Construction of unmodified oligonucleotide-based microarrays in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: screening of the candidates for circadianly expressed genes

Abstract

DNA microarrays with unmodified oligonucleotide probes are a cost-effective and high-performance alternative to cDNA microarrays. We searched every gene in the genome of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 for 45-mer oligonucleotide probes with optimal nucleotide sequences, and found such probes in 90% of the genes. Using the probes, we constructed a microarray that represented 2,397 genes (95% of total genes). We detected only low signals in the negative control probes whose nucleotide sequences are not contained in the T. elongatus genome, demonstrating that specific hybridization occurred. To evaluate the reliability of the measurements obtained by the oligonucleotide microarray, we performed microarray experiments using RNA samples from two different time points of circadianly synchronized cultures, LL2 (early sub-jective day) and LL14 (early subjective night). Measurements obtained from the two independent microarray hybridizations were highly concordant (correlation coefficient [r] > 0.8). Northern blot analyses of 20 genes confirmed that expression changes detected by the microarrays were correct (r = 0.832). We identified 143 candidate clock-controlled genes whose expression levels at LL2 and LL14 were significantly different. Expression of 69 of them was enhanced at LL14 while expression of the other 74 was enhanced at LL2. The physiological functions of the genes were diverse and included metabolism, translation, transcription, membrane transport, DNA replication and repair, and cell growth and death.