Marine Ecosystems Research Department - jamstec japan agency ...

More documents

Recommendations

Info

Japan Marine Science and Technology Center Frontier Research System for Extremophiles ciated with alkaliphily. First of all, we prepared the DNA chip containing , genes identified in the B. halodurans C- genome in this study, and then we analyzed the transcriptome with the mRNAs isolated from both alkaline and neutrality cultures at the exponential growth phase. It was observed that a number of the genes were induced apparently under alkaline pH conditions. The results for the expression pattern of some genes were also confirmed by Northern hybridization analysis. Since most of the alkaliphilic Bacillus strains require sodium ions for growth, we also carried out transcriptome analysis with DNA chips under various concentrations of NaCl. First, we attempted to make NDA chips. The specific -mer oligonucleotide sequence for each gene was designed through BLAST search analysis. The oligonucleotides designed were spotted in duplicate on the silane-treated glass slides by a pen type spotting robot. Bacteria were grown in Horikoshi II medium (pH. or .) in jar fermentor, and harvested during the logarithmic growth phase. Total RNAs from alkaline and neutral culture were isolated and purified. It was quantified by measuring absorbance at nm. The purity of RNA was confirmed by an agarose gel electrophoresis. The mRNAs were converted to cDNAs by coincident labeling with Cy-dUTP or Cy-dUTP. Random hexamer (MDI Inc., Tokyo) was used for the labeling reaction. The hybridization for each sample was duplicated with the probes labeled by reversal fluor. The hybridization was carried out at ˚C for hours with the hybridization buffer. After washing and drying, array images were scanned using GenPix A laser microarray scanner (Axon, Inc). The analysis of the density of each spot and calculation of the expression ratio for each spot were performed using the analysis software. Calculation of the expression ratio between experiments allowed pairwise comparisons of the relative transcript levels for each gene under the two growth conditions. Thus, the changes observed in the DNA chips were not substantially influenced by the difference in bacteria densities. Only those genes whose expression levels differed by a ratio of at least . were evaluated. As shown in Fig. and Table , genes were sig- DNA Microarry Images pH7:pH9.5=Cy3:Cy5 Sample 1 pH7:pH9.5=Cy5:Cy3 Sample 2 Fig.2 Gene expression analysis using DNA microarray. RNA from alkaline and neutral cultures are compared. To ensure consistency, the two samples are labeled with different fluors. In sample 1, a red fluor for the mRNA from alkaline and a green fluor for the mRNA from the neutral cultures. In sample 2, we labeled RNA awaps. The colour was showed log 10 (expression ratio) as: (1) red when the red-labeled RNA is upregulated relative to the green-labeled RNA; (2) green when the red-labeled RNA is downregulated relative to the green-labeled RNA; (3) Yellow when the red-labeled RNA is regulated as equal levels as the green-labeled RNA; (4) black when the log 10 (expression ratio) is close to zero. 71
JAMSTEC 2002 Annual Report Frontier Research System for Extremophiles Table 1 Expression profiles of B. halodurans C-125 when grown under alkaline condition (pH7: pH9.5) BH No. gene name size (a.a) Dir product BH BH + unknown BH spoVG + stage V sporulation protein G BH rplL + ribosomal protein L/L BH BH + two-component sensor histidine kinase BH BH + two-component response regulator BH BH - lantibiotic cytolysin precursor BH BH + lantibiotic mersacidin precursor BH BH + unknown BH BH + unknown conserved protein BH psaA + surface adhesin A precursor BH groEL + class I heat-shock protein (chaperonin) BH BH + unknown conserved protein in others BH BH + unknown conserved protein in others BH BH + unknown BH BH + unknown conserved protein in others BH BH + unknown BH BH - unknown conserved protein in others BH BH + unknown conserved protein in others BH fhuB - ferrichrome ABC transporter (permease) BH alaS + alanyl-tRNA synthetase BH BH - unknown conserved protein BH gltA + glutamate synthase (large subunit) BH BH + cytosine permease BH cotX + spore coat protein X (insoluble fraction) BH BH - unknown BH pyrE - orotate phosphoribosyltransferase BH pyrF - orotidine '-phosphate decarboxylase BH spoVE - stage V sporulation protein E BH BH - unknown conserved protein BH BH - unknown conserved protein in B. subtilis BH BH + unknown conserved protein in others BH BH - glycolate oxidase subunit BH BH - glycolate oxidase subunit BH BH - C-dicarboxylate transport system BH bfmBAA - branched-chain alpha-keto acid dehydrogenase E BH argB - N-acetylglutamate -phosphotransferase (EC ...) BH BH - maltose/maltodextrin transport system (permease) BH mreC - cell-shape determining protein BH BH - unknown 72
Page 2 and 3:
JAMSTEC 2002 Annual Report J apan M
Page 4 and 5:
Japan Marine Science and Technology
Page 6 and 7:
Page 8 and 9:
Page 10 and 11:
Page 12 and 13:
Page 14 and 15:
Page 16 and 17:
Page 18 and 19: Japan Marine Science and Technology
Page 26 and 27: JAMSTEC 2002 Annual Report Marine T
Page 48 and 49: JAMSTEC 2002 Annual Report Marine E
Page 112 and 113: JAMSTEC 2002 Annual Report Frontier
Page 118 and 119:
JAMSTEC 2002 Annual Report Frontier
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Page 154 and 155:
Page 156 and 157:
Page 158 and 159:
1000 Japan Marine Science and Techn
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
JAMSTEC 2002 Annual Report Mutsu Re
Page 180 and 181:
JAMSTEC 2002 Annual Report Mutsu Re
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
JAMSTEC 2002 Annual Report Research
Page 196 and 197:
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Page 218 and 219:
Page 220 and 221:
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Page 228 and 229:
Page 230 and 231:
Page 232 and 233:
Page 234 and 235:
Page 236 and 237:
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
Page 244 and 245:
Page 246 and 247:
Page 248 and 249:
Page 250 and 251:
Page 252 and 253:
Page 254 and 255:
Page 256 and 257:
Page 258 and 259:
Page 260 and 261:
Page 262 and 263:
Page 264 and 265:
Page 266 and 267:
Page 268 and 269:
Page 270 and 271:
Page 272 and 273:
Page 274 and 275:
Page 276 and 277:
JAMSTEC 2002 Annual Report PATENT .
Page 278:
JAMSTEC Japan Marine Science and Te
show all

Marine Ecosystems Research Department - jamstec japan agency ...

Create successful ePaper yourself

Delete template?

Save as template?