Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

Abstract

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

Fig. 1.

Relative number of genes whose expression into mRNA was detectable (threshold > 0.734) by the 36-mer tiling experiment reported here (blue, total; red, detected; green, undetected). The left set of bars shows the relative count from all genes reported in GenBank V5 release, the middle set represents the count from those genes that were previously verified by ESTs or cDNAs, and the right set shows the subset of genes that were predicted.

Fig. 2.

Hybridization intensities for the gene At1g01300, encoding an aspartyl protease family protein previously verified by EST and cDNA, are shown here. (Upper) This study. (Lower) Previous study (11). Red, flower; green, root; blue, suspended cell; magenta, cells in the presence of light. The gene is located on Watson strand of chromosome 1 and has only one exon marked by solid black line near y = 0. For long exons, this study obtained signals above cutoff for all or most probes, whereas the signals measured by the previous study more often fell below the cutoff. This result could be due to the difference in choices of probe lengths or to other differences in hybridization protocol between the two experiments.

Fig. 3.

Overlap among sets of genes expressed in T87 cell line (white circle) and suspended cell line of the previous study (gray circle) (11).

Fig. 4.

Plot of hybridization intensities (T87 cell line) for the 594 RT-PCR positive (X) and 353 RT-PCR negative (♦) genes. Strong hybridization intensities observed for some RT-PCR negative genes (near gene no. 350) were artifacts due to ORF-based primer choices. Seventeen of 20 RT-PCR negative genes in this region were later confirmed positive by a measurement that chose different sets of primers.

Fig. 5.

Hybridization signals in a region on chromosome 1 (Crick strand) are shown. Upper and Lower present data from this study and the suspended cell line of previous study (11), respectively. The figure illustrates close correlation in hybridization patterns between two measurements, even though they are conducted with different platforms and probe lengths. The large peak between bases 296,400–296,500 is a putative expressed intergenic region identified in this study.

Fig. 6.

tRNA abundance vs. amino acid usage. One outlier (P-orig) in the graph for proline was caused by 25 proline identical tRNAs located closely on chromosome 1 that most likely measure the same signal many times due to cross-hybridization. They were excluded from the analysis.