Dataset Information

LC-MS/MS analysis of immunoprecipitation samples of Tetrahymena meiotic proteins Melg1, Melg2, Melg3, and Tass1

ABSTRACT: In most eukaryotes, the meiotic chromosomal bouquet (comprising clustered chromosome ends) provides an ordered chromosome arrangement that facilitates pairing and recombination between homologous chromosomes. In the protist Tetrahymena thermophila, the meiotic prophase nucleus stretches enormously and chromosomes assume a bouquet-like arrangement in which telomeres and centromeres are attached to opposite poles of the nucleus. We have identified and characterized three meiosis-specific genes (MELG1-3) that control nuclear elongation and centromere and telomere clustering. Hence, to find out potential interactions, we did LC-MS/MS analysis for Melg1, Melg2, Melg3, and Tass1 (a partner of Melg3) immunoprecipitation samples.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Tetrahymena Thermophila

SUBMITTER: Miao Tian

LAB HEAD: Josef Loidl

PROVIDER: PXD021813 | Pride | 2020-11-23

REPOSITORIES: Pride

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Publications

Spatial constraints on chromosomes are instrumental to meiotic pairing.

Tian Miao M Agreiter Christiane C Loidl Josef J

Journal of cell science 20201130 22

In most eukaryotes, the meiotic chromosomal bouquet (comprising clustered chromosome ends) provides an ordered chromosome arrangement that facilitates pairing and recombination between homologous chromosomes. In the protist <i>Tetrahymena thermophila</i>, the meiotic prophase nucleus stretches enormously, and chromosomes assume a bouquet-like arrangement in which telomeres and centromeres are attached to opposite poles of the nucleus. We have identified and characterized three meiosis-specific g ...[more]

PMID: 33172984

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Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

Dataset Information

LC-MS/MS analysis of immunoprecipitation samples of Tetrahymena meiotic proteins Melg1, Melg2, Melg3, and Tass1

Publications

Spatial constraints on chromosomes are instrumental to meiotic pairing.

Similar Datasets

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