Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: Histone morphology of the T. brucei NCP alters its interactions with DNA. ( A ) Estimated diameters of H. sapiens and T. brucei NCP models from averaged measurements in angstrom ( Å ) between the phosphate atom of nucleotide –19 and nucleotide –59 in each DNA strand using ChimeraX . ( B ) Magnified view of the horizontal compression of the T. brucei NCP at SHL6 and SHL2. ( C ) Poorer ordering and asymmetry of DNA ends (colored black) in the T. brucei NCP. ( D ) Distinct 3D classes from cryo-EM processing showing different DNA conformations of the T. brucei NCP (Class 1 = 45974 particles, Class 2 = 47 192 particles, and Class 3 = 47 429 particles).

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques: Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: The T. brucei nucleosome core particle is evolutionarily divergent. ( A ) Phylogenetic trees constructed from histone sequences of selected model organisms and kinetoplastid species. Scale bars on the right indicate pairwise percentage identity (%ID) to T. brucei colored from light to dark (range 40–100%). A comparison of T. brucei and H. sapiens histone sequences in terms of %ID, percentage similarity (%S), and predicted isoelectric points (pI) is shown below. ( B ) Polyacrylamide gels of in-vitro reconstituted T. brucei and H. sapiens NCPs or component histones in their native (DNA stain) and denatured (protein stain + SDS) states. ( C ) Mean melting temperatures ( T m ) of T. brucei and H. sapiens NCPs from three independent experiments (T m values were calculated from the first derivative of melting curves shown in Figure ). ( D ) 3.3 Å cryo-EM density map of the T. brucei NCP coloured according to density attributed to histones and DNA.

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques: Construct, Comparison, In Vitro, Staining, Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: The T. brucei NCP is less stable and has a monophasic disassembly pathway. ( A–D ) Magnified views comparing key amino acid interactions at histone-histone interfaces in the T. brucei and H. sapiens NCPs . Apostrophes indicate the second copy of a histone if two histones of the same type are present. ( E ) Biphasic thermal denaturation curves of H. sapiens NCPs (darker blue) and monophasic denaturation curves of H. sapiens H3–H4 tetrasomes (lighter blue) ( n = 6). ( F ) Monophasic thermal denaturation curves of T. brucei NCPs (darker pink) and T. brucei H3-H4 tetrasomes (lighter pink) ( n = 6). Data points in (E). and (F) are normalized average values ± SD.

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques:

Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: Protein-DNA contacts in the T. brucei NCP have a different distribution and result in weaker DNA binding. ( A ) Frequencies of different amino acids forming hydrogen bonds with DNA in the T. brucei and H. sapiens (PDB: 7XD1) NCPs predicted by ePISA are shown on the left. The total number of interacting residues for each NCP (n) is stated below. On the right, the DNA contacting residues predicted for T. brucei are mapped onto the model of the NCP as spheres. SHL annotations are colored according to reduced (black), increased (pink) or similar (grey) interactions with the DNA phosphate backbone. ( B–E ) Reduced protein-DNA interactions at SHL0, 1.5, 2.5 and 4.5 in the T. brucei NCP compared to the H. sapiens NCP. ( F ) Multiple sequence alignment of the N-terminal tail of histone H2A at SHL 4.5, with DNA-contacting residues from part (E) highlighted. ( G ) Native polyacrylamide gels stained for DNA showing DNA unwrapping in T. brucei and H. sapiens NCPs after incubation at different NaCl concentrations for 1h on ice. Quantification of the percentage of NCP ( H. sapiens = blue, T. brucei = red) and DNA (black) present in each lane, derived from three biological repeats. The NaCl concentration at which free DNA overtakes wrapped NCPs is indicated in grey.

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques: Binding Assay, Sequencing, Staining, Incubation, Derivative Assay, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: A uniqdue mode of DNA binding at SHL 3.5 in the T. brucei NCP. ( A ) Surface electrostatics showing that T. brucei histone octamers have a higher density of positive charge at SHL 3.5 compared to H. sapiens . ( B ) On the left, predicted p K a values of residues contributing to the electrostatic potential at SHL 3.5 in T. brucei and H. sapiens NCPs. On the right, the same residues shown as spheres mapped onto the structures of the T. brucei and H. sapiens H2A-H2B dimers. ( C ) Graphical representation of the predicted overall molecular dipole moments of T. brucei (∼1093 Debye) and H. sapiens (∼294 Debye) histone octamers (positive end = blue, negative end = red). ( D ) Limited micrococcal nuclease digestion of DNA alone, H. sapiens NCPs, T. brucei NCPs, and H. sapiens NCPs containing mutations in H2A and H2B that mimic the positively charged SHL 3.5 interface in T. brucei H2A and H2B (‘ H. sapiens + Tb SHL3.5′ H2A R32T K36R E41R & H2B E35R K85R R86K S87R). ( E ) Scaled up MNase digest of H. sapiens and T. brucei NCPs used for the sequencing reaction. ( F ) Start and end positions of sequenced H. sapiens MNase digestion products from (E) with fractions of reads present in peaks P1, P2 and P3 displayed in a pie chart. ( G ) The same analysis as in (F) repeated for T. brucei MNase digestion products.

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Histone divergence in trypanosomes results in unique alterations to nucleosome structure

doi: 10.1093/nar/gkad577

Figure Lengend Snippet: The T. brucei acidic patch is highly atypical and refractory to well-characterized binders. ( A ) Multiple sequence alignments of acidic patch regions in H2A and H2B, where residues are highlighted and coloured Glu = orange, Asp = red, Lys = purple, Asn = grey. and the residue numbers for T. brucei (above) and H. sapiens (below) are indicated. ( B ) The acidic patch region differs when visualizing the electrostatic surface representation of the T. brucei (top) and H. sapiens (PDB: 6T79 , bottom) NCPs. ( C ) The T. brucei NCP with a magnified view of the acidic patch and compared to the H. sapiens acidic patch below. Acidic patch residues are shown as spheres and coloured using same scheme as (A). The 2-residue insertion in Loop 2 of T. brucei H2A is indicated in black. ( D ) Fluorescence polarization assay showing binding of a FITC-tagged LANA peptide and a non-binding mutated LANA peptide (L8A R9A S10A, ‘LANA LRS’) to H. sapiens and T. brucei NCPs (K D for H. sapiens NCPs = ∼2.38 μM, others ND).

Article Snippet: Experiments were performed with H. sapiens, T. brucei WT and T. brucei Hs H3 NCPs in triplicate and the disappearance of NCP band intensities were quantified with the BioRad Image Lab software.

Techniques: Sequencing, Residue, Fluorescence, Binding Assay

Journal:

Article Title: Pre-M Phase-promoting Factor Associates with Annulate Lamellae in Xenopus Oocytes and Egg Extracts

doi: 10.1091/mbc.E02-08-0511

Figure Lengend Snippet: Enrichment of nuclear pore proteins in HSP-2. Twenty micrograms (as determined by Bradford assay) of each fraction obtained by serial centrifugation of prophase extracts (Figure ​(Figure1)1) was loaded onto different percentages of gels depending upon the antibody to be used, transferred to nitrocellulose, and probed with the antibodies indicated. HSP-1b was highly enriched in ER protein, GRP94 (A, lane 2), whereas HSP-1a was enriched in the mitochondrial protein VDAC (C, lane 3). HSP-2 contained three nuclear pore proteins detected by the anti-NUP antibody: NUP214, NUP153, and p62 (arrowheads in D, lane 5). HSP-2 was also enriched in γ-tubulin (B, lane 5). In all immunoblots, lane 1 contains an unfractionated HSS-1 and lane 4 contains the HSS-2 made by dilution and recentrifugation of HSS-1.

Article Snippet: Transfers onto nitrocellulose paper (Shleicher & Schuell, Keene, NH) were carried out in 0.025 M Tris/0.192 M glycine/15% methanol at 100 V for 60 min. For immunodetection, the protocol was exactly as in Pérez-Mongiovi et al. , (2000) .

Techniques: Bradford Assay, Centrifugation, Western Blot