Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.     

Mapping behaviorally relevant neural circuits with immediate-early gene expression

Immediate-early genes have gained widespread popularity as activity markers for mapping neuronal circuits involved in specific behaviors in many different species. In situ immediate early gene detection methods provide cellular level resolution, a major benefit for mapping neuronal networks. Recent advances using fluorescence in situ hybridization also afford temporal resolution, enabling within-animal activity maps for two distinct behaviors. Moreover, use of transgenic mice with fluorescent reporter proteins driven by immediate early gene promoters is enabling repeated measurements, over long time scales, of cortical activity within the same animal. These methodological innovations, coupled with recent advances in fluorescence imaging and probe development, will enable large scale mapping of behaviorally relevant circuits with temporal and three-dimensional spatial resolution in experimental animals.     

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.     

Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production

The effect of human cytomegalovirus (HCMV) gene expression on beta interferon (IFN-beta) expression was examined. We demonstrate that the HCMV immediate-early 2 (IE2) gene product IE86 can effectively block the induction of IFN-beta during HCMV infection. IE86 also efficiently blocked the induction of IFN-beta following Sendai virus infection, demonstrating that IE86's ability to block induction of IFN-beta is not limited to HCMV infection, identifying IE2 as an IFN-beta antagonist.     

The human cytomegalovirus major immediate-early distal enhancer region is required for efficient viral replication and immediate-early gene expression

The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.     

Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.     

Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol

cAMP/PKA signaling transientlystimulates mRNA expression of immediate-early genes, including IL-6 andc-fos. We confirmed that these mRNAs are transientlystimulated by parathyroid hormone (PTH) in ROS 17/2.8 osteoblasticcells. Consistent with the role for cAMP/PKA signaling in thisresponse, PTH induces transient cAMP elevation, PKA activation, andcAMP-responsive element-binding protein (CREB) phosphorylation. Ourgoal was to determine whether termination of immediate-early geneexpression is due to receptor desensitization or cAMP degradation. Theapproaches used were 1) inhibition of PTH receptordesensitization with G protein-coupled receptor kinase 2 (GRK2)antisense oligonucleotides or antisense plasmids, 2)sustained activation of adenyl cyclase with forskolin, and3) inhibition of cAMP degradation with3-isobutyl-1-methylxanthine. These experiments show that mechanismsdownstream of receptor desensitization and cAMP degradation areprimarily responsible for termination of PKA activity, CREBphosphorylation, and immediate-early gene expression. Similarconclusions were also obtained in response to PTH in a secondosteoblastic cell line (MC3T3-E1) and in response to isoproterenol inNIH3T3 fibroblasts. This conclusion may therefore reflect a generalmechanism for termination of immediate-early gene expression afterinduction by cAMP/PKA.

     

Osmostress-induced changes in yeast gene expression

When Saccharomyces cerevisiae cells are exposed to high concentration of NaCl, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCl) by a previous treatment with 0.7 M NaCl, but not by a previous heat shock. Two-dimensional analysis of [3H]-leucine-labelled proteins from salt-shocked cells (0.7 M NaCl) revealed the elevated rate of synthesis of nine proteins, among which were the heat-shock proteins hsp12 and hsp26. Northern analysis using gene-specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol-3-phosphate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso-osmolar NaCl concentration (0.7 M).