scholarly journals Biochemical characterization of mouse brain necdin

1996 ◽  
Vol 314 (3) ◽  
pp. 895-901 ◽  
Author(s):  
Etsuko MARUYAMA
Keyword(s):  
Dna Binding ◽  
Affinity Chromatography ◽  
Mouse Brain ◽  
Silver Staining ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Micrococcal Nuclease ◽  
Dependent Manner ◽  
Chromatography Column

Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on micrococcal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits a pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (> 200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.

scholarly journals The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Antoine Hocher ◽  
Maria Rojec ◽  
Jacob B Swadling ◽  
Alexander Esin ◽  
Tobias Warnecke
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coli Strain ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromatin Architecture ◽  
Archaeal Histone

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae

2007 ◽  
Vol 53 (1) ◽  
pp. 195-200
Author(s):  
Javier Vernal ◽  
Viviane I. Serpa ◽  
Carolina Tavares ◽  
Emanuel M. Souza ◽  
Fábio O. Pedrosa ◽  
...  
Keyword(s):  
Dna Binding ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Herbaspirillum Seropedicae ◽  
Single Stranded Dna

scholarly journals Unique Properties of the Alpha-Helical DNA-Binding Protein KfrA Encoded by the IncU Incompatibility Group Plasmid RA3 and Its Host-Dependent Role in Plasmid Maintenance

2020 ◽  
Vol 87 (2) ◽  
Author(s):  
Ewa Lewicka ◽  
Monika Mitura ◽  
Kamil Steczkiewicz ◽  
Justyna Kieracinska ◽  
Kamila Skrzynska ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Regulatory Function ◽  
Broad Host Range ◽  
Dependent Manner ◽  
Incompatibility Group ◽  
Plasmid Maintenance ◽  
Helical Protein

ABSTRACT KfrA, encoded on the broad-host-range RA3 plasmid, is an alpha-helical DNA-binding protein that acts as a transcriptional autoregulator. The KfrARA3 operator site overlaps the kfrA promoter and is composed of five 9-bp direct repeats (DRs). Here, the biological properties of KfrA were studied using both in vivo and in vitro approaches. Localization of the DNA-binding helix-turn-helix motif (HTH) was mapped to the N29-R52 region by protein structure modeling and confirmed by alanine scanning. KfrA repressor ability depended on the number and orientation of DRs in the operator, as well as the ability of the protein to oligomerize. The long alpha-helical tail from residues 54 to 355 was shown to be involved in self-interactions, whereas the region from residue 54 to 177 was involved in heterodimerization with KfrC, another RA3-encoded alpha-helical protein. KfrA also interacted with the segrosome proteins IncC (ParA) and KorB (ParB), representatives of the class Ia active partition systems. Deletion of the kfr genes from the RA3 stability module decreased the plasmid retention in diverse hosts in a species-dependent manner. The specific interactions of KfrA with DNA are essential not only for the transcriptional regulatory function but also for the accessory role of KfrA in stable plasmid maintenance. IMPORTANCE Alpha-helical coiled-coil KfrA-type proteins are encoded by various broad-host-range low-copy-number conjugative plasmids. The DNA-binding protein KfrA encoded on the RA3 plasmid, a member of the IncU incompatibility group, oligomerizes, forms a complex with another plasmid-encoded, alpha-helical protein, KfrC, and interacts with the segrosome proteins IncC and KorB. The unique mode of KfrA dimer binding to the repetitive operator is required for a KfrA role in the stable maintenance of RA3 plasmid in distinct hosts.

Preparation of the DNA-binding protein HU from Escherichia coli by immuno-affinity chromatography

FEBS Letters ◽  
1978 ◽  
Vol 96 (2) ◽  
pp. 291-294 ◽  
Author(s):  
Bernard Laine ◽  
Jean-Pierre Kerckaert ◽  
Pierre Sautiere ◽  
Gérard Biserte
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Affinity Chromatography ◽  
Binding Protein ◽  
Dna Binding Protein

scholarly journals Adenovirus DNA Binding Protein Interacts with the SNF2-Related CBP Activator Protein (SrCap) and Inhibits SrCap-Mediated Transcription

2001 ◽  
Vol 75 (21) ◽  
pp. 10033-10040 ◽  
Author(s):  
Xiequn Xu ◽  
Isaac Chackalaparampil ◽  
M. Alexandra Monroy ◽  
Maria T. Cannella ◽  
Elizabeth Pesek ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein A ◽  
Dna Binding Protein ◽  
Cellular Protein ◽  
Protein Family ◽  
Dependent Manner ◽  
Transcription Activity ◽  
Activator Protein

ABSTRACT The SNF2-related CBP activator protein, SrCap (pronounced “sir cap”), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.

scholarly journals Purification and characterization of thyroid transcription factor 2

1994 ◽  
Vol 304 (3) ◽  
pp. 981-985 ◽  
Author(s):  
D Civitareale ◽  
A Saiardi ◽  
P Falasca
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
Thyroid Tissue ◽  
Dna Binding Protein ◽  
Purification And Characterization ◽  
Thyroid Transcription Factor

Thyroid transcription factor 2 binds to the promoters of both thyroglobulin and thyroperoxidase genes, two markers of thyroid tissue differentiation, and its binding modulates the activity of both promoters. In this paper we describe the purification of thyroid transcription factor 2 essentially to homogeneity and demonstrate that it is a thyroid-specific DNA-binding protein. Furthermore, we provide a biochemical characterization suggesting that thyroid transcription factor 2 binds to DNA as a dimer and that it is a zinc-finger DNA-binding protein regulated in vitro by the redox state.

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