scholarly journals Characterization of hCINAP, a Novel Coilin-interacting Protein Encoded by a Transcript from the Transcription Factor TAFIID32 Locus

2005 ◽  
Vol 280 (43) ◽  
pp. 36429-36441 ◽  
Author(s):  
Niovi Santama ◽  
Stephen C. Ogg ◽  
Anna Malekkou ◽  
Spyros E. Zographos ◽  
Karsten Weis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Essential Gene ◽  
Single Copy ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Binding Motif

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

scholarly journals Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

2003 ◽  
Vol 69 (12) ◽  
pp. 7480-7491 ◽  
Author(s):  
Isabelle Hautefort ◽  
Maria José Proença ◽  
Jay C. D. Hinton
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Single Copy ◽  
Green Fluorescent Protein Gene ◽  
Gene Fusions ◽  
Bacterial Gene ◽  
Green Fluorescent

ABSTRACT We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as β-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

scholarly journals Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

2018 ◽  
Vol 19 (12) ◽  
pp. 3767 ◽  
Author(s):  
Qian Wang ◽  
Jian Fang ◽  
Qihua Pan ◽  
Yizhou Wang ◽  
Ting Xue ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Transgenic Fish ◽  
Early Gene ◽  
Drug Selection ◽  
Baculovirus System ◽  
Fish Cells

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

2018 ◽  
Vol 194 ◽  
pp. 29-39 ◽  
Author(s):  
Fatemeh Motevalli ◽  
Azam Bolhassani ◽  
Shilan Hesami ◽  
Sepideh Shahbazi
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions

2012 ◽  
Vol 23 (6) ◽  
pp. 1095-1103 ◽  
Author(s):  
Sylvie Urbé ◽  
Han Liu ◽  
Sebastian D. Hayes ◽  
Claire Heride ◽  
Daniel J. Rigden ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
A549 Cells ◽  
Binding Motif ◽  
Vitro Assay ◽  
Microtubule Binding ◽  
Functional Studies ◽  
Physiological Processes ◽  
Associated Functions ◽  
Green Fluorescent

Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 deubiquitinases (DUBs) encoded in the human genome, of which 79 are predicted to have catalytic activity. We tagged 66 DUBs with green fluorescent protein and systematically surveyed their subcellular distribution, identifying enzymes specific to the nucleus, plasma membrane, and secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule-binding motif encompassed within amino acids 59–75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule- and centrosome-associated physiological processes: Depletion of USP21 in A549 cells compromises the reestablishment of a radial array of microtubules during recovery from cold-induced depolymerization and also reduces the probability of primary cilium formation, whereas USP21 knockdown in PC12 cells inhibits nerve growth factor–induced neurite outgrowth.

scholarly journals Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes.

1993 ◽  
Vol 13 (12) ◽  
pp. 7267-7277 ◽  
Author(s):  
A R Fattaey ◽  
E Harlow ◽  
K Helin
Keyword(s):  
Transcription Factor ◽  
Protein Complexes ◽  
Susceptibility Gene ◽  
Cyclin A ◽  
Cellular Proteins ◽  
Adenovirus E1a ◽  
E2f Transcription Factor ◽  
E1a Gene

The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.

Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library 1 1Edited by K. Nayai

2000 ◽  
Vol 301 (4) ◽  
pp. 783-793 ◽  
Author(s):  
Fanyi Jiang ◽  
Susanne Wisén ◽  
Mikael Widersten ◽  
Birgitta Bergman ◽  
Bengt Mannervik
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
In Vitro Selection ◽  
Binding Motif ◽  
Random Library ◽  
Selection Of

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

Synergistic activation of a human promoter in vivo by transcription factor Sp1

1991 ◽  
Vol 11 (4) ◽  
pp. 1935-1943
Author(s):  
G M Anderson ◽  
S O Freytag
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Single Copy ◽  
Relative Contribution ◽  
Human Promoter ◽  
Transcription Factor Sp1 ◽  
Synergistic Activation ◽  
Cellular Context

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.

Gabp Transcription Factor Is Required for the Development of Mouse Megakaryocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2604-2604
Author(s):  
Zhong-Fa Yang ◽  
Timothy M Chlon ◽  
John Crispino ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Dna Ploidy ◽  
Conflicts Of Interest ◽  
Knock Out ◽  
Megakaryocytic Differentiation ◽  
Platelet Counts ◽  
Redundant Role

Abstract Abstract 2604 GABP transcription factor has been implicated in the regulation of genes that are required for normal megakaryocytic differentiation. Megakaryocytes express several related ets factors, including Fli-1, ets2, and GABP, and it has been unclear if any single ets factor plays a non-redundant role in these cells. The tetrameric GABP transcription factor complex contains two molecules of GABPα, which binds DNA, and two molecules of GABPβ, which encodes the transcription activation domain. We created mice with loxP recombination sites which flank exons that encode Gabpa ets-related DNA-binding domains (floxed Gabpa, or Gabpa fl/fl), and bred them to mice that carry Mx1-Cre. In response to injection with the synthetic polynucleotide, pIC, these mice express Cre recombinase and efficiently delete Gabpa; these animals are referred to as knock-out (KO) mice. Control mice, which carry floxed Gabpa but lack Mx1-Cre, were treated identically with pIC. Platelet counts of KO mice declined to less than 50,000 within nine days, while platelet counts in control mice were unaffected. One half of KO mice died within two weeks of Gabpa deletion due to widespread visceral hemorrhage. Histologic examination of the bone marrow and spleen reveals a loss of megakaryocytes in KO mice, compared to control animals. Residual megakaryocytes in KO mice exhibit increased expression of platelet-specific antigens, CD41 and CD42, and a significant increase of DNA ploidy. Because Gabpa KO mice died with a striking loss of megakaryocytes and platelets, yet megakaryocytic differentiation appeared to be unimpaired, we sought to better define the nature of this defect. Bone marrow from Gabpa fl/fl mice was infected with a retrovirus that expresses Cre and green fluorescent protein (GFP), or control virus that expresses only GFP; grown for three days in liquid culture conditions that foster megakaryocytic differentiation; and analyzed for CD41 and CD42 expression, ploidy, and apoptosis. Gabpa was efficiently deleted by the Cre-bearing virus, and Gabpa deletion was associated with increased expression of CD41 and CD42, and increased DNA ploidy. However, Gabpa deletion was also associated with increased megakaryocytic-associated apoptosis, and in vitro megakaryocyte colony formation was dramatically reduced in Gabpα null cells. In summary, deletion of Gabpa in mice is associated with plummeting platelet counts, widespread visceral hemorrhage, and a loss of splenic and bone marrow megakaryocytes. In vitro analysis demonstrates intact megakaryocytic differentiation and a profound loss of megakaryocytic progenitor cells. The increased expression of megakaryocytic antigens and DNA ploidy may indicate that Gabpa deletion enhances megakaryocytic differentiation or, alternatively, it may represent selective loss of more immature megakaryocytic cells following Gabpa disruption. Data that directly test these alternative hypotheses will be presented. In summary, we demonstrate that GABP plays a non-redundant role in megakaryocyte development, that GABP is required for the proliferation of committed megakaryocytic progenitors, but that GABP is not required for the later stages of megakaryocytic maturation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

1997 ◽  
Vol 17 (9) ◽  
pp. 5288-5298 ◽  
Author(s):  
N H Kuldell ◽  
S Buratowski
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Large Subunit ◽  
Small Subunit ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol Ii

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

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