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(1/10948) Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.

The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry.  (+info)

(2/10948) Oligomerization and scaffolding functions of the erythropoietin receptor cytoplasmic tail.

Signal transduction by the erythropoietin receptor (EPOR) is activated by ligand-mediated receptor homodimerization. However, the relationship between extracellular and intracellular domain oligomerization remains poorly understood. To assess the requirements for dimerization of receptor cytoplasmic sequences for signaling, we overexpressed mutant EPORs in combination with wild-type (WT) EPOR to drive formation of heterodimeric (i.e. WT-mutant) receptor complexes. Dimerization of the membrane-proximal portion of the EPOR cytoplasmic region was found to be critical for the initiation of mitogenic signaling. However, dimerization of the entire EPOR cytoplasmic region was not required. To examine this process more closely, we generated chimeras between the intracellular and transmembrane portions of the EPOR and the extracellular domains of the interleukin-2 receptor beta and gammac chains. These chimeras allowed us to assess more precisely the signaling role of each receptor chain because only heterodimers of WT and mutant receptor chimeras form in the presence of interleukin-2. Coexpression studies demonstrated that a functional receptor complex requires the membrane-proximal region of each receptor subunit in the oligomer to permit activation of JAK2 but only one membrane-distal tail to activate STAT5 and to support cell proliferation. Thus, this study defines key relationships involved in the assembly and activation of the EPOR signal transduction complex which may be applicable to other homodimeric cytokine receptors.  (+info)

(3/10948) Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.  (+info)

(4/10948) Chlamydia infections and heart disease linked through antigenic mimicry.

Chlamydia infections are epidemiologically linked to human heart disease. A peptide from the murine heart muscle-specific alpha myosin heavy chain that has sequence homology to the 60-kilodalton cysteine-rich outer membrane proteins of Chlamydia pneumoniae, C. psittaci, and C. trachomatis was shown to induce autoimmune inflammatory heart disease in mice. Injection of the homologous Chlamydia peptides into mice also induced perivascular inflammation, fibrotic changes, and blood vessel occlusion in the heart, as well as triggering T and B cell reactivity to the homologous endogenous heart muscle-specific peptide. Chlamydia DNA functioned as an adjuvant in the triggering of peptide-induced inflammatory heart disease. Infection with C. trachomatis led to the production of autoantibodies to heart muscle-specific epitopes. Thus, Chlamydia-mediated heart disease is induced by antigenic mimicry of a heart muscle-specific protein.  (+info)

(5/10948) The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.

Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.  (+info)

(6/10948) Multiple oligodeoxyribonucleotide syntheseson a reusable solid-phase CPG support via the hydroquinone-O,O'-diacetic acid (Q-Linker) linker arm.

A strategy for oligodeoxyribonucleotide synthesis on a reusable CPG solid-phase support, derivatized with hydroxyl groups instead of amino groups, has been developed. Ester linkages, through a base labile hydroquinone- O, O '-diacetic acid ( Q-Linker ) linker arm, were used to couple the first nucleoside to the hydroxyl groups on the support. This coupling was rapidly accomplished (10 min) using O -benzotriazol-1-yl- N, N, N ', N '-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole as the activating reagents. Oligodeoxyribonucleotide synthesis was performed using existing procedures and reagents, except a more labile capping reagent, such as chloro-acetic anhydride, methoxyacetic anhydride or t-butylphenoxyacetic anhydride, was used instead of acetic anhydride. After each oligodeoxyribonucleotide synthesis, the product was cleaved from the support with ammonium hydroxide (3 min) and deprotected as usual. Residual linker arms or capping groups were removed by treatment with ammonium hydroxide/methylamine reagent and the regenerated support was capable of reuse. Up to six different oligodeoxyribonucleotide syntheses or up to 25 cycles of nucleoside derivatization and cleavage were consecutively performed on the reusable support. This method may provide a significant cost advantage over conventional single-use solid supports currently used for the manufacture of antisense oligodeoxyribonucleotides.  (+info)

(7/10948) Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.  (+info)

(8/10948) Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe).

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.  (+info)