RESUMEN
Histone H1 proteins bind to DNA and are important in formation and maintenance of chromatin structure. Little is known about differences among variant H1 histones in their interactions with DNA. We examined the effects of histones H1(0) and H1t on thermal denaturation of several DNA species. One of the DNA molecules was a 214-base-pair fragment from the plasmid pBR322, which contains an AT-rich and a GC-rich region. Both H1(0) and H1t bound preferentially to one region of the DNA fragment, a region that is relatively GC-rich. This result indicates that histones H1(0) and H1t are not totally nonspecific but rather bind with some sequence preference to DNA. This conclusion was supported by studies of other DNA species, including two 92-base-pair fragments derived from the two regions of the 214-mer, and several synthetic homocopolymers of DNA. Data obtained with the homocopolymers suggested that the binding preference was not simple preference for GC base pairs. The binding of the two H1 variants was not identical: there appear to be differences in binding site sizes, affinities, and sequence selectivities between H1t and H1(0).
Asunto(s)
Proteínas de Unión al ADN/química , Histonas/química , Animales , Composición de Base , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Escherichia coli , Ratones , Modelos Químicos , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Polinucleótidos/químicaRESUMEN
We amplified the coding regions of the previously cloned H1 genes H1-1, H1 zero and H1t and inserted them into the expression vector pET-11d. The synthesis of the H1 histones can be induced in the appropriate strains of bacteria, and the H1 histones can be readily purified. We report detailed protocols for the purification of the expressed proteins using combinations of ion-exchange and reverse-phase HPLC. Sufficient amounts of each pure variant protein can be obtained for use in physical studies of H1-DNA interactions.