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Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase.

Heidrich K, Fraenkel DG - BMC Genet. (2002)

Bottom Line: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons.Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston MA 02115, USA. kheid@server3.medizin.uni-leipzig.de

ABSTRACT

Background: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.

Results: In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot.

Conclusion: An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

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Glucose metabolism at 23°C after pretreatment in growth medium at 37°C. The control cultures, left hand panels, were similarly handled, but at 23°C. Left hand axes are for glucose or ethanol, mM in medium; right hand axes for fructose-1,6-P2 (Fru-1,6-P2) and glycerate-3-P (Gta-3-P), mM in cells.
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Figure 1: Glucose metabolism at 23°C after pretreatment in growth medium at 37°C. The control cultures, left hand panels, were similarly handled, but at 23°C. Left hand axes are for glucose or ethanol, mM in medium; right hand axes for fructose-1,6-P2 (Fru-1,6-P2) and glycerate-3-P (Gta-3-P), mM in cells.

Mentions: Glucose metabolism, at 23°C, in resting cells [3,7] was first assessed with cells grown at 23°C but pretreated in growth medium for an additional period of 30 min at 37°C (or, control, 23°C). As shown in Fig. 1, from both temperatures the wild type strain showed similar glucose metabolism and the usual low glycerate-3-P concentration. By contrast, lower panels, the 37°C-pretreated mutant suspension was considerably lowered in its rate of glucose metabolism and glycerate-3-P was accumulated to high level, as in a phosphoglycerate mutase mutant [3]; the control cells not exposed to 37°C showed slightly elevated levels of this metabolite. The decrease in glucose flux, v, in the test situation of non-growing cells at 23°C, depended on time of previous exposure to 37°C, with, e.g., values of 0.76 (no exposure to 37°C), 0.24 (30 min exposure), 0.09 (60 min) and 0.03 (90 min). Thus it appears that inactivation of the reaction in vivo was stopped by restoration to the lower temperature.


Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase.

Heidrich K, Fraenkel DG - BMC Genet. (2002)

Glucose metabolism at 23°C after pretreatment in growth medium at 37°C. The control cultures, left hand panels, were similarly handled, but at 23°C. Left hand axes are for glucose or ethanol, mM in medium; right hand axes for fructose-1,6-P2 (Fru-1,6-P2) and glycerate-3-P (Gta-3-P), mM in cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117797&req=5

Figure 1: Glucose metabolism at 23°C after pretreatment in growth medium at 37°C. The control cultures, left hand panels, were similarly handled, but at 23°C. Left hand axes are for glucose or ethanol, mM in medium; right hand axes for fructose-1,6-P2 (Fru-1,6-P2) and glycerate-3-P (Gta-3-P), mM in cells.
Mentions: Glucose metabolism, at 23°C, in resting cells [3,7] was first assessed with cells grown at 23°C but pretreated in growth medium for an additional period of 30 min at 37°C (or, control, 23°C). As shown in Fig. 1, from both temperatures the wild type strain showed similar glucose metabolism and the usual low glycerate-3-P concentration. By contrast, lower panels, the 37°C-pretreated mutant suspension was considerably lowered in its rate of glucose metabolism and glycerate-3-P was accumulated to high level, as in a phosphoglycerate mutase mutant [3]; the control cells not exposed to 37°C showed slightly elevated levels of this metabolite. The decrease in glucose flux, v, in the test situation of non-growing cells at 23°C, depended on time of previous exposure to 37°C, with, e.g., values of 0.76 (no exposure to 37°C), 0.24 (30 min exposure), 0.09 (60 min) and 0.03 (90 min). Thus it appears that inactivation of the reaction in vivo was stopped by restoration to the lower temperature.

Bottom Line: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons.Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston MA 02115, USA. kheid@server3.medizin.uni-leipzig.de

ABSTRACT

Background: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead.

Results: In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot.

Conclusion: An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

Show MeSH
Related in: MedlinePlus