Circular Dichroism of Avian‐Erythrocyte Chromatin and Ethidium Bromide Bound to Chromatin

Abstract

The circular dichroism of mature gander erythrocyte chromatin was compared with that of partially dehistonized chromatin and deproteinized DNA. The ellipticity at 275 nm for native chromatin was found to be three-times less than that for DNA. The 275-nm transition was gradually increased in magnitude upon removal of histones. Selective removal of the very lysinerich histone I and the erythrocyte-specific histone V, but not histone I alone, resulted in a significant increase in transcription in vitro and ethidium bromide primary binding properties of chromatin. Concomitant with these changes a "blue shift" of the positive DNA band from 282 nm for native chromatin to 275 nm for purified DNA was observed.Optimal conditions for circular dichroism studies in ethidium bromide bound to DNA and native chromatin were determined.The circular dichroic spectra of ethidium bromide bound to native chromatin and partially deproteinized chromatin were found to be essentially the same as that exhibited by ethidiumbromide . DNA complexes. However, the ellipticity of the dye bound to native chromatin was smaller than that when ethidium bromide was bound to DNA. The 310-nm dye transition was increased after the removal of chromosomal proteins. Selective removal of histone I and V was found to generate the most notable increment in ellipticity.The represent results are consistent with recent interpretations on how ethidium bromide interacts with chromatin ; native chromatin has fewer ethidium bromide primary binding sites than deproteinized DNA ; the mode of this dye interaction in these two systems is nearly the same, if not identical.Several circular dichroic spectral studies on chromatin isolated from various tissues have been made [l-71. The results generally indicate that the conformation of DNA in chromatin is significantly different from that of deprotcnizcd DNA.The use of the intercalative dye ethidium bromide [8,9] as a probe for studying the state of DNA in the isolated chromatin or nucleoprotein complexes of interphase chromosomes has recently been considered for calf thymus [lo] and avian erythrocytes [ll]. Both studies have demonstrated a decrease in ethidium bromide primary or strong binding to chromatin in comparison to deproteinized DNA. However, it appears that chromatin from biologically inactive erythrocytes binds much less ethidium bromide than chromatin from calf thymus, and that the erythrocyte-specific histone V, or f2c [il-141 is fundamental in the process which Abbreviations. CD, circular dichroism ; DNA-P, DNAEnzyme. Micrococcus luteus DNA-dependent RNA polyphosphate. merase (EC 2.7.7.6).limits the extent of ethidium bromide primary binding.Although fluorescence data suggest that ethidium bromide binding sites in chromatin and in DNA are very much alike [lo], additional evidence is required to further establish the nature of the DNA available for ethidium bromide binding in chromatin. Dalgleish et al. [15] have shown that ethidium bromide acquires optical activity upon binding to DNA, the magnitude of the 3...

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin

“…In the region of the spectrum above 240 nm there is a progressive transition with increasing HCHO concentration from a native chromatin spectrum (Simpson and Sober, 1970;Shih and Fasman, 1970;Boffa et al, 1971;Fric and Sponar, 1971;Hanlon et al, 1972;Johnson et al, 1972;Williams et al, 1972), to one which closely resembles the spectra of films of the Li salt of naked DNA2 (TunisSchneider and Maestre, 1970). For purposes of discussion in this paper we shall refer to this as the "suppressed" spect r~m .~ Below 240 nm, where the major contributor to CD is protein , there is little change in the chromatin spectrum upon fixation with HCHO.…”

Water-Soluble Lysine-containing Polypeptides. II. The Interaction of Several Sequential Lysine–Glycine Polypeptides with DNA. A Circular Dichroism Study of DNA Conformation in Annealed Complexes

“…At lysine/DNA-phosphate ratios of approximately 0.5 it was noted that in each case the D N A portion of the spectrum was reminiscent of spectra seen in chromatin isolated from various sources (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). That is, the single band a t 275 nm in the spectrum of the native DNA shifted over to a smaller positive band a t approximately 282 nm upon complex formation with polymers I11 and IV (Figs.…”

Interaction of Acridine Drugs With Dna and Nucleotides

“…Angerer et al (1974) showed that there are at least two classes of highly fluorescent strong binding sites in deoxyribonucleoprotein (DNP) as compared to only one class in DNA. The restriction of the ethidium bromide-binding capacity of D N P appears to be due to t h e f l lysine-rich histone for calf thymus D N P (Angerer et al, 1974) and to the lysine-rich histone P and the histone V for chickblood D N P (Williams et a!., 1972).…”