scholarly journals hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

1999 ◽  
Vol 19 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Min-Yuan Chou ◽  
Nanette Rooke ◽  
Christoph W. Turck ◽  
Douglas L. Black
Keyword(s):  
Nuclear Extract ◽  
Peptide Sequence ◽  
Binding Assays ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Shift Analysis ◽  
Hnrnp F ◽  
Gel Mobility Shift ◽  
Splicing Enhancer

ABSTRACT The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

scholarly journals Negative Regulation of the Gene for Fe-Containing Superoxide Dismutase by an Ni-Responsive Factor inStreptomyces coelicolor

1999 ◽  
Vol 181 (23) ◽  
pp. 7381-7384 ◽  
Author(s):  
Hye-Jung Chung ◽  
Jae-Hyun Choi ◽  
Eun-Ja Kim ◽  
You-Hee Cho ◽  
Jung-Hye Roe
Keyword(s):  
Superoxide Dismutase ◽  
Streptomyces Coelicolor ◽  
Dna Binding Protein ◽  
Negative Regulation ◽  
Superoxide Dismutases ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Gel Mobility Shift

ABSTRACT In Streptomyces coelicolor, transcription of thesodF genes, encoding Fe-containing superoxide dismutases, is negatively regulated by nickel. Gel mobility shift assays withsodF1 promoter fragments and cell extracts from the A3(2) strain indicate the presence of a nickel-responsive DNA-binding protein, most likely a transcriptional repressor. The boundary for the Ni-responsive cis-acting region was identified both in vitro and vivo. Ni does not regulate the level of the putative repressor but only the binding competence of this protein.

Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions

1993 ◽  
Vol 13 (10) ◽  
pp. 6336-6345 ◽  
Author(s):  
S Meyers ◽  
J R Downing ◽  
S W Hiebert
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Consensus Sequence ◽  
Myelogenous Leukemia ◽  
Chromosome 21 ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Shift Analysis

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.

scholarly journals Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

1993 ◽  
Vol 13 (10) ◽  
pp. 6336-6345 ◽  
Author(s):  
S Meyers ◽  
J R Downing ◽  
S W Hiebert
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Consensus Sequence ◽  
Myelogenous Leukemia ◽  
Chromosome 21 ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Shift Analysis

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

A C. elegans E/Daughterless bHLH protein marks neuronal but not striated muscle development

Development ◽  
1997 ◽  
Vol 124 (11) ◽  
pp. 2179-2189 ◽  
Author(s):  
M. Krause ◽  
M. Park ◽  
J.M. Zhang ◽  
J. Yuan ◽  
B. Harfe ◽  
...  
Keyword(s):  
Muscle Development ◽  
Striated Muscle ◽  
Bhlh Protein ◽  
E Proteins ◽  
Mobility Shift ◽  
C Elegans ◽  
Helix Loop Helix ◽  
Neuronal Precursors ◽  
Gel Mobility Shift

The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. We report here the characterization of a related C. elegans protein, CeE/DA, which has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described Achaete-scute-like gene hlh-3. In contrast, we have been unable to detect CeE/DA in CeMyoD-positive striated muscle cells. In vitro gel mobility shift analysis detects dimerization of CeE/DA with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species.

scholarly journals Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

1992 ◽  
Vol 12 (8) ◽  
pp. 3490-3498 ◽  
Author(s):  
N Hosokawa ◽  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Aoike ◽  
...  
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Mobility Shift ◽  
Human Colon Cancer ◽  
Heat Shock Element ◽  
Mrna Accumulation ◽  
Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

scholarly journals ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence

2009 ◽  
Vol 77 (7) ◽  
pp. 2832-2839 ◽  
Author(s):  
Francois Lebreton ◽  
Eliette Riboulet-Bisson ◽  
Pascale Serror ◽  
Maurizio Sanguinetti ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Animal Experiments ◽  
Opportunistic Pathogen ◽  
Transcriptional Analysis ◽  
Binding Assays ◽  
Mobility Shift ◽  
Reverse Transcription Quantitative Pcr

ABSTRACT Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

scholarly journals Role of Pseudomonas aeruginosa dinB-Encoded DNA Polymerase IV in Mutagenesis

2006 ◽  
Vol 188 (24) ◽  
pp. 8573-8585 ◽  
Author(s):  
Laurie H. Sanders ◽  
Andrea Rockel ◽  
Haiping Lu ◽  
Daniel J. Wozniak ◽  
Mark D. Sutton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Polymerase ◽  
Opportunistic Pathogen ◽  
Binding Assays ◽  
Mutator Phenotype ◽  
Cell Extracts ◽  
Dna Polymerase Iv ◽  
Mechanisms Of Mutagenesis ◽  
Y Family

ABSTRACT Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB Pa ). Our results indicate that DinB Pa is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB Pa from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB Pa has a propensity to promote C→A transversions and −1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA + and ΔlexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA Pa ), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB Pa gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB Pa in translesion DNA synthesis over N 2 -dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB Pa function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

scholarly journals Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

2000 ◽  
Vol 74 (11) ◽  
pp. 5053-5065 ◽  
Author(s):  
Jeannie F. Spagnolo ◽  
Brenda G. Hogue
Keyword(s):  
Protein Interactions ◽  
Helper Virus ◽  
Host Protein ◽  
Genome Replication ◽  
Mobility Shift ◽  
Bovine Coronavirus ◽  
Replication Assay ◽  
Infected Cells ◽  
Gel Mobility Shift

ABSTRACT RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.

scholarly journals The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

1997 ◽  
Vol 17 (10) ◽  
pp. 5923-5934 ◽  
Author(s):  
H Lu ◽  
R P Fisher ◽  
P Bailey ◽  
A J Levine
Keyword(s):  
Transcription Factor ◽  
Excision Repair ◽  
Binding Activity ◽  
Mobility Shift ◽  
Trimeric Complex ◽  
Dna Binding Activity ◽  
Terminal Amino ◽  
Gel Mobility Shift ◽  
Kinase Phosphorylation

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

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