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

doi:10.1073/pnas.0408203102

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

^*Genome Research Facility, National Aeronautics and Space Administration Ames Research Center, Moffett Field, CA 94035; ^†Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; ^¶Systemix Institute, Cupertino, CA 94035; ^∥Eloret Corporation at National Aeronautics and Space Administration Ames Research Center, Moffett Field, CA 94035; and ^**Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, WI 53706

Edited by Sidney Altman, Yale University, New Haven, CT, and approved January 28, 2005 (received for review November 4, 2004)

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.

Footnotes

↵ § To whom correspondence may be addressed. E-mail: vstolc{at}mail.arc.nasa.gov or manoj.samanta{at}systemix.org.
↵ ‡ V.S. and M.P.S. contributed equally to the work.
↵ †† M.R.S. is a cofounder of NimbleGen Systems, Inc., the company that is commercializing the maskless array synthesizer technology described in this article.
Author contributions: V.S., M.P.S., and W.T. designed research; V.S., M.P.S., W.T., H.S., D.C.N., A.H., C.N., D.R., S.B., E.U., Q.Z., R.L.W., and C.S.N. performed research; V.S., M.P.S., W.T., S.L., B.G.F., G.N.P., J.L.M., and M.R.S. contributed new reagents/analytic tools; V.S., M.P.S., W.T., and H.S. analyzed data; V.S., M.P.S., and M.R.S. wrote the paper; and D.C.N. proofread the manuscript.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: miRNA, microRNA.