basis for the analysis of promoter elements in fission yeast.

An Integrating Vector for Tunable, High Copy, Stable Integration into the Dispersed Ty δ Sites of Saccharomyces cerevisiae

“…The relationship between G418 resistance and pITy2 copy number was explored (Figure 3). The G418 growth inhibition level was measured by using square media plates with a linear gradient of G418 concentration across one dimension (Lang‐Hinrichs et al, 1990). The G418 resistance level is measured as the distance across the gradient to which cell growth is visually observed.…”

“…G418 gradient plates were made by pouring 30 mL of YPD into a sloped, square petri dish. After solidification, the maximum desired concentration of G418 was added to another 30 mL of YPD which was then poured on top of the solidified agar into the same dish laying flat (Lang‐Hinrichs et al, 1990). The G418 was allowed to diffuse to equilibrium vertically for >24 h before use.…”

Biocontrol activity in a nonpathogenic REMI mutant of <i>Fusarium oxysporum</i> f. sp. <i>conglutinans</i> and characterization of its disrupted gene

“…REMI10 and FOC were transformed with pNEO-EGFP and pHYG-EGFP, respectively. pHYG-EGFP carrying egfp under the control of A. nidulans gpd promoter and terminator was provided by Dr. T. Motoyama (RIKEN, Wako, Saitama, Japan), and pNEO-EGFP was constructed by replacing hph in pHYG-EGFP in a geneticin-resistant gene (NPTII ) 23) from pII99, which was provided by Dr. T. Tsuge and Dr. I. Imazaki (Nagoya University, Nagoya, Japan).…”

Overexpression of an archaeal protein in yeast: Secretion bottleneck at the ER

“…Approximate gene copy numbers of pITY4-b transformants were determined through G418 resistance as described previously (Parekh et al, 1996). Gradient plates (Lang-Hinrichs et al, 1990) were used to determine the approximate resistance levels, after which liquid cultures containing G418 were used to more accurately determine resistance levels as described below. Transformants were inoculated into culture tubes of YPD [2% glucose (Fisher Scienti®c), 2% Bacto peptone (Difco), 1% yeast extract (Fisher Scienti®c)] from freshly transformed colonies on YPD plates.…”