Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Application of cDNA microarrays in determining molecular phenotype in cardiac growth, development, and response to injury.

BACKGROUND: Normal myocardial development and the tissue response to cardiac stress are accompanied by marked changes in gene expression; however, the extent of these changes and their significance remain to be fully explored. We used cDNA microarrays for gene expression profiling in rat cardiac tissue samples to study developmental transitions and the response to myocardial infarction (MI). METHODS AND RESULTS: Microarrays with rat cDNAs for 86 known genes and 989 anonymous cDNAs obtained by molecular subtraction (representational difference analysis) of mRNA from sham-operated and 6-week post-MI samples were used in 2-color hybridization experiments. Twelve known genes previously associated with myocardial development were identified together with 10 uncharacterized expressed sequence tags and 36 genes not previously associated with cardiac development. After MI, genes associated with myocardial stress and wound healing exhibited differences in magnitude and expression kinetics, and 14 genes not previously associated with MI were identified. In situ hybridization revealed mRNA localization characteristic of wound healing and vascular and cardiomyocyte reactivity. CONCLUSIONS: Tissue analysis of gene expression with cDNA microarrays provides a measure of transcriptional or posttranscriptional regulation and cellular recruitment. Our results demonstrate the complexity of gene regulation in the developing myocardium and show that cDNA microarrays can be used to monitor the evolution of the cardiac stress-inducible phenotype.

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

The active form of the KorB protein encoded by the Streptomyces plasmid pIJ101 is a processed product that binds differentially to the two promoters it regulates.

The korB gene of Streptomyces lividans plasmid pIJ101 is known to encode an autoregulated protein that also represses transcription of a gene, kilB, implicated in pIJ101 transfer and in spreading of the plasmid along mycelia of the recipient. Earlier work has indicated that the primary gene product of korB is a 10-kDa protein predicted from the gene sequence (D.S. Stein and S.N. Cohen, Mol. Gen. Genet. 222:337-344, 1990; S. Zamen H. Richards, and J. Ward, Nuleic Acids Res. 20:3693-3700, 1992). We report here that the 10-kDa KorB protein product is processed in vivo into a 6-kDa peptide that has a 20-fold-greater binding affinity for its operator-promoter target; in addition, the 6-kDa peptide binds differentially to the regulatory regions of the two genes it controls, showing 50-fold-greater affinity for the kilB sequence. While both the processed and unprocessed forms of KorB were observed in Escherichia coli following korB gene expression under control of the bacteriophage T7 promoter, only the 6-kDa peptide was found in S. lividans containing pIJ101, implying that this peptide is normally the biologically active form of KorB. The footprint resulting from KorB binding to the korB operator sequence overlaps the sti locus, which affects pIJ101 copy number and incompatibility as well as the size of zones of inhibited recipient cell growth ("pocks") that form around donor cells during mating. The observed ability of the korB gene product to interact with both sti sequences and the kilB promoter region suggests that it may have a role in coordinating the replication and intramycelial spread of plasmids during and/or following bacterial mating.

Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission.

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.

Large scale gene expression analysis of cholesterol-loaded macrophages.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.