In vitro reconstitution of Artemia satellite chromatin.
We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.
Motta
M C
MC
Dipartimento di Biologia Stutterale e Funzionale III Facoltá di Scienze, Universitá di Milano, 21100 Varese, Italy.
Landsberger
N
N
Merli
C
C
Badaracco
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001156:Artemia / Q000235:genetics*; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism*; D004276:DNA, Satellite / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data
D002843:Chromatin; D004276:DNA, Satellite; D000255:Adenosine Triphosphate; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium