This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.IL-8 ͉ genomics ͉ high-throughput screening ͉ transducer of regulated cAMP response element-binding protein

A 340 nucleotide element within the 3¢ untranslated region of Vg1 mRNA determines its localization to the vegetal cortex of Xenopus oocytes. To identify protein factors that bind to this region, we screened a cDNA expression library with an RNA probe containing this sequence. Five independent isolates encoded a protein (designated Prrp for proline-rich RNA binding protein) having two RNP domains followed by multiple polyproline segments. Prrp and Vg1 mRNAs are co-localized to the vegetal cortex of stage IV oocytes, substantiating an interaction between the two in vivo. Prrp also associates with VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. The proline-rich domain of Prrp interacts with pro®lin, a protein that promotes actin polymerization. Prrp can also associate with the EVH1 domain of Mena, another micro®lament-associated protein.Since the anchoring of Vg1 mRNA to the vegetal cortex is actin dependent, one function of Prrp may be to facilitate local actin polymerization, representing a novel function for an RNA binding protein.

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.Gene expression in bacteria has been traditionally analyzed by transcriptional or translational fusions to promoterless "reporter" genes (e.g., lacZ, cat, and gus) or by direct detection of transcripts using Northern blotting or reverse transcription-PCR (RT-PCR). With the completion of many bacterial genomes and the development of large-scale analysis tools such as DNA genomic arrays, however, researchers have increasingly applied genomics tools in their research. Measurements of mRNA levels using genome arrays for Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, and Haemophilus influenzae (10,11,12,17,23,24,29,32,38,39,41) have been found to offer many advantages to traditional gene-monitoring methods. Since the structure of bacterial genomes is relatively simple, containing ca. 4,000 genes and few repetitive sequences, DNA arrays can monitor transcript levels of an entire genome in a single hybridization with high sensitivity. This can lead to the elucidation of complex interactions among genetic networks, which then can be coupled with results from other newer technologies that analyze global protein synthesis (proteome) and metabolite levels (metabolome) to provide a comprehensive picture of the physiology of the bacterium (13,14,18,34,35,42).Using the public B. subtilis genome sequence (20), we developed an oligonucleotide B. subtilis genome microarray using Affymetrix GeneChip technology (21,40). This technology offers high sensitivity, high specificity, and excellent reproducibility (19). We show that the microarray can monitor gene expression changes in response to transition from the exponential to the stationary growth phases and exposure to three different vitamins that repress expression of biosynthetic genes. Moreover, we also present evidence that the microarray can be used to detect novel transcripts within intergenic regions and on the opposite strand of known genes, leading in some cases to the identification of previous unreported coding regions. MATERIALS AND METHODSMicroarray design. An "antisense" oligonucleotide array complementary to the Baci...

A Xenopus oocyte expression library was screened for proteins that bind to the 340-nucleotide localization element of Vg1 mRNA. Four different isolates encoded a Xenopus homolog of the human transcription factor, FUSE-binding protein 2 (FBP2). This protein has been independently identified as the splicing regulatory factor KSRP. The only significant difference between the Xenopus protein, designated VgRBP71, and KSRP is the absence of a 58 amino acid segment near the N-terminal of the former. In vivo binding assays show that VgRBP71 is associated with mRNAs localized to either the vegetal or animal hemispheres, but was not found with control mRNAs.Unlike other factors that bind to the localization element of Vg1 mRNA, VgRBP71 does not accumulate at the vegetal cortex with the mRNA; rather, it is present in the nucleus and throughout the cytoplasm at all stages of oogenesis. Cytoplasmic VgRBP71 appears to be most concentrated at the cell cortex. VgRBP71 interacts with Prrp, another protein that binds to the Vg1 localization element; this association does not require the presence of Vg1 mRNA.

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