scholarly journals Semiquantitative Comparison of the DNA-Binding Activity of In Vitro-Synthesized Proteins

BioTechniques ◽  
1997 ◽  
Vol 23 (5) ◽  
pp. 792-794 ◽  
Author(s):  
Patrick Chène
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Dna Binding Activity

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes

1989 ◽  
Vol 9 (5) ◽  
pp. 2018-2024
Author(s):  
D L Johnson ◽  
S L Wilson
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Iii ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Trna Genes ◽  
Dna Binding Activity ◽  
Transcription In Vitro ◽  
Affinity Interaction ◽  
Dna Complex

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 245-255 ◽  
Author(s):  
M. Van Doren ◽  
H.M. Ellis ◽  
J.W. Posakony
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Protein Complexes ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Function ◽  
Biochemical Basis ◽  
Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

DNA Binding Activity of Marine Shrimp LvProfilin

2021 ◽  
Author(s):  
Yanisa Laoong-u-thai ◽  
Warapond Wanna ◽  
Autaipohn Kaikaew
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
3D Structure ◽  
Binding Activity ◽  
Shrimp Farming ◽  
Binding Prediction ◽  
3D Protein Structure ◽  
Dna Binding Activity ◽  
Marine Shrimp

Shrimp farming is an important business in Thailand and worldwide. The study of molecular biology and biochemical pathway of the key molecules controlling muscle growth is an essential to improve shrimp livestock. Profilin is a pivotal protein in muscle formation, especially actin protein. Its nuclear function has been reported in many species for gene regulation. Here in this work, we characterized the function of LvProfilin, a marine shrimp profilin from Litopenaeus vannamei, both in silico and in vitro. The phylogenetic tree of LvProfilin among organisms and its 3D protein structure showed that LvProfilin was highly conserved among shrimp and arthropods. The homology modeling of its 3D structure revealed 3 alpha-helices and 6 beta-strands similar to most eukaryotic profilins. To interpret its possible function, the gene expression of LvProfilin in various tissues was performed. We found that this gene was expressed in various tissues. This result may imply that LvProfilin could share a common function in all tissues. Nuclear activity has been a promising function of LvProfilin. We performed a DNA/RNA binding prediction analysis using DRNApred. The result indicated that Lysine-90 and Threonine-91 were the putative DNA-binding sites with the probability of 63.12% and 54.16%, respectively. Its binding activity was confirmed in vitro which bound stronger to single strand DNA than double strand DNA. To our best knowledge, this is the first report of DNA binding activity of profilin in invertebrates.

Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells

1997 ◽  
Vol 272 (3) ◽  
pp. L504-L511 ◽  
Author(s):  
I. Jaspers ◽  
E. Flescher ◽  
L. C. Chen
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Epithelial Cells ◽  
Inflammatory Cells ◽  
Binding Activity ◽  
Ozone Exposure ◽  
Air Exposure ◽  
Dna Binding Activity ◽  
Nf Kappab

Ozone, one of the most reactive oxidant gases to which humans are routinely exposed, induces inflammation in the lower airways. The airway epithelium is one of the first targets that inhaled ozone will encounter, but its role in airway inflammation is not well understood. Expression of inducible genes involved in the inflammatory response, such as interleukin (IL)-8, is controlled by transcription factors. Expression of the IL-8 gene is regulated by the transcription factors nuclear factor (NF)-kappaB, NF-IL-6, and possibly activator protein-1 (AP-1). Type II-like epithelial cells (A549) were grown on a collagen-coated membrane and exposed in vitro to 0.1 ppm ozone or air. Exposure to ozone induced DNA-binding activity of NF-kappaB, NF-IL-6, and AP-1. IL-8 mRNA and IL-8 protein levels were also increased after ozone exposure. These results link ozone-induced DNA-binding activity of transcription factors and the production of IL-8 by epithelial cells thus demonstrating a potential cellular cascade resulting in the recruitment of inflammatory cells into the airway lumen.

scholarly journals The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

2004 ◽  
Vol 85 (7) ◽  
pp. 2001-2013 ◽  
Author(s):  
Koen W. R. van Cleef ◽  
Wendy M. A. Scaf ◽  
Karen Maes ◽  
Suzanne J. F. Kaptein ◽  
Erik Beuken ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Replication ◽  
Deletion Mutant ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

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