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At least 235 records · Page 13Linked to original sources
PubMed1998

The Bacillus subtilis DinR binding site: redefinition of the consensus sequence.

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB, dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5' and 3' of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5'-CGAACRNRYGTTYC-3'. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

Amino Acid Sequence↗
PubMed1986

Two or more copies of Drosophila heat shock consensus sequence serve to activate transcription in yeast.

A synthetic oligonucleotide bearing the Drosophila heat shock consensus sequence confers heat inducibility on a CYC1-lacZ gene in Saccharomyces cerevisiae. This sequence CTGGAATTTTCTAGA was inserted in place of the upstream activation sites of the CYC1 promoter adjacent to CYC1 TATA boxes. These constructs were transformed into yeast and found to be heat-inducible when two or more inserts were present. The level of inducibility seemed to increase with the number of inserted sequences: however, the orientations of these sequences relative to each other did not have much effect.

Animals↗
PubMed1995

Structure determination of a tetradecapeptide mimicking the RXVRG consensus sequence recognized by a Xenopus laevis skin endoprotease: an approach based on simulated annealing and 1H NMR.

The tetradecapeptide of sequence H-Asp-Val-Asp-Glu-Arg5-Asp-Val-Arg-Gly9-Phe-Ala-Ser-Phe-Leu- NH2 is recognized by a putative maturation endoprotease of the Xenopus laevis skin, which cleaves between Arg8 and Gly9. A conformational search has been performed on this peptide by simulated annealing calculations. Two different models in agreement with the NMR data were found. The conformational difference between the two types of model is located in the consensus sequence, i.e., from Arg5 to Gly9.

Amino Acid Sequence↗
PubMed2004

A consensus sequence in the endothelin-B receptor second intracellular loop is required for NHE3 activation by endothelin-1.

Endothelin-1 (ET-1) increases the activity of Na(+)/H(+) exchanger 3 (NHE3), the major proximal tubule apical membrane Na(+)/H(+) antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ET(B)) and not in cells expressing the endothelin-A (ET(A)) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca(2+)] in ET(A)- and ET(B)-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ET(B) receptor. The present studies used ET(A) and ET(B) receptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ET(A) receptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ET(B) receptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

Amino Acid Sequence↗
PubMed1999

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.

Calcitonin↗
PubMed2005

Four differentially expressed cDNAs in Callinectes sapidus containing the Rebers-Riddiford consensus sequence.

Decapod crustaceans such as Callinectes sapidus, the blue crab, provide unique opportunities to study proteins involved in biomineralization. Subsequent to each molt, the previously deposited soft cuticle is calcified while the postecdysial layers are simultaneously deposited and mineralized. Though the majority of the exoskeleton hardens, morphologically similar cuticle at the joints, called arthrodial membrane, remains flexible. It seems reasonable that hypodermal cells producing these cuticle types should be synthesizing proteins that regulate mineralization. Data presented here are consistent with this hypothesis, showing that transcripts coding for proteins containing the chitin-binding Rebers-Riddiford (RR) consensus sequence (Gx(8)Gx(7)YxAxExGYx(7)Px(2)P) are differentially expressed. Two RR-containing transcripts, CsAMP8.1 and CsAMP6.0, are found only in arthrodial membrane and are expressed uniformly both before and after ecdysis. They have high sequence homology with RR-containing proteins from uncalcified portions of the cuticle of Cancer pagurus, Penaeus japonicus, and Homarus americanus. The other two transcripts, CsCP8.5 and CsCP8.2, are expressed solely in premolt and in hypodermis depositing calcifying cuticle rather than arthrodial membrane. They have high sequence homology with calcification-associated peptides containing the RR sequence obtained from the calcified cuticle of Procambarus clarkii. This suggests possible involvement in the postmolt mineralization of the pre-ecdysial cuticle.

Amino Acid Sequence↗
PubMed2004

Determination of HIV type 1 CRF01_AE gag p17 and env-V3 consensus sequences for HIV/AIDS vaccine design.

A molecular epidemiological study of the gag p17 and env-V3 regions on HIV-infected drug users and blood donors was carried out in northern Thailand from 1998 through 2002 to determine the predominant subtype and consensus sequence (CS) for circulating HIV-1 strains. CRF01_AE was concluded to be a predominant strain and the nucleotide CSs in gag p17 and env-V3 showed only 1.26% and no difference from CS in the Los Alamos database, respectively. Our env-V3 CS was identical to the previously published CSs, suggesting that the CS was very conserved from 1990 through 2002 in Thailand. Gag p17 and env-V3 nucleotide sequences of seroconvertors in our subjects were quite similar to the CS and conserved for at least 9 and 6 years postinfection, respectively. These results suggest that the CS approach to the HIV-1 antigen design could overcome HIV diversity and help us develop an effective HIV/AIDS vaccine.

AIDS Vaccines↗
PubMed1994

Evidence that the heparin-binding consensus sequence of vitronectin is recognized by Staphylococcus aureus.

Binding of heparin-binding form of vitronectin to Staphylococcus aureus was inhibited completely by heparin or by the same form of vitronectin. The binding was inhibited only to about 50% by the non-heparin-binding form of vitronectin, indicating an apparent involvement of the heparin-binding properties in the interaction between vitronectin and S. aureus. This was supported by experiments in which a synthetic peptide (Ala347-Arg361, comprising heparin-binding consensus sequences) was found to partly inhibit bacterial adherence to immobilized vitronectin. A bacterial cell surface protein could bind to the quinquedecapeptide, but not to the highly charged peptides consisting entirely of arginine or lysine, immobilized on microtiter plates and the binding could be competitively inhibited by an excess of soluble peptide. Direct binding of radiolabeled peptide to bacterial cells was also demonstrated, which was rapid, saturable, and pH-dependent. Furtherly a bacterial surface protein having molecular mass of 60 kDa was isolated by affinity chromatography on a quinquedecapeptide-HiTrap-NHS column. Our data suggest that the heparin-binding properties of vitronectin play a role in bacterial recognition.

Amino Acid Sequence↗
PubMed2000

An IFN-gamma-inducible transcription factor, IFN consensus sequence binding protein (ICSBP), stimulates IL-12 p40 expression in macrophages.

IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-gamma/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-gamma/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP-/- mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-gamma/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP-/- cells, even in the absence of IFN-gamma/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-gamma/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-gamma and LPS. Taken together, our results show that ICSBP induced by IFN-gamma/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.

Animals↗
PubMed2004

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

Amino Acids↗
PubMed2002

IFN consensus sequence binding protein/IFN regulatory factor-8 guides bone marrow progenitor cells toward the macrophage lineage.

IFN consensus sequence binding protein (ICSBP; IFN regulatory factor-8) is a transcription factor of the IFN regulatory factor family. Disruption of this gene results in a leukemia-like disease in mice. To investigate the role of ICSBP in myeloid cell development, lineage marker-negative (Lin(-)) bone marrow progenitor cells were purified from ICSBP(+/+) and ICSBP(-/-) mice and tested for gene expression and colony-forming ability. ICSBP was expressed in Lin(-) progenitor cells, and its levels were markedly increased by IFN-gamma. The colony-forming potential of ICSBP(-/-) progenitor cells was grossly abnormal, as they gave rise to a disproportionately high number of granulocyte colonies and many fewer macrophage colonies. IFN-gamma inhibited colony formation, while promoting macrophage maturation in ICSBP(+/+) cells. In contrast, the effects of IFN-gamma were completely absent in ICSBP(-/-) progenitors. By retrovirus transduction we tested whether reintroduction of ICSBP restores a normal colony-forming potential in -/- progenitor cells. The wild-type ICSBP, but not transcriptionally defective mutants, corrected abnormal colony formation by increasing macrophage colonies and decreasing granulocyte colonies. Taken together, ICSBP plays a critical role in myeloid cell development by controlling lineage selection and is indispensable for IFN-gamma-dependent modulation of progenitor cell maturation.

Animals↗
PubMed1996

Molecular cloning and characterization of the yellowtail GH gene and its promoter: a consensus sequence for teleost and avian Pit-1/GHF-1 binding sites.

The gene and 5' flanking promoter region for yellowtail (Seriola quinqueradiata) GH (yGH) have been cloned, sequenced and characterized. The yGH gene spans approximately 4.6 kb and consists of six exons and five introns, as has been observed with rainbow trout, Atlantic salmon and tilapia GH genes. This result suggests that the structure of six exons and five introns is a dominant form in fish GH genes. A typical TATA box exists 26 bp upstream from the transcription start site, and Pit-1/GHF-1 (Pit-1) binding site-homologous regions were found in the promoter region of the yGH gene. In a gel shift assay, however, a single shifted band was detected with the fragments containing a region from -128 to -90 of the yGH 5' flanking region when they were incubated with yellowtail pituitary nuclear extracts. The bound fragments contained an octamer base sequence similar, but not identical, to mammalian consensus Pit-1 binding element. A consensus octamer sequence is also proposed in this report for the binding of teleost and avian Pit-1 transcription factors.

Amino Acid Sequence↗
PubMed2005

Cross-clade protection induced by human immunodeficiency virus-1 DNA immunogens expressing consensus sequences of multiple genes and epitopes from subtypes A, B, C, and FGH.

The correlate of protection in human immunodeficiency virus (HIV) infection is not known, but preclinical and clinical studies support the involvement of both antibodies and cellular immunity. In addition, the existence of multiple HIV clades makes HIV vaccine design especially challenging. We have constructed a vaccine platform with an HIV-1 subtype B DNA immunogen expressing full length consensus sequences from HIV-1 rev, nef, tat, and gag with additional cellular epitope clusters from the env and pol regions. Furthermore, this platform has been extended to three additional plasmids expressing the same immunogens but originating from subtypes A or C consensus or FGH ancestral sequences. Immunogenicity in BALB/c mice, by gene gun or intramuscular delivery, revealed strong IFN-gamma production in response to in vitro re-stimulation with a H-2d restricted gag peptide (AMQMLKETI) or even stronger toward an env epitope (RGPGRAFVTI). Weak humoral immunity was detected. Gene gun immunization with a cocktail of all four plasmids induced pre-challenge cellular immunity in C57Bl6/A2.01 mice and subsequently a robust frequency of protection (11/12 animals) after experimental challenge with subtype A or B HIV-1/Murine Leukemia Virus (HIV-1/MuLV). The cross-clade protection observed in this challenge experiment demonstrates that these multigene/multiepitope HIV DNA immunogens are likely to be potent immunogens also against the HIV-infection of human beings.

AIDS Vaccines↗
PubMed1999

The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.

Cold Temperature↗
PubMed1999

Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence.

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.

Amino Acid Sequence↗
PubMed2002

Accumulation of c-Cbl and rapid termination of colony-stimulating factor 1 receptor signaling in interferon consensus sequence binding protein-deficient bone marrow-derived macrophages.

Mice deficient for the transcription factor interferon consensus sequence binding protein (ICSBP) are immunodeficient and develop granulocytic leukemia. Further analyses indicated that ICSBP is a molecular switch factor directing the differentiation of bipotential myeloid precursors to the monocytic lineage. To reveal the molecular mechanisms responsible for the deregulation of myelopoiesis, we examined the signaling of the colony-stimulating factor 1 receptor (CSF-1R) in bone marrow-derived macrophages (BMMs) from ICSBP(-/-) mice. We found that in the absence of ICSBP, CSF-1R signaling is attenuated as seen from an accelerated termination of Erk phosphorylation and reduced cell growth. This finding coincides with an increased CSF-1R ubiquitination and an enhanced accumulation of c-Cbl. c-Cbl is an ubiquitin-ligase known to down-regulate activated CSF-1R by targeting it to the endocytic pathway. Our results indicate that upon CSF-1R activation, c-Cbl itself is partly proteolytically degraded in ICSBP(+/+) but not in ICSBP(-/-) BMMs. Congruently, the expression of a major endosomal/lysosomal protease, cathepsin B, is strongly reduced in ICSBP(-/-) BMMs.

Animals↗
PubMed2000

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.

Animals↗