Article Figures & Data
Figures
- Fig. 1.
Alignment of amino acid sequences of several theta-class GSTs. Each amino acid sequence was deduced from the cloned-DNA sequence (GST Y-2 [25], maize GST I [21], maize GST III [11], P. mirabilis GST [17], and E. coli GST [13]). Amino acid positions are indicated on the right. The amino acid residues conserved with those of GST Y-1 are shaded. Asterisks indicate the amino acids completely conserved in the six GSTs. The double dagger indicates conserved serine residues in theta-class GSTs.
- Fig. 2.
Northern blot analysis. Twenty-microgram portions of total RNA extracted from I. orientalis cultured with (induced [I]) or without (control [C]) 200 μM DNB were loaded onto 1% agarose gels and, after electrophoresis, stained with ethidium bromide (bottom). After proteins were blotted onto Hybond N+ nylon membranes, hybridization was performed with32P-labeled GST Y-1 (top left) or Y-2 (top right) cDNA probe (2 × 106 cpm/ml). The positions of 25S and 18S rRNAs are indicated on the right.
- Fig. 3.
Effects of DNB on cell growth and GST gene expression.I. orientalis cells grown in YPD medium to late exponential phase were inoculated into a jar fermentor containing 20 liters of YPD medium, and the cells were further cultivated after DNB was added to 200 μM. (A and B) Before and after DNB addition, cell cultures were sampled at the indicated times and samples were subjected to Northern blot analysis. Levels of GST Y-1 (top) and GST Y-2 (middle) mRNAs are shown. Total RNA was loaded onto agarose gels, electrophoresed, and stained with ethidium bromide (bottom). (A) Cells cultured for 0 to 12 h; (B) cells cultured for 32 to 78 h. (C and D) Cell growth and DNB concentration (conc.) in the culture medium were also analyzed. (C) Cells cultured for 0 to 12 h; (D) cells cultured for 32 to 78 h. OD600nm, optical density at 600 nm.
Tables
- Table 1.
Purification of GST Y-1 and Y-2
Fraction Amt of protein obtained (mg) Total activity (mU) Sp act (mU/mg of protein) Purification (fold) Yield (%) Protamine 1,000 9,700 9.7 100 DEAE-cellulose 300 8,310 27.7 2.8 85.7 GSH-agarose 0.57 1,230 2,160 223 12.7
