Deviation of carbohydrate metabolism by the SIT4 phosphatase in Saccharomyces cerevisiae.
Willy Jablonka¶*, Simón Guzmán £*, Jorge Ramírez £, and Mónica Montero-Lomelí¶¥
¶Departamento de Bioquímica Médica, ICB-CCS, Universidade Federal do Rio de Janeiro, Brazil , £Departamento de Genética Molecular, IFC, Universidad Nacional Autónoma de México
Printing of arrays. Yeast 50-mer oligo library from MWGBiotech Oligo Sets (http:/www.mwgbiotech.com) were resuspended to 40mM in Micro Spotting solution (Telechem International Inc.). SuperAmine coated slides 25x75 mm (TeleChem International INC) were printed in duplicate, and fixed at 80°C for 4 hours. For pre-hybridization the slides were re-hydrated with water vapor at 60°C, and fixed with two cycles of UV light (1200J). After boiling for two minutes at 92°C, slides were washed with 95% ethanol for one minute and prehybridzed in 5X SSC, 0.1% SDS and 1% BSA for one hour at 42°C .The slides were washed and dried for further hybridization.
Probe preparation and hybridization to arrays. 10 mg of total RNA were used for cDNA synthesis incorporating dUTP-Cy3 or dUTP-Cy5 employing the CyScribe Firs-Strand cDNA labeling kit (Amersham). Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Cy3 and 655 for Cy5. Equal quantities of labeled cDNA were hybridized using hybridization solution HybIT2 (TeleChem International INC), to the collection of yeast arrays for 14 h at 42°C. For duplicate experiments RNA was used from independent cultures and fluorophore used was dUTP-Cy5 for FY833 and dUTP-Cy3 for FY833-Dsit4 strain and in the second experiment labeling was switched.
Data acquisition and analysis of array images. Acquisition and quantification of array images was performed in ScanArray 4000 with its accompanying software ScanArray 4000 from Packard BioChips. All images were captured using 60% PMT gain, 70 to 75% laser power and 10µm resolution at 50% scan rate. For each spot the density mean value (D), background mean value (B), normalized signal mean value(S), which is the result from (D-B) and inertial diameter (I) were calculated with software ArrayPro Analyzer from Media Cibernetics. The normalized signal mean value (S) for each spot was then normalized using as 100% the total mean signal value obtained from all spots of the array.
Signal and noise selection. To select significant changes the ratio between the mean density from all spots to the mean background of all spots of the array was calculated (Dmean / Bmean). Good signal from Cy3 or Cy5 spots were selected if DCyX / BCyX of each spot was >= (Dmean / Bmean). Good spot size was selected if Cy3 inertial diameter (ICy3) to Cy5 inertial diameter (ICy5) ratio was >= to 0.9 and <= 1.1.
Selection of regulated genes in individual arrays. The Log2 Cy3/Cy5 signal ratio for each spot was obtained. Up-regulated genes were selected if Log2 (SCy3/SCy5) was > 0.6 (1.5 fold up-regulated). Down-regulated genes were selected if Log2 (SCy3/SCy5) was < -0.6 (1.5 fold down-regulated). As each array contains a duplicate for each gene the average and standard deviation of both spots signals was calculated. The spots were selected as positive only if the average value between them was two times the standard deviation. The conditions to be met for up-regulated genes were:DCy3 / BCy3 >= DmeanCy3 / BmeanCy3; ICy3 / ICy5 >= 0.9; ICy3 / ICy5 <= 1.1; Log2 (SCy3/SCy5) > 0.6 and Saverage > 2 X StDev
The conditions to be met for down-regulated genes were: DCy5 / BCy5 >= DmeanCy5 / BmeanCy5; ICy3 / ICy5 >= 0.9; ICy3 / ICy5 <= 1.1; Log2 (SCy3/SCy5) < -0.6; Saverage > 2 X StDev
Similar results were obtained by freeware BRB array tools (linus.nci.nih.gov).Final fold changes were calculated by using the average of three individual. Genes were considered as having a changed expression if average (Sfinal) between experiments of Log2 (SCyx/SCyx) < -0.6 or Log2 (SCyx/SCyx) > 0.6 and Sfinal=2X standard deviation.
Gene functions were annotated in february 2005 according to SGD (http://genome-www.stanford.edu/Saccharomyces/). Clusters were originated using the SGD Gene Ontology Term Finder (http://genome-www4.stanford.edu/cgi-bin/SGD/GO/goTermFinder) applying a cut-off of p < 0.01. Over-represented gene groups with common biological processes were annotated.
Microarray primary data
Intensity values for each gene included in the MWGBiotech
Oligo set (http:/www.mwgbiotech.com), the corresponding background balue for
each gene, signal intensity and expression is included in each table as
tab-delimited text file. Three independent experiments for cells grown on
galactose 2% are included (Galactose 1, galactose 2 and galactose 3).
Changed genes in sit4 mutant
versus wild type glucose grown cell Tables with ORFs with changed expression are shown here as tab-delimited
We thank Felipe Figueiredo for his help in designing this page.
Changed genes in sit4 mutant versus wild type glucose grown cell
Tables with ORFs with changed expression are shown here as tab-delimited