Marine Ecosystems Research Department - jamstec japan agency ...

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.
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