The growth of secondary xylem and phloem depends on the division of cells in the vascular cambium and results in an increase in the diameter of the root and stem. Very little is known about the genetic mechanisms that control cambial activity and the differentiation of secondary xylem and phloem cell types. To begin to identify new genes required for vascular cell differentiation and function, we performed genome-wide expression profiling of xylem and phloem-cambium isolated from the root-hypocotyl of Arabidopsis (Arabidopsis thaliana). Gene expression in the remaining nonvascular tissue was also profiled. From these transcript profiles, we assembled three sets of genes with expression significantly biased toward xylem, phloem-cambium, or nonvascular tissue. We also assembled three two-tissue sets of genes with expression significantly biased toward xylem/phloem-cambium, xylem/nonvascular, or phloem-cambium/nonvascular tissues. Localizations predicted by transcript profiles were supported by results from promoter-reporter and reverse transcription-polymerase chain reaction experiments with nine xylem- or phloem-cambium-biased genes. An analysis of the members of the phloem-cambium gene set suggested that some genes involved in regulating primary meristems are also regulators of the cambium. Secondary phloem was implicated in the synthesis of auxin, glucosinolates, cytokinin, and gibberellic acid. Transcript profiles also supported the importance of class III HD ZIP and KANADI transcription factors as regulators of radial patterning during secondary growth, and identified several members of the G2-like, NAC, AP2, MADS, and MYB transcription factor families that may play roles as regulators of xylem or phloem cell differentiation and activity.

Phosphorylation of the transcription factor CREB leads to the recruitment of the coactivator, CREB binding protein (CBP). Recent studies have suggested that CBP recruitment is not sufficient for CREB function, however. We have identified a conserved protein-protein interaction motif within the CBP-binding domains of CREB and another transcription factor, SREBP (sterol-responsive element binding protein). In contrast to CREB, SREBP interacts with CBP in the absence of phosphorylation. We have exploited the conservation of this interaction motif to test whether CBP recruitment to CREB is sufficient for transcriptional activation. Substitution of six nonconserved amino acids from SREBP into the activation domain of CREB confers high-affinity, phosphorylation-independent CBP binding. The mutated CREB molecule, CREB DIEDML , activates transcription in F9 teratocarcinoma and PC12 cells even in the absence of protein kinase A (PKA). Addition of exogenous CBP augments the level of transcription mediated by CREB DIEDML , and adenovirus 12S E1A blocks transcription, implicating CBP in the activation process. Thus, recruitment of CBP to CREB is sufficient for transcriptional activation. Addition of PKA stimulates transcription induced by CREB DIEDML further, suggesting that a phosphorylation event downstream from CBP recruitment augments CREB signaling.The signaling mechanism that activates genes through the cyclic AMP (cAMP)-regulated enhancer (CRE) (23) represents one of the most intensively studied transcriptional pathways. This pathway consists of protein kinase A (PKA), the transcription factor CREB, and the coactivator CREB binding protein (CBP) (3,7,18). CBP has been shown to participate in many additional transcriptional pathways as well (11), but the mechanism by which it activates gene expression remains uncertain. CBP and its homologue p300 interact with the basal transcription factors TFIID and TFIIB, as well as with the RNA polymerase II holoenzyme component, RNA helicase A (9, 24, 34), suggesting that one function of this coactivator is to stabilize the preinitiation complex. Other studies have argued that transcriptional activation through CBP/p300 occurs only in the context of chromatin, however (17). The involvement of chromatin in CBP function is consistent with the finding that CBP, and several associated proteins including P/CAF, steroid receptor coactivator 1, and p/CIP, have the ability to acetylate the amino-terminal tails of histone proteins (6,31,36,38). These and other posttranslational modifications of chromatin components induced by the cAMP signaling cascade may stimulate transcription through nucleosome remodeling.Of all transcription factor-CBP associations, only the interaction with phosphorylated CREB has been characterized in detail. Our lab has studied this association by using a fluorescence polarization binding assay and a genetic interaction assay in yeast (18,30). These studies indicated that CREB phosphorylated at Ser 133 binds to CBP with an affinity of approximately 350 nM and that...

Brucella spp. are facultative intracellular bacteria that cause brucellosis in humans and other animals. Brucella spp. are taken up by macrophages, and the outcome of the macrophage-Brucella interaction is a basis for establishment of a chronic Brucella infection. Microarrays were used to analyze the transcriptional response of the murine macrophage-like J774.A1 cell line to infection with virulent Brucella melitensis strain 16M. It was found that most significant changes in macrophage gene transcription happened early following infection, and global macrophage gene expression profiles returned to normal between 24 and 48 h postinfection. These findings support the observation that macrophages kill the majority of Brucella cells at the early infection stage, but the surviving Brucella cells are able to avoid macrophage brucellacidal activity inside replicative phagosomes at the later infection stage. At 4 h postinfection, macrophage genes involved in cell growth, metabolism, and responses to endogenous stimuli were down-regulated, while the inflammatory response (e.g., tumor necrosis factor alpha and Toll-like receptor 2), the complement system, the responses to external stimuli, and other immune responses were up-regulated. It is likely that the most active brucellacidal activity happened between 0 and 4 h postinfection. Mitochondrion-associated gene expression, which is involved in protein synthesis and transport, electron transfer, and small-molecule transfer, and many other mitochondrial functions were significantly down-regulated at 4 h postinfection. Although there were both proand antiapoptosis effects, B. melitensis 16M appears to inhibit apoptosis of macrophages by blocking release of cytochrome c and production of reactive oxygen species in the mitochondria, thus preventing activation of caspase cascades.Brucellosis in humans and other animal species is caused by facultative intracellular bacteria belonging to the genus Brucella. Unlike many pathogenic bacteria, the brucellae lack classical virulence factors, such as invasive proteases, exotoxins, endotoxic lipopolysaccharide (LPS), capsules, fimbriae, pili, virulence plasmids, and lysogenic phages (29,48). Brucella virulence relies on the ability of the organism to survive and replicate within vacuolar phagocytic compartments of macrophages (37). The host macrophage-Brucella interaction is critical for establishment of chronic Brucella infections. For example, both the type IV secretion system encoded by the virB operon (7) and the two-component regulatory system encoded by the bvrRS operon (44) are necessary for successful replication of Brucella inside macrophages. Smooth Brucella strains with intact LPS O side chains are virulent and invade macrophages through lipid rafts (56). Immediately after entry into macrophages, Brucella strains reside in an acidified compartment that fuses with components of the early endosomal pathway (56). The majority of Brucella strains are killed at the early infection stage (14, 56). However, a subpopulation of virule...

The cAMP response element-binding protein (CREB) 1 is a 43-kDa basic leucine zipper (bZIP) transcription factor that couples gene activation to a wide variety of cellular signals (for review, see Ref. 1). Initially identified as a mediator of the cAMP pathway, CREB is now recognized to respond to calcium/ calmodulin and growth factor pathways as well (2-4). The prototypical target sequence for CREB is the palindromic cAMP response element (CRE; 5Ј-TGACGTCA-3Ј), first identified in the neuropeptide somatostatin gene (5). Consensus CRE sequences, or slight variants of this sequence, have since been identified in hundreds of cellular and viral genes. In many instances, functional studies have indicated that these sequences are required for second messenger-directed transcriptional responses (for review, see Ref. 6). Whether variations of the CRE from the consensus sequence have functional consequences has not been determined.Two general categories of factors can recognize the CRE (1, 7, 8). The first is composed of factors that can dimerize with CREB. This group includes the CRE modulator CREM␣ (and the multitude of splice variants that contain the CREM␣ DNA binding domain) and the activating transcription factor-1 (ATF-1). CREM␣ and ATF-1 recognize the CRE as homodimers and as heterodimers with CREB (9 -11). The second category of CRE binding proteins includes factors that cannot heterodimerize with CREB, such as c-Jun, some of the other ATFs, and members of the CAAT/enhancer-binding protein gene family (12, 13). In addition to their inability to heterodimerize with CREB, these factors appear to be capable of recognizing DNA sequences that are distinct from the CRE (14). This promiscuity in DNA binding is not entirely unexpected, because some of these distinct elements, such as the AP-1 site, differ from the CRE by only a single nucleotide. CREB family members are notable in that they recognize the CRE exclusively, or nearly so. One caveat in this characterization, however, is that the defining features of the CRE have not been established unequivocally.CREB binds as a dimer to the CRE with an affinity of ϳ1-2 nM (15). Dimerization and DNA binding are mediated by the adjacent basic and leucine zipper domains (7,8). At the extreme C-terminal end of the protein is the leucine zipper, characterized by a conserved heptad repeat of seven residues, denoted a-g. The residues at positions a and d typically form a hydrophobic interface, with conserved leucines at position d. As with other bZIP proteins, this configuration in CREB allows for the formation of a two-stranded parallel coiled-coil and, along with charged residues at positions e and g, presents a plane for dimerization specificity (12, 16 -19). In addition, dimerization of the CREB monomers apposes the basic regions in a parallel orientation to the DNA, allowing the dimer to bind at a right angle to the DNA helical axis. DNA binding elicits an ␣-helical conformation of the basic region that facilitates DNA recognition. The basic region of CREB abuts the amino ...

Overexposure to corticosteroid hormones is harmful to hippocampal neuronal integrity, likely by perturbation of calcium homeostasis. To identify molecular mechanisms at the singlecell level, we characterized mRNA expression corresponding to voltage-and ligand-gated Ca channels in individual dissociated CA1 neurons in response to long-term corticosterone (CORT) exposure. Predominant mineralocorticoid receptor occupation (ADC-LO group) resulted in low levels of P/Q-and L-type Ca channel mRNAs, high levels of GluR-2 versus GluR-1, and a high ratio of NMDAR-2A to NMDAR-2B mRNA. Corresponding alterations in protein expression were consistent with the restriction of Ca influx. In contrast, additional glucocorticoid receptor occupation (ADC-HI group) altered the expression of these mRNAs in a manner consistent with enhanced Ca influx; interestingly, qualitatively similar alterations were seen in control ADX neurons. Electrophysiological data from the same neurons indicate that Ca current amplitudes also are modulated by CORT, although on a shorter time scale. Finally, principal components analysis (PCA) suggests that neuronal AMPA and NMDA receptor composition may be regulated by MR and GR activation in a complex manner. Therefore, our data implicate molecular events by which CORT may regulate Ca influx into CA1 hippocampal neurons.

Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.

The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetrahydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations.

The teratogenic potential of valproic acid has been well established both in experimental models and in human clinical studies. As with all human teratogens, there are genetically determined differences in individual susceptibility to the induction of congenital defects. Using a mouse model of valproate‐induced neural tube defects, a study was undertaken to examine differential changes in gene expression for selected transcription factor (Pax‐3, Emx‐1, Emx‐2, c‐fos, c‐jun, creb) and cell cycle checkpoint genes (bcl‐2, p53, wee‐1) during neural tube closure. In general, exposure to teratogenic concentrations of valproic acid elicited GD 9:12 control levels of transcription factor mRNA expression in GD 9:0 embryos of both strains. This accelerated developmental profile is marked by significant elevation of Emx‐1, Emx‐2, c‐fos, c‐jun, and creb expression. There was also a significant overexpression of the cell cycle genes p53 and bcl‐2 in the LM/Bc embryos in response to the teratogenic insult. Examination of the ratio of expression of these genes clearly favored bcl‐2, which supports the hypothesis that altered neuroepithelial cell proliferation rates, rather than increased apoptosis, is the underlying mechanism by which valproic acid alters normal neural tube morphogenesis. An investigation into interactive effects of these genes on the molecular profile of GD 9:0 embryos further validated this observation. That is, the overall proliferative state among the control embryos was prematurely modified into a more differentiated state following teratogenic insult. These results suggest that alterations in the expression of multiple genes are most likely responsible for valproic acid‐induced neural tube defects. Teratology 54:284–297, 1996. © 1997 Wiley‐Liss, Inc.