Construction of plasmids suitable for in vitro synthesis of full-length mRNAs having a 3′-poly(A)+tail

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

E4F1, a Novel Estrogen-Responsive Gene in Possible Atheroprotection, Revealed by Microarray Analysis

Self-disruptive Escherichia coli that produces foreign target protein was developed. E. coli was co-transformed with two vector plasmids, a target gene expression vector and a lysis gene expression vector. The lytic protein was produced after the expression of the target gene, resulting in simplification of the cell disruption process. In this study, the expression of cloned T4 phage gene e or t was used for the disruption of E. coli that produced beta-glucuronidase (GUS) as a model target protein. The expression of gene e did not lead to prompt cell disruption but weakened the cell wall. Resuspension with deionized water facilitated cell lysis, and GUS activity was observed in the resuspended liquid. Expression of gene e at mid logarithmic growth phase was the optimal induction period for GUS production and release. On the other hand, the expression of gene t induced immediate cell lysis, and intracellular GUS was released to the culture medium. Maximum GUS production was obtained when gene t was induced at late logarithmic growth phase.

We report a novel post-transcriptional control of the ptsG gene encoding the major glucose transporter IICB(Glc). We demonstrate that the level of IICB(Glc) is markedly reduced when the glycolytic pathway is blocked by a mutation in either the pgi or pfkA gene encoding phosphoglucose isomerase or phosphofructokinase, respectively. This down-regulation of ptsG is not exerted at the transcriptional level. Both northern blot and S1 analyses demonstrate that the mutation dramatically accelerates the degradation of ptsG mRNA. The degradation of ptsG mRNA occurs in wild-type cells when alpha-methylglucoside, a non- metabolizable analog of glucose, is present in the medium. The addition of any one of the glycolytic intermediates downstream of the block prevents the degradation of ptsG mRNA. The rapid degradation of ptsG mRNA is eliminated when RNase E is thermally inactivated. We conclude that the glycolytic pathway controls ptsG expression by modulating RNase E-mediated mRNA degradation. This is the first instance in which the glycolytic flux has been shown to affect the expression of a specific gene through mRNA stability.

Structure of vaccinia virus late promoters

Natural plant extracts and their isolated phenolic substances have been shown to exhibit several functions including the anti-inflammatory activity in various tissues. EGCG, curcumin and resveratrol have been identified as possible anti-inflammatory agents while little is known about extracts of Boswellia serrata (BS) and Rosmarinus officinalis (RO). In this thesis project, several methods were applied in order to identify the anti-inflammatory effects of aforementioned substances and extracts in “inflamed” intestinal epithelial cells. The colon cancer cell lines, HT29 and T84, were stimulated with TNF-α in the presence of various concentrations of EGCG, BS, RO, curcumin and resveratrol. Their effects were analyzed at different levels: (1) on the expression of multiple genes involved in inflammatory responses; (2) on the generation of metabolites such as PGE2, and production of chemokines (e.g., IL-8 and MIP-3α); (3) and on the protein expression. The expression of genes was determined by quantitative RT-PCR and microarray technology. The production of IL-8, MIP-3α and PGE2 were measured by ELISA. The expression of proteins belonging to the NF-κB pathway was analyzed by immunoblots. In addition, NF-κB DNA binding was carried out using EMSA. Treatment of activated cells with EGCG, BS, RO, curcumin and resveratrol reduced the expression of most genes related to the inflammatory response of HT29 and T84 cells. Furthermore, EGCG, curcumin and resveratrol diminished the generation of PGE2 in HT29 cells. In addition, the secretion of IL-8 and MIP-3α were dose-dependently inhibited by candidate compounds in HT29 cells. EGCG reduced the production of these chemokines also in T84 cells while curcumin and resveratrol only diminished MIP-3α synthesis. Moreover, the expression of IκBα and NF-κB p65 were analyzed in HT29 cells. BS was not able to prevent IκBα degradation and subsequent NF-κB translocation whereas RO interfered with IκBα degradation, however, it did not impede the activation of NF-κB. Additionally, RO reduced NF-κB DNA binding tested by EMSA. Collectively, the presented data show that phenolic substances or whole plant extracts have the capacity to attenuate inflammatory features by modulating genes involved in inflammatory responses; by reducing the chemoattraction of immunocytes through the inhibition of IL-8 and MIP-3α; and by affecting specific pathways involved in inflammation such as the NF-κB. Furthermore, phenolic compounds might have a preventive and/or therapeutic utility in chronic inflammation such as IBD, and therefore, they should be further analyzed in in vivo studies to consolidate these findings.

The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.

The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3'-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.

A lipase gene from B. subtilis WRRL-B558 was cloned in Escherichia coli JM109 using pBluescript as a vector plasmid. Two methods were combined to screen for the lipase-producing clone. The first was done by overlaying the screening plates with β-naphthylacetate and Fast Blue BB dye. Positive clones were then confirmed by a second method using 1% (v/v) tributyrin agar plates. Positive clones which formed clear zones on the tributyrin agar plates were selected and analysed by restriction mapping, Southern blot hydridization and deletion studies to locate the lipase gene on a 2.2 kb HindIII fragment insert. A subclone harbouring a plasmid with a 0.9 kb DNA fragment between the HindIII and EcoRI sites that still exhibited lipase activity was used for sequencing. The nucleotide sequence showed a single open reading frame which contained 636 nucleotides (212 deduced amino acids). A conserved pentapeptide postulated to be the catalytic site was Ala-X-Ser-X-Gly instead of Gly-X-Ser-X-Gly. The deduced protein was found to have a molecular weight of 21 kDa which was similar to that obtained from the recombinant plasmid as determined by SDS-PAGE. Expression of the Bacillus lipase gene was found to be high in recombinant E. coli.

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins

Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo

Thymic stromal lymphopoietin (TSLP) transgenic mice develop cryoglobulin-associated membranoproliferative glomerulonephritis, characterized by renal monocyte/macrophage infiltration, marked expansion of extracellular matrix, and variable intraluminal and mesangial deposits of cryoglobulins. A microarray approach was used to study global gene expression in glomerular RNA obtained from these mice, as well as from combined TSLP transgenic and Fcγ receptor IIb null mice (TSLP/FcIIb−/−), which develop aggravated membranoproliferative glomerulonephritis. Protease nexin-1 (PN-1) and tissue plasminogen activator (tPA), two potential regulators of fibrosis that are involved in the fibrinolytic and coagulation pathways, were dramatically upregulated in TSLP mice compared with wild-type controls. In situ hybridization revealed minimal expression of PN-1 mRNA in the glomeruli of wild-type mice, increased expression in TSLP mice, and the greatest expression in the mesangial cells of TSLP/FcIIb−/− mice. Immunohistochemistry demonstrated greater expression of PN-1, tPA, and PAI-1 in the mesangial cells of TSLP mice compared with wild-type and the greatest in TSLP/FcIIb−/− mice. In cultured mesangial cells, incubation with cryoglobulins induced an upregulation of PN-1 mRNA; increased expression of PN-1, tPA, and PAI-1 proteins; and stimulated secretion of TGF-β1. It is concluded that PN-1, tPA, PAI-1, and TGF-β1 are likely important mediators of murine cryoglobulinemic glomerulonephritis and that the cryoglobulins may directly upregulate their expression.

Regulation of Intercellular Adhesion Molecule-1 Gene Expression Involves Multiple mRNA Stabilization Mechanisms: Effects of Interferon-γ and Phorbol Myristate Acetate

Mutations that alter muscle contraction lead to a large array of diseases, including muscular dystrophies and cardiomyopathies. Although the molecular lesions underlying many hereditary muscle diseases are known, the downstream pathways that contribute to disease pathogenesis and compensatory muscle remodeling are poorly defined. We have recently identified and characterized mutations in Myosin Heavy Chain (Mhc) that lead to hypercontraction and subsequent degeneration of flight muscles in Drosophila. To characterize the genomic response to hypercontraction-induced myopathy, we performed expression analysis using Affymetrix high density oligonucleotide microarrays in Drosophila Mhc hypercontraction alleles. The altered transcriptional profile of dystrophic Mhc muscles suggests an actin-dependent remodeling of the muscle cytoskeleton. Specifically, a subset of the highly up-regulated transcripts is involved in actin regulation and structural support for the contractile machinery. In addition, we identified previously uncharacterized proteins with putative actin-interaction domains that are up-regulated in Mhc mutants and differentially expressed in muscles. Several of the up-regulated proteins, including the dystrophin-related protein, MSP-300, and the homolog of the neuronal activity-regulated protein, ARC, localize to specific subcellular muscle structures that may provide key structural sites for cytoskeletal remodeling in dystrophic muscles. Defining the genome-wide transcriptional response to muscle hypercontraction in Drosophila has revealed candidate loci that may participate in the pathogenesis of muscular dystrophy and in compensatory muscle repair pathways through modulation of the actin cytoskeleton.

Brassinosteroids (BRs) are steroid hormones that are essential for the development of plants. A tight control of BR homeostasis is vital for modulating their impact on growth responses. Although it is recognized that the rapid adaptation of de novo synthesis has a key role in adjusting required BR levels, our knowledge of the mechanisms governing feedback control is limited. In this study, we identify the transcription factor CESTA as a regulator of BR biosynthesis. ces-D was isolated in a screen of Arabidopsis mutants by BR over-accumulation phenotypes. Loss-of-function analysis and the use of a dominant repressor version revealed functional overlap among CESTA and its homologues and confirmed the role of CESTA in the positive control of BR-biosynthetic gene expression. We provide evidence that CESTA interacts with its homologue BEE1 and can directly bind to a G-box motif in the promoter of the BR biosynthesis gene CPD. Moreover, we show that CESTA subnuclear localization is BR regulated and discuss a model, in which CESTA interplays with BEE1 to control BR biosynthesis and other BR responses.