Biomedical subjects

C Thiriet

Publications and source records attributed to C Thiriet.

5 recordsLinked to original sources

PubMed1999

Histone proteins in vivo: cell-cycle-dependent physiological effects of exogenous linker histones incorporated into Physarum polycephalum.

We detail a method which allows biochemical quantities of histone proteins to be introduced into a living eukaryotic cell. This method involves absorption of purified proteins into macroplasmodia of Physarum polycephalum. Further, since Physarum macroplasmodia exist as syncitial culture with completely synchronous nuclei with respect to cell cycle events, proteins may be introduced at specific points during the eukaryotic cell cycle. We show that a linker histone is absorbed whole into these cells and are properly transported to the nuclei of the cell. Furthermore, we also show incorporation of linker histone H5 inhibits the transcriptional activities occurring during the G2 phase in Physarum. This method will make it possible to introduce histones modified with structural probes into chromatin naturally assembled in vivo.

Animals↗

PubMed1999

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum.

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.

Animals↗

PubMed1998

Functionally relevant histone-DNA interactions extend beyond the classically defined nucleosome core region.

We demonstrate that core histones can affect the accessibility of a DNA element positioned outside of the classically defined nucleosome core region. The distance between a well positioned nucleosome and the binding site for the 5 S-specific transcription factor TFIIIA was systematically varied and the relative binding affinity for TFIIIA determined. We found that core histone-DNA interactions attenuate the affinity of TFIIIA for its cognate DNA element by a factor of 50-100-fold even when the critical binding region lies well outside of the classically defined nucleosome core region. These results have implications for the validity of parallels drawn between the accessibility of general nucleases to DNA sequences in chromatin and the activity of actual sequence-specific DNA binding factors.

Animals↗

PubMed1997

Antisera directed against anti-histone H4 antibodies recognize linker histones. Novel immunological probes to detect histone interactions.

We introduce a novel immunological approach to detect structural interactions between chromosomal proteins. Antigenically pure core histone H4 was prepared from chicken erythrocytes and used to produce anti-histone H4 antisera. IgG fractions were isolated from purified anti-H4 antibodies and used as antigens to produce "second generation" antisera. Epitopes cross-reacting with the second generation antisera were then identified within chromosomal proteins. These epitopes were presumed to mimic the complementary molecular surface of the original anti-H4 antibodies, and thus proteins containing these epitopes were putatively identified as specific ligands of H4 in chromatin. Surprisingly, we found this immunoreactivity was predominantly directed against H1 compared with H5 from chicken erythrocytes. Further, the immunoreactive epitopes were located within the C-terminal tail domain of the linker histones. These results suggest similar complementary interactions occur between H4 and the C-terminal tail domain of H1s in native chromatin. This could occur either within a single nucleosome as suggested by a previous report (Banères, J.-L., Essalouh, L., Jariel-Encontre, I., Mesnier, D., Garrod, S., and Parello, J. (1994) J. Mol. Biol., 243, 48-59) or between neighboring nucleosomes within the condensed chromatin fiber. The implications of these results with regard to the structure of the chromatin fiber and the future utility of this technique are discussed.

Animals↗

PubMed1995

Rapid and effective western blotting of histones from acid-urea-Triton and sodium dodecyl sulfate polyacrylamide gels: two different approaches depending on the subsequent qualitative or quantitative analysis.

An improved method for the electrophoretic transfer of histones from sodium dodecyl sulfate (SDS) and acetic acid-urea-Triton X-100 (AUT) polyacrylamide gels onto nitrocellulose membranes is described. In the case of SDS-gels, it was not essential to equilibrate them before transfer while for the AUT-gels, an equilibration step is essential to prevent the interference of Triton X-100 with the binding of histones to nitrocellulose. Transfer efficiency was different for different histone classes. Two procedures were developed: (1) one suitable for qualitative studies, and (ii) another for quantitative transfer.

Acetates↗