To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

Parathyroid hormone (PTH) binds to its receptor PTH1R (parathyroid hormone 1 receptor) in osteoblastic cells to regulate bone remodeling and calcium homeostasis. While prolonged exposure to PTH causes increased bone resorption, intermittent injections of PTH have an anabolic effect on bone. The molecular mechanisms regulating these processes are still largely unknown. Here, we present our results on gene expression profile changes in the PTH-treated osteoblastic cell line, UMR 106-01, using DNA microarray analysis. A total of 125 known genes and 30 unknown expressed sequence tags (ESTs) were found to have at least 2-fold expression changes after PTH treatment at 4, 12, and 24 h. 14 genes were previously known to be PTH-regulated but many were unknown to be regulated by PTH prior to our experiments. Real-time reverse transcriptase-PCR confirmed that 90 and 50% of the genes are regulated more than 2-fold by PTH in UMR 106-01 and rat primary osteoblastic cells, respectively. Most genes belong to the following protein families: hormones, growth factors, and receptors; signal transduction pathway proteins; transcription factors; proteases; metabolic enzymes; structural and matrix proteins; transporters; etc. These results provide a comprehensive and deeper knowledge about PTH regulation of osteoblastic gene expression. Next, we designed a computational method to extract information about transcription factors likely involved in regulating these genes. These factors include those previously known to be involved in PTH signaling (AP-1 and the cAMP response element-binding protein), those that were identified by microarray data (C/EBP), and some novel transcription factors (AP-2, AP-4, SP1, FoxD3, etc.). Our results suggest that a reliable bioinformatics approach can be easily applied for other systems.

Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 mol of SUL. The plasma concentration occurred at 1 h and peaked around 20 M at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.

We have completed the first large-scale gene expression study of acute spinal cord injury (SCI) in rat. Oligonucleotide microarrays containing 1,200 gene-specific probes were used to quantify mRNA levels, relative to uninjured controls, in spinal cords injured using a standard contusion model. Our results revealed a marked loss of neuron-specific mRNAs at the injury site. The surviving cells showed a characteristic inflammatory response that started at the injury site and spread to the distal cord. Changes in several mRNA levels were associated with putative regenerative responses in the spinal cord. Notably, phosphodiesterase 4, nestin, glia-derived neurite promoting factor, and GAP-43 mRNAs increased significantly. Other mRNAs clustered temporally and spatially with these regeneration-associated genes. Thus we have described global patterns of gene expression following acute SCI, and we have identified targets for future study and possible therapeutic intervention.

Control of RNA turnover is a major, but poorly understood, aspect of gene regulation. In multicellular organisms, progress toward dissecting RNA turnover pathways has been made by defining some cis-acting sequences that function as either regulatory or cleavage targets (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993). However, the identification of genes encoding proteins that regulate or cleave target RNAs has been elusive (C. A. Beelman and R. Parker, Cell 81:79-183, 1995); this gap in knowledge has made it difficult to identify additional components of RNA turnover pathways. We have utilized a modified expression cloning strategy to identify a developmentally regulated gene from Drosophila melanogaster that encodes a RNase that we refer to as Clipper (CLP). Significant sequence matches to open reading frames encoding unknown functions identified from the Caenorhabditis elegans and Saccharomyces cerevisiae genome sequencing projects suggest that all three proteins are members of a new protein family conserved from lower eukaryotes to invertebrates. We demonstrate that a member of this new protein family specifically cleaves RNA hairpins and that this activity resides in a region containing five copies of a previously uncharacterized CCCH zinc finger motif. CLP's endoribonucleolytic activity is distinct from that associated with RNase A (P. Blackburn and S. Moore, p. 317-433, in P. D. Boyer, ed., The Enzymes, vol. XV, part B, 1982) and is unrelated to RNase III processing of rRNAs and tRNAs (J. G. Belasco and G. Brawerman, Control of Messenger RNA Stability, 1993, and S. A. Elela, H. Igel, and M. Ares, Cell 85:115-124, 1995). Our results suggest that CLP may function directly in RNA metabolism.