Isolation and properties of yeast mutants with highly efficient thymidylate utilization

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biochemistry

Background:

• Saccharomyces cerevisiae possesses an inherent ability to utilize exogenous deoxythymidine-5 -monophosphate (5 -dTMP).
• Specific mutations (typ, tup, tmp, per, tum) can significantly enhance this wild-type capability.

Purpose of the Study:

• To develop a screening method for isolating yeast mutants with highly efficient 5 -dTMP utilization.
• To characterize the phenomenon of 5 -dTMP utilization in yeast.
• To investigate the genetic and physiological factors influencing 5 -dTMP uptake and sensitivity.

Main Methods:

• Screening procedure for isolating yeast mutants (typ tir) with enhanced 5 -dTMP utilization.
• Analysis of wild-type and mutant yeast strains for 5 -dTMP incorporation and utilization.
• Mathematical modeling to describe the observed levels of 5 -dTMP utilization.

Main Results:

• Identified yeast mutants capable of over 50% utilization of exogenous 5 -dTMP.
• Demonstrated that 5 -dTMP utilization is an amplified wild-type trait, not solely mutation-dependent.
• Quantified eight distinct stages of 5 -dTMP utilization, fitting a "2n x 1.5" mathematical series.
• Found that optimal 5 -dTMP uptake requires acidic extracellular conditions (pH ≤ 6).
• Observed 5 -dTMP-mediated growth inhibition in sensitive mutants, dependent on thymidylate biosynthesis.
• Characterized extracellular dephosphorylation of 5 -dTMP to thymidine by a phosphatase in typ/tlr mutants.

Conclusions:

• Specific yeast mutations act as amplifiers of a pre-existing wild-type 5 -dTMP utilization capacity.
• The uptake and utilization of 5 -dTMP are complex processes influenced by genetic alterations, environmental pH, and thymidylate biosynthesis.
• Extracellular phosphatase activity contributes to 5 -dTMP metabolism but is distinct from uptake and sensitivity mechanisms.