1. Volume 13, Issue 6, Pages 771-781 (March 2004)
Yeast Adapt to Near-Freezing Temperatures by STRE/Msn2,4-Dependent Induction of Trehalose Synthesis and Certain Molecular Chaperones 
Olga Kandror, Nancy Bretschneider, Evgeniy Kreydin, Duccio Cavalieri, Alfred L Goldberg 
Molecular Cell 
Volume 13, Issue 6, Pages (March 2004)
DOI: /S (04)

2. Figure 1
Increase in TPS1 and TPS2 mRNA Content after Temperature Shift from 30°C to 10°C, 4°C, and 0°C
Cultures were grown to mid-log phase and shifted to the indicated temperatures. Before and at different times after the shift, samples were taken, and total RNA was isolated and analyzed by Northern blot.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2
Synthesis and Cellular Content of Tps1 and Tps2 Proteins Increase at 0°C
(A) Wild-type cells were grown at 30°C in minimal media lacking methionine until mid-log phase, when half of the culture was transferred to 0°C. Thirty milliliter aliquots were labeled for 15 min at 30°C or for 6 hr after 24 hr incubation at 0°C. Thirty microliters (0.3 mCi) of methionine (Amersham) was used in each labeling experiment. Cells were harvested by centrifugation, washed with 50 ml ice-cold water, and stored at −80°C. Equal amounts of cell extracts (by radioactivity) were loaded on the gel and analyzed by PAGE, followed by Western blot with anti-Tps antiserum and autoradiography.
(B and C) Cells were grown until mid-log phase at 30°C and shifted to 0°C. Cell extracts were prepared before the shift and after 48 hr at 0°C. Equal amounts of the extracts (by protein) were loaded onto PAGE and analyzed by Western blot with anti-Tps or anti-Hsp104 antiserum.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3
Yeast Growth and Trehalose Accumulation at Different Temperatures
Wild-type cells were grown at 30°C to mid-log phase and shifted to designated temperatures, and growth rate (circles) and trehalose content (triangles) were followed for up to 100 hr after the shift (0′ time).
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
A Number of Near-Freezing mRNAs Are Induced at 0°C and Disappear within Minutes upon Return to 30°C
(A) Increase in TPS1, TPS2, and HSP104 mRNA levels at 0°C is Msn2,4 dependent. Wild-type and ΔGCN, ΔYAP1, ΔYAP1,2,5, and ΔMSN2,4 deletion strains were grown until mid-log phase and shifted to 0°C. Before and 24 hr after the shift, samples were taken, and total RNA was isolated and analyzed by Northern blot with TPS1, TPS2, and HSP104 probes. ACT1 was used as a control.
(B) A number of near-freezing mRNAs are rapidly degraded upon return to 30°C. Wild-type cells were grown until mid-log phase at 30°C, shifted for 2 days to 0°C, and then returned to 30°C. Samples were taken after 2 days at 0°C, and at different times after the shift to 30°C, total RNA was isolated and analyzed by Northern blot with TPS1, TPS2, HSP104, NTH1, and ACT1 (control) probes. Similar results were obtained when 1,10-phenanthroline (100 μg/ml) was added before the shift to 30°C to block mRNA synthesis.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5
Mutant Strains Carrying Deletions in TPS1,2 or MSN2,4 Genes Are Less Viable at 0°C (A) and −20°C (B)
(A) Exponentially growing wild-type and mutant cultures (ΔTPS1,2, ΔHSP104, ΔSSA4, ΔMSN2,4, ΔYAP1,2,5, ΔGCN4) were shifted to 0°C. Trehalose levels were determined after 3 days at 0°C. In ΔHSP104, ΔSSA4, ΔYAP1,2,5, and ΔGCN4, they were similar to those of the wild-type. Before and every 7 days after the shift, aliquots of suitable dilutions were plated onto YPGal agar plates. After incubation at 30°C for 3 days for a wild-type and 5 days for mutant, colonies were counted, and the percent of viable cells was measured.
(B) Wild-type and mutant strains were grown at 30°C until mid-log phase. Equal amounts of cells (OD600) were spotted onto sterile filter paper (1 cm2) and shifted to −20°C. Before freezing, and then 60 min, 1 day, and 3 days after freezing, filters were incubated for 15 min in 2 ml YPGal media and plated on YPGal plates, and the percent of viable cells was determined. Viability of ΔHSP104, ΔSSA4, ΔYAP1,2,5, and ΔGCN4 mutants at 0°C and −20°C was similar to that of the wild-type.
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
Freeze Tolerance Closely Correlates with Cellular Trehalose Content
(A) Adaptation of wild-type (but not ΔTPS1,2) cells to 4°C increases their resistance to freezing. Wild-type and ΔTPS1,2 cells were grown until mid-log phase at 30°C, and each culture divided in two. One portion was shifted to 4°C, and after 2 days, cells were frozen at −20°C, and the percent of viable cells was determined after different times at −20°C (as in Figure 5). The other portion was transferred from 30°C directly to −20°C, and the percent of viable cells was determined.
(B) Trehalose content in the wild-type and ΔNTH1 mutant at 30°C and after 2 days at 0°C.
(C) Trehalose content and acquired freeze tolerance decrease rapidly in the wild-type (but not in ΔNTH1 mutant) upon return to 30°C. The wild-type and ΔNTH1 mutant were grown until mid-log phase at 30°C, shifted to 4°C for 2 days, and then returned to 30°C. Samples were taken for assay of trehalose content and sensitivity to freezing (as in Figure 5) before the shift to 4°C, after 2 days at 4°C, and at various times after the shift to 30°C. The increase in the amount of cells resistant to freezing due to adaptation at 4°C (termed “acquired freeze tolerance”) was taken as 100%.
Molecular Cell  , DOI: ( /S (04) )