Repressive effect of Sp1 on the C/EBPalpha gene promoter: role in adipocyte differentiation.
(41/6278)
Expression of C/EBPalpha is required for differentiation of 3T3-L1 preadipocytes into adipocytes. Previous investigations indicated that transcription of the C/EBPalpha gene is sequentially activated during differentiation, initially by C/EBPbeta and C/EBPdelta and later by C/EBPalpha (autoactivation). These events are mediated by a C/EBP regulatory element in the promoter of the C/EBPalpha gene. This article presents evidence that members of the Sp family, notably Sp1, act repressively on the C/EBPalpha promoter prior to the induction of differentiation. Sp1 was shown to bind to a GC box at the 5' end of the C/EBP regulatory element in the C/EBPalpha promoter and, in so doing, to competitively prevent binding to and transactivation of the promoter by the C/EBPs. One of the differentiation inducers methylisobutylxanthine (a cAMP phosphodiesterase inhibitor) or Forskolin, both of which increase the cellular cAMP level, causes down-regulation of Sp1. This decrease in Sp1 level early in the differentiation program appears to facilitate access of C/EBPbeta and/or C/EBPdelta to the C/EBP regulatory element and, thereby, derepression of the C/EBPalpha gene. (+info)
BseSI, a restriction endonuclease from Bacillus stearothermophilus Jo 10-553, which recognizes the novel hexanucleotide sequence 5'-G(G/T)GC(A/C)C-3'.
(42/6278)
A new restriction endonuclease Bse SI has been isolated from Bacillus stearothermophilus Jo10-553. Bse SI recognizes a degenerate hexanucleotide sequence 5'-G(G/T)GC(A/C)C-3' and cleaves DNA to produce 3[prime]-protruding tetranucleotide ends. (+info)
An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases.
(43/6278)
DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides. A proof of the concept is obtained using pseudo-thymidine (psiT), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psiT arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation. From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psiT. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psiT are presented. This is the first time that PCR has been performed with a C-nucleoside. (+info)
The effects of secondary structure and O2 on the formation of direct strand breaks upon UV irradiation of 5-bromodeoxyuridine-containing oligonucleotides.
(44/6278)
BACKGROUND: 5-Bromodeoxyuridine is a radiosensitizing agent that is currently being evaluated in clinical trials as an adjuvant in the treatment of a variety of cancers. gamma-Radiolysis and UV irradiation of oligonucleotides containing 5-bromodeoxyuridine result in the formation of direct strand breaks at the 5'-adjacent nucleotide by oxidation of the respective deoxyribose. We investigated the effects of DNA secondary structure and O2 on the induction of direct strand breaks in 5-bromodeoxyuridine-containing oligonucleotides. RESULTS: The efficiency of direct strand break formation in duplex DNA is dependent upon O2 and results in fragments containing 3'-phosphate and the labile 3'-ketodeoxyadenosine termini. The ratio of the 3'-termini is also dependent upon O2 and structure. Deuterium product isotope effects and tritium-transfer studies indicate that hydrogen-atom abstraction from the C1'- and C2'-positions occurs in an O2- and structure-dependent manner. CONCLUSIONS: The reaction mechanisms by which DNA containing 5-bromodeoxyuridine is sensitized to damage by UV irradiation are dependent upon whether the substrate is hybridized and upon the presence or absence of O2. Oxygen reduces the efficiency of direct strand break formation in duplex DNA, but does not affect the overall strand damage. It is proposed that the sigma radical abstracts hydrogen atoms from the C1'- and C2'-positions of the 5'-adjacent deoxyribose moiety, whereas the nucleobase peroxyl radical selectively abstracts the C1'-hydrogen atom from this site. This is the second example of DNA damage amplification by a nucleobase peroxyl radical, and might be indicative of a general reaction pattern for this family of reactive intermediates. (+info)
Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina.
(45/6278)
AIMS: To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat. METHODS: Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 microl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV). RESULTS: Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue. CONCLUSIONS: These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV. (+info)
Enhancement of antigen-presenting cell surface molecules involved in cognate interactions by immunostimulatory DNA sequences.
(46/6278)
Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-gamma receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcgamma receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a Th1 phenotype in vitro. Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity. (+info)
Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway.
(47/6278)
To determine whether the systemic immune activation by CpG DNA could alter airway inflammation, we pretreated mice with either i.v. bacterial DNA (bDNA) or oligonucleotides with or without CpG motifs, exposed these mice to LPS by inhalation, and measured the inflammatory response systemically and in the lung immediately following LPS inhalation. Compared with non-CpG oligonucleotides, i. v. treatment with CpG oligonucleotides resulted in higher systemic concentrations of polymorphonuclear leukocytes, IL-10, and IL-12, but significantly reduced the concentration of total cells, polymorphonuclear leukocytes, TNF-alpha, and macrophage inflammatory protein-2 in the lavage fluid following LPS inhalation. The immunoprotective effect of CpG-containing oligonucleotides was dose-dependent and was most pronounced in mice pretreated between 2 and 4 h before the inhalation challenge, corresponding to the peak levels of serum cytokines. bDNA resulted in a similar immunoprotective effect, and methylation of the CpG motifs abolished the protective effect of CpG oligonucleotides. The protective effect of CpG oligonucleotides was observed in mice with either a disrupted IL-10 or IFN-gamma gene, but release of cytokines in the lung was increased, especially in the mice lacking IFN-gamma. In contrast, CpG DNA did not protect mice with a disrupted IL-12 gene against the LPS-induced cellular influx, even though CpG DNA reduced the release of TNF-alpha and macrophage inflammatory protein-2 in the lung. These findings indicate that CpG-containing oligonucleotides or bDNA are protected against LPS-induced cellular airway inflammation through an IL-12-dependent pathway, and that the pulmonary cytokine and cellular changes appear to be regulated independently. (+info)
Predicting regional mutability in antibody V genes based solely on di- and trinucleotide sequence composition.
(48/6278)
Somatic mutations are not distributed randomly throughout Ab V region genes. A sequence-specific target bias is revealed by a defined hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA. Here we report that the di- and trinucleotide mutability preference pattern is shared by mouse intronic JH and Jkappa clusters and by human VH genes, suggesting that a common mutation mechanism exists for all Ig V genes of both species. Using di- and trinucleotide target preferences, we performed a comprehensive analysis of human and murine germline V genes to predict regional mutabilities. Heavy chain genes of both species exhibit indistinguishable patterns in which complementarity-determining region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be more mutable than FR1 and FR2. This prediction is borne out by empirical mutation data from nonproductively rearranged human VH genes. Analysis of light chain genes in both species also revealed a common, but unexpected, pattern in which FR2 is predicted to be highly mutable. While our analyses of nonfunctional Ig genes accurately predicts regional mutation preferences in VH genes, observed relative mutability differences between regions are more extreme than expected. This cannot be readily accounted for by nascent mRNA secondary structure or by a supplemental gene conversion mechanism that might favor nucleotide replacements in CDR. Collectively, our data support the concept of a common mutation mechanism for heavy and light chain genes of mice and humans with regional bias that is qualitatively, but not quantitatively, accounted for by short nucleotide sequence composition. (+info)