Abstract
Limited micrococcal nuclease action on trout testis nuclei yields two mononucleosome subsets, MN1 and MN2, enriched in transcribed DNA sequences. MN1 is soluble and MN2 insoluble in 0.1 M NaCl. Electrophoresis of intact MN1 and MN2 mononucleosomes in 4% polyacrylamide gels shows that each fraction is heterogeneous and four major and several minor subcomponents can be resolved. Dissociation of the protein components of these nucleosomal particles by two-dimensional electrophoresis shows that the major component of MN1 possesses stoichiometric amounts of the four inner histones and of the high mobility group protein H6, associated with DNA fragments of 140 base pairs in length. The major component of MN2 possesses equimolar amounts of the four inner histones plus 1 molecule of H1 and H6, complexed with DNA fragments of 220 base pairs in size.
Highlights
Limited micrococcal nuclease action on trout testis mM EDTA, 10%glycerol and 0.025%’bromphenol blue were added, nuclei yields two mononucleosome subsets, MNI and MN2,enriched in transcribed DNA sequences
Electrophoresis was for 25 h at 200 volts during which time the protamine migrated through the region occupied bv the DNP bands in the tube gel and dissociated the DNA-associated proteins
MNIand MNr are Heterogeneous-When samples of MNI and MNr were analyzed by electrophoresis on 4% polyacrylamide gels a t low ionic strength, we observed thatboth fractions separated into several bands (Fig. 2)
Summary
U broadness of the bands, etc. as hasbeen discussed in detail by Kornberg [8].Component A of MNI consists of a DNA fragment -100 base pairs long. As hasbeen discussed in detail by Kornberg [8].Component A of MNI consists of a DNA fragment -100 base pairs long. Component C is -145 base pairs long (125 to 170). Component D is -155 base pairs long (130 to 190) (Fig. 3). The amountsof each of the protein bands were quantitated as described under “Methods.”. Particular attention should be paid to component C This componentcomprises 38%of the total MNIand contains, in addition to equimolar amounts of the four inner nucleosomal histones, the high mobility group protein H6, in. H2B is not subject to the limitations listed above and, was the best choice for standardization A extinction coefficientsof all of the histones andH6 are approximately the same overa linear range of concentrations (0 to 20 p g )
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