Preparation of fluorescently labelled hybrid histone octamers.
Labelling hybrid histone octamers (the Cys variant of histone H4 replaced histone H4 in the chicken erythrocyte octamer) with the fluorescent probe 5-(2(iodoacetyl)aminoethyl)aminonapthalene- 1-sulfonic acid, IAEDANS, resulted in significant non-specific incorporation of label. Fluorescently labelled hybrid histone octamers were prepared by reconstitution methodology after labelling the isolated histone Cys-H4 and separation of specifically and non-specifically labelled histone. Core particles prepared from these octamers have identical thermal denaturation to unlabelled core particles demonstrating that the incorporation of a fluorescent probe at this site has no overall effect on either histone-histone or histone-DNA interactions. DNase 1 digestion of 32P end-labelled fluorescent core particles yielded the anticipated asymmetric cutting pattern with a 10 bp interval between fragments. Comparison of the cutting pattern with those previously obtained in these laboratories for both polyglutamic acid reconstituted and 'native' core particles demonstrated that fluorescent core particles had an enhanced susceptibility to digestion at site 7.
Lindsey
G G
GG
Department of Biochemistry, University of Cape Town, Private Bag, Rondebosch Republic of South Africa.
Thompson
P
P
D000596:Amino Acids; D000818:Animals; D002645:Chickens; D002845:Chromatography; D004247:DNA / Q000648:ultrastructure; D005456:Fluorescent Dyes; D006657:Histones / Q000648:ultrastructure*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation
D000596:Amino Acids; D005456:Fluorescent Dyes; D006657:Histones; D004247:DNA