Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

YEAST STRAINS AND MEDIA.

S. pombe bearing the sod2 gene disruption (sod2::ura4) was used as a host for all transformations and as a control strain. It was maintained on yeast extract adenine (YEA) or low sodium KMA medium using standard procedures. KMA medium contained (per liter): potassium hydrophtalate, 3 g; K2HPO4, 3 g; yeast nitrogen base without amino acids, 7 g; glucose, 20 g; adenine, 100 mg, and histidine, 100 mg. Leucine (200 mg/ml) was added to maintain the sod2::ura4 strain that was not transformed with leu2 containing plasmids. SITE-DIRECTED MUTAGENESIS. Site-directed mutagenesis of residues H12, H54, H67, H98, H233, H376, H424, H429, D241, D266, D267 of sod2 was performed using the TransformerTM Site-Directed Mutagenesis Kit (ClonTech, version 2) as recommended by the manufacturer. All mutations were designed to create or remove a restriction enzyme site that could be easily detected in the subsequent analysis. The following oligonucleotides were used to produce the desired gene alterations the names correspond to the mutations - mutated bases are shown in bold and the alteration in the restriction enzyme site follows. The oligonucleotides are: H12R, 5'-A GAC AAA GTC Cgc TTA GCT TTA ATA GTG-3', new Dde I site; H54R, 5'-A TTT GGG CCT Cgc GCT GCT AAA CTC GTA GA-3', new Hha I site; H67R, 5'-C CCT TTT TCC TGG GGT GAC CgT GGA GAT TAC TTG-3', removed a Nco I site; H98R, 5'-GCA TAT TTT CAg Cgc AAT TTT CGA AGC ATC ATT G-3', new Hha I site; H233Rr, 5'-TTT CTG AGC GcG CTT TAA AAT GAA CG-3', new Hha I site; H367Rr, 5'-TAT TGG TCC GAA AcG gCC AAC GAA AAG GGC-3', new Hae III site; H424/429Rr, 5'-CA AAT CAC TAA TAC AcG GAT ACT GAA ACC gcG cAC AAT GAT TG-3', (double mutation), new Hha I site; H367Dr, 5'-CCC TAT TGG gCC GAA ATc TCC AAC GAA AAG GGC-3', new Hae III site; H367Ar, 5'-CC TAT TGG TCC GAA Agc TCC AAC GAA AAG GGC-3', Alu I site; D241Nr, 5'-G CGG AAG GGA ATA ATA gCT AAT AGC ATt AAT TAA ACG GTA T-3', new Alu I site; and D266,267N, 5'-GGA ACT ATT ATT GGA GTT aAc aAC CTG TTG ATG TCC TTT TTT GC-3', (a double mutation), with a new Hinc II site. Oligonucleotides with the "r" designation indicate that the oligonucleotide encodes for the complementary DNA strand. The template for mutagenesis was the plasmid pKS-sod2 that contains the sod2 gene as a 2.3 kb Hind III fragment together with the 187-bp upstream and 692-bp downstream flanking regions (Jia et al., 1992). Mutated double-stranded DNA was transformed into the mutS strain of E. coli (BMH 71-18). The standard trans oligo Sca I/Stu I (ClonTech) was used as a selection primer in all mutagenesis reactions.

After selective digestion of the mixed plasmid pool with Sca I, the plasmid was propagated in E. coli DH5a. The transformant clones were screened by digestion with enzymes whose sites were created/eliminated by the mutagenic primers. Plasmid DNA from the selected clones was isolated and digested with following enzymes to excise the minimal sod2 gene fragments containing the desired mutation. Bsg I and BspE I were used to obtain a 430 bp fragment with the H367 mutation and the H424,429R mutation. Nco I and Nhe I were used to obtain a 549 bp fragment containing the D241N, H67R, H98R and H233R mutations. The combination of Nhe I with BspE I excised a 312 bp insert containing the double D266,267N mutation. The enzymes Sal I and Nco I were used to obtain a 476 bp insert with the H12R or H54R mutation. Mutated fragments were then used to replace the respective sequences in pSK-sod2. After verification by DNA sequencing, the HA tag was introduced at the 3'-terminus of the sod2 gene as described below. HA tagged variants were made for the wild type sod2, as well as for the mutants H367A; H367D; H367R; D241N; and D266,267N. ADDITION OF THE HEMAGGLUTININ (HA) TAG TO THE 3'-TERMINUS OF THE SOD2 GENE. The plasmid pARTA was made by an exchange of Swa I - Swa I fragments that added the HA tag to the wild-type sod2 cloned in pSOD2.11 where it is controlled by the strong ADH promoter. The purpose of this construct was to have a positive control for immunoblotting experiments since we found that expression from the endogenous sod2 promoter was only at low levels. In this work, the 813-bp Swa I-Swa I fragment containing the HA tag, was ligated into a series of pSK-sod2 vectors carrying the wild-type or mutated form of sod2. Finally, tagged variants of the sod2 gene were cloned into the pWH5 shuttle vector as Hind III - Hind III inserts for expression in the S. pombe sod2::ura4 strain.

GROWTH RATE ASSESSMENT.

The lithium tolerance of various transformants was determined at different pHs in liquid KMA media that was supplemented with the indicated amounts of LiCl. Leucine (200 mg/ml) was added for growth of the sod2::ura4 strain. To assess growth 2 x 106 cells were inoculated into 2 ml of medium and incubated at 30oC with vigorous aeration for 24 hours or where specified for 48 hours. Growth was then assessed by measuring the increase in absorbance of the cell suspension at 600 nm. The pH of the culture medium was assessed after cell growth and in all cases the growth medium was not acidified more than 0.10-0.15 pH units. The results presented are typical of at least three experiments for each S. pombe strain and the standard deviation was approximately 15%.

DETERMINATION OF INTRACELLULAR pH.

Cytoplasmic pH in S. pombe sod2::ura4/pWH5-sod2 (i.e., possessing the wild-type sod2) was determined using pH-sensitive fluorescent dye, carboxy-seminaphthorhodafluor-1. The standard deviation for these measurements was less than 5%.

22Na TRANSPORT.

Cells were grown in KMA medium and harvested at a cell density of approximately 5 x 106 cells/ml. They were washed twice with double distilled water, and resuspended in loading buffer (10 mM NaCl, 20 mM Mes-Tris, pH 7.0) at 109 cells/ml. After addition of 0.5 mCi/ml carrier-free 22Na, cells (1 ml) were gently rotated at room temperature for 1 hour. 22Na-loaded cells were briefly pelleted, washed with the same volume of loading buffer without isotope, and diluted quickly into 20 ml of 22Na-free loading buffer of pH 4.0. At the indicated times, 1.5 ml aliquots of the cell suspension were withdrawn, filtered immediately through the 0.8 mm pore-size Millipore AA filters, and washed with 5.0 ml of double distilled water. The radioactivity of filters was measured with a b-counter. Cells treated with 0.2% saponin for 10 minutes, were used to subtract nonspecific absorption of the isotope.

MEASUREMENT OF PROTON UPTAKE.

Approximately 107 cells/ml in logarithmic phase of growth were washed twice with double distilled water. They were then resuspended at a density of 5 x 108 cells/ml in 20 mM Mes/Tris (pH 6.1) with or without 100 mM NaCl. After 2 hours of incubation at room temperature, cells were briefly pelleted in a microfuge and concentrated 5-fold in 0.5 ml of 1 mM Mes/Tris (pH 6.1). They were quickly added to 2 ml of the same buffer in a stirred cuvette and the pH was measured using a Fisher Scientific Accumet 925 pH meter. Data were collected with an Apple Macintosh SE computer.

MEMBRANE ISOLATION.

Yeast cells were grown aerobically in KMA medium to an OD600 of 1.5 at 30oC and the following procedures were carried out at 0-4oC. After pelleting and washing with double distilled water, cells were resuspended at a concentration of 5 X 109 cells/ml in disruption buffer (50 mM Tris/HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 1 mM EGTA, 5 mg/ml bovine serum albumin, protease inhibitor cocktail, and 1 mM dithiothreitol) and passed through a French Press at 20,000 p.s.i. Unbroken cells were pelleted by centrifugation at 3,500 x g for 5 min, and the supernatant was centrifuged at 14,000 x g for 20 min. Membranes were then pelleted at 200,000 x g for 1 hour and resuspended in 1 mM EGTA-Tris (pH 7.5), 10% glycerol using a glass homogenizer with a Teflon plunger. The membrane suspension was centrifuged as before, and finally resuspended in a small volume of 1 mM EGTA-Tris (pH 7.5), 10% glycerol.

ELECTROPHORETHIC METHODS.

Yeast membranes were pre-extracted and solubilized using dodecylmaltoside. Proteins were resolved using a 12% polyacrylamide gel and were visualized using Coomassie Blue staining. Western blotting and immunoblotting were performed essentially as described erlier. Anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim, Laval, Que., Canada) was used at a dilution of 1:500, and peroxidase-conjugated goat anti-mouse antibody (Bio/Can, Mississaugua, ON) was used at a dilution of 1:2000. Immunoreactive proteins were visualized using the Amersham Enhanced Chemiluminescence kit as described by the manufacturer.

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