Journal:

Article Title: Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

doi: 10.1093/emboj/20.8.2015

Figure Lengend Snippet: Fig. 1. A functional homologue of p150 CAF-1 in Xenopus. (A) Sequence alignment between human (hp150), mouse (mp150) and Xenopus p150 (xp150) CAF-1 obtained using ClustalW and Boxshade programs (BCM and ISREC web sites). The amino acid identity is black boxed and similarity is shown by grey boxes. The position of the KER and ED boxes (Kaufman et al., 1995) is indicated on the side. (B) Comparative schematic representation of the domain organization of human and Xenopus p150. The percentage similarity/identity in the N- and C-terminal ends is indicated above the arrows delineating areas of comparison. Residues delimiting domains are indicated for each species. P, PEST domain; KER, KER domain; ED, ED domain (Kaufman et al., 1995). (C) Depletion of xp150 impairs chromatin assembly coupled to DNA repair. Top: western blot analysis of a Xenopus egg extract (HSE) depleted of xp150. Anti-xp150 antibody (serum 566, 1/1000) and anti-PCNA antibody (PC10, DAKO) were used for detection. Lane 1, HSE depleted with control IgG; lane 2, HSE depleted with pre-immune serum; lane 3, HSE depleted with affinity-purified anti-xp150 antibody; lane 4, HSE depleted with anti-xp150 serum; lane 5, HSE diluted 1/10; lane 6, undiluted HSE equivalent to the depleted extract. Bottom: analysis of chromatin assembly by supercoiling on control and UV-irradiated DNA. The pBscript plasmid mock treated (–) or UV irradiated (+) (500 J/m2) (Gaillard et al., 1996) was incubated for 3 h at 23°C in HSE, mock-depleted HSE or HSE depleted with anti-xp150 antibody. Alternatively, the DNA was incubated for 3 h at 37°C in S100 human cytosolic extract (Smith and Stillman, 1989) or S100 extract complemented with HSE treated as indicated. [α-32P]dCTP was added to all samples to follow DNA repair synthesis. The migration of relaxed/nicked (Ir,II) and supercoiled DNA (I) is indicated. (D) Xenopus p150 complements S100 extracts for chromatin assembly. As in (A), DNA is incubated for 3 h at 37°C in S100 extract (–), S100 extract complemented with HeLa nuclear extract (NE) (Martini et al., 1998), or in vitro translated hp150 or xp150 proteins (TnT reticulocyte lysate system, Promega).

Article Snippet: Immunological procedures Polyclonal anti-xp150 antibody (serum 566) and anti-hp150 antibody (serum 573) were obtained after injections of purified GST–xp150(540– 896) and His 6 -hp150(620–938) proteins in rabbits (AgroBio, Villeny).

Techniques: Functional Assay, Sequencing, Western Blot, Affinity Purification, Irradiation, Plasmid Preparation, Incubation, Migration, In Vitro

Journal:

Article Title: Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

doi: 10.1093/emboj/20.8.2015

Figure Lengend Snippet: Fig. 2. Inter- and intraspecies p150 dimerization: identification of a critical domain and role in chromatin assembly. (A) p150 self-association by GST pull-down assay. Left: GST–xp150(540–896) was co-expressed in bacteria (Studier et al., 1990) containing hp150(620–938) or hp150(678–938) tagged with His6. Middle: His6-tagged hp150(620–938) was co-expressed in bacteria with either GST–hp150(620–938) or GST–hp150(678–938). Right: equal volumes of separate bacterial extracts containing the full-length His6-xp150 and GST–xp150(540–986) were mixed at 2 M NaCl; the salt concentration was lowered by sequential dilution to 350 mM NaCl, and glutathione–Sepharose resin was then added. Protein production and purification on glutathione–Sepharose resin were performed essentially as described (Moggs et al., 2000). Co-purification of His-tagged hp150 proteins was detected by western blot probed with anti-His6. The xp150 proteins were detected using the specific anti-xp150 serum 566. I, input; B, bound fraction; B*, 1 M NaCl-resistant bound fraction. The positions of His6-hp150(620–938) and His6-hp150(678–938) are indicated. Asterisks mark degradation products. (B) Glycerol gradient sedimentation of p150 proteins. Bacterially produced recombinant proteins wild-type hp150, hp150Δ(642–678) and wild-type xp150 were sedimented on a linear 10–40% glycerol gradient. Every second fraction of the gradient was run on SDS–PAGE. The proteins were detected by the specific anti-hp150 and anti-xp150 sera on western blots. The sedimentation positions of the molecular weight standards for each gradient are indicated above the gel: BSA (66 kDa) and catalase (258 kDa). Arrows mark the mean position of the proteins. The salt concentrations used in the gradients are as indicated. (C) Chromatin assembly analysis by supercoiling in the presence of hp150Δ(642–678). pBscript control (–) and UV-irradiated (500 J/m2) (+) were incubated as in Figure 1D with S100 extract (–), or S100 extract complemented by HeLa nuclear extract (NE) or in vitro translated hp150 wild-type (lanes 5–6), hp150Δ(642–678) and xp150 wild type. The positions of the relaxed/nicked (Ir,II) and supercoiled DNA (I) are indicated.

Article Snippet: Immunological procedures Polyclonal anti-xp150 antibody (serum 566) and anti-hp150 antibody (serum 573) were obtained after injections of purified GST–xp150(540– 896) and His 6 -hp150(620–938) proteins in rabbits (AgroBio, Villeny).

Techniques: Pull Down Assay, Concentration Assay, Purification, Copurification, Western Blot, Sedimentation, Produced, Recombinant, SDS Page, Molecular Weight, Irradiation, Incubation, In Vitro

Journal:

Article Title: Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

doi: 10.1093/emboj/20.8.2015

Figure Lengend Snippet: Fig. 3. Human p150 expressed in Xenopus embryos associates with Xenopus p150. (A) Scheme of the experiment: Xenopus embryos at the two-cell stage prepared as described in Vize et al. (1991) were injected into one blastomere using a nanoject injector (Drummond) with 1 ng of RNA encoding hp150 CAF-1. Embryos collected at the gastrula stage were processed for immunoprecipitation assay. (B) Immunoprecipitation of extracts from embryos injected with hp150 RNA using anti-xp150 antibody, anti-hp150 antibody or control antibody as indicated on top. Western blot of the different fractions: I, input; S, supernatant; P, pellet. hp150 and xp150 were detected by the polyclonal 573 and 566 antibodies, respectively; PCNA by the PC10 monoclonal antibody; nucleoplasmin by the monoclonal PAC35 antibody; and histone H4 acetylated at Lys5 by the polyclonal anti-H4AcK5 antibody.

Article Snippet: Immunological procedures Polyclonal anti-xp150 antibody (serum 566) and anti-hp150 antibody (serum 573) were obtained after injections of purified GST–xp150(540– 896) and His 6 -hp150(620–938) proteins in rabbits (AgroBio, Villeny).

Techniques: Injection, Immunoprecipitation, Western Blot

Journal:

Article Title: Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

doi: 10.1093/emboj/20.8.2015

Figure Lengend Snippet: Fig. 5. The dimerization domain of p150 is required to perturb Xenopus early development. (A) Xenopus embryos were injected with buffer or RNA encoding the full-length hp150 (WT) and truncated forms of hp150 RNA: (1–649), ΔED, (620–938), (678–938) and Δ(642–678), as indicated on the side. Images were recorded under a dissecting microscope at stages 9 and 36 of development (as judged from control embryos). The white arrowheads point to the area of the embryo where the defect is first visible. Scale bars are indicated. (B) Injections were as in (A) but with RNA encoding the full-length xp150 (WT) or xp150(540–896). On the right, a schematic representation of hp150 and xp150 proteins and a sequence alignment of dimerization domain for each species are shown.

Article Snippet: Immunological procedures Polyclonal anti-xp150 antibody (serum 566) and anti-hp150 antibody (serum 573) were obtained after injections of purified GST–xp150(540– 896) and His 6 -hp150(620–938) proteins in rabbits (AgroBio, Villeny).

Techniques: Injection, Microscopy, Sequencing