Cleavage of eukaryotic translation initiation factor 4G by exogenously added hybrid proteins containing poliovirus 2Apro in HeLa cells: effects on gene expression.
(17/8235)
Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated. (+info)
Transcriptional regulation of interleukin-2 gene expression is impaired by copper deficiency in Jurkat human T lymphocytes.
(18/8235)
Copper deficiency reduces secretion of the cytokine interleukin-2 (IL-2) by activated rodent splenocytes, human peripheral blood mononuclear cells and Jurkat cells, a human T lymphocyte cell line. Previous studies showed that low Cu status also decreased the level of IL-2 mRNA in activated Jurkat cells by 50%. Synthesis of this cytokine is regulated by alterations in transcription of the IL-2 gene and the stability of IL-2 mRNA. To determine if Cu status influenced promoter activity of the IL-2 gene, Jurkat cells were transfected with a luciferase reporter gene construct containing the entire 300 bp human IL-2 promoter/enhancer sequence. Cu deficiency was induced by incubating stably transfected cells with the Cu chelator 2,3,2-tetraamine for 35 h prior to activating cells with phytohemagglutinin-P and phorbol myristate acetate. Luciferase activity in lysates of Cu-deficient cells was approximately 50% lower in several multiclonal and clonal cell lines of stably transfected cells than in replicate cultures that were not exposed to chelator. The relative levels of endogenous IL-2 bioactivity and luciferase activity were highly correlated in the transfected cell lines. The chelator-mediated reduction in reporter gene activity was dose-dependent at levels of 5-40 micromol 2,3,2-tetraamine/L. The addition of a slight molar excess of Cu, but not Zn or Fe, to medium containing 2,3,2-tetraamine prevented the decline in luciferase activity. IL-2 mRNA stability in parental Jurkat cells was independent of Cu status. These data indicate that decreased cellular Cu attenuates IL-2 synthesis in T lymphocytes by inhibiting transcription of the IL-2 gene. (+info)
Topical gene delivery to murine skin.
(19/8235)
We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations > or =0.25 microg per microl were required to achieve maximal expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders. (+info)
The host-cell architectural protein HMG I(Y) modulates binding of herpes simplex virus type 1 ICP4 to its cognate promoter.
(20/8235)
The productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site in in vitro assays and to enhance the activity of this protein in short-term transient expression assays. (+info)
Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides.
(21/8235)
The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non-dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH-sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected lack of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo. (+info)
HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus.
(22/8235)
Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain. (+info)
Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad.
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Transcription controls active at the initial stages of meiosis are clearly key elements in the regulation of germinal differentiation. Transcription of the Sycp1 gene (synaptonemal complex protein 1) starts as early as the leptotene and zygotene stages. Constructs with Sycp1 5' upstream sequences directed the expression of reporter genes to pachytene spermatocytes in transgenic mice. A short fragment encompassing the transcription start (n.t. -54 to +102) was sufficient for stage-specific expression in the adult male and for temporal regulation during development. Upstream enhancer element(s) quantitatively regulating expression were localized in the region between -54 and -260. The gene is normally expressed both in the male and female gonads, but none of the promoter sequences active in the testis allowed the expression of reporter genes during meiosis in the ovary. (+info)
Genomic organization and promoter analysis of a mouse homeobox gene, Hex.
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A homeobox gene, Hex, is mainly expressed in haematopoietic cells and hepatocytes. It is assumed to play a role in the early stage of differentiation of these cells. To understand the mechanisms involved in the regulation of the Hex gene expression in hepatocytes, we cloned and characterized the mouse Hex gene. The gene consists of four exons and three introns, and spans about 5.7 kb. All the exon-intron boundaries are consistent with the "GT-AG" rule. A single transcription start site was identified by primer extension and S1 mapping analyses. Although the 5'-flanking region is G/C rich (69%), it contains probable "TATA and CCAAT" boxes. Potential binding sequences for transcriptional regulatory proteins including Sp1 and AP-2 are also present in this region. Functional analysis of the Hex promoter was performed by transfecting MH1C1, HeLa, COS-7, and Caco-2 cells with Hex promoter region-luciferase constructs. We found three possible positive regulatory regions, comprising of nucleotides -199 and -172, -154 and -133, and -105 and -68, respectively, required for Hex gene expression in MH1C1 cells by analyses of a series of 5'-deletion constructs of the fusion genes. The activities of these constructs were extremely low in HeLa, COS-7, and Caco-2 cells suggesting that they possess cell-type specificity. Further analysis revealed two GC boxes, GC box1 and GC box2, at nucleotides -197 to -188 and -176 to -167, respectively, necessary for Hex gene expression. Thus, multiple regulatory elements contribute to the Hex gene expression in hepatocytes. (+info)