Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Abstract

The structure of the chicken adult beta‐globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM‐papers and probed with labelled DNA fragments spanning the adult beta‐globin gene and its 5′‐ and 3′‐flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta‐globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta‐globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta‐globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5′ moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

“…It is not known why, in an earlier study of the 13 -globin gene in chicken erythrocyte nuclei, somewhat different anomalous ladders were obtained (46). All of the nucleosome oligomers generated by MNase in that study appeared to be about 100 bp shorter than corresponding oligomers generated from the inactive ovalbumin gene.…”

“…For example, the 180-bp 1-globin spacing in chick erythroid cells (49), in which this gene is expressed, corresponds to the 180-bp spacing obtained in vitro. Similarly, the 196-bp spacing for the chicken ovalbumin gene in hen oviduct, in which this gene is expressed (3,46), corresponds to the in vitro value obtained (30). The chicken ovalbumin gene is not restricted to this value because in erythrocytes, it has a 210-bp nucleosome spacing periodicity, the bulk chromatin repeat (46,49) (Fig.…”

“…Moreover, activation in some cases appears to entail packaging into a large and stable nuclease-sensitive chromatin domain that exceeds by far the region of DNA undergoing transcription (6, 48). Additionally, active chromatin often contains irregular or altered nucleosome arrays (3,12,37,46,49) or exhibits a loss of nucleosome positioning with respect to DNA (7). It has not been adequately explained, however, how a localized activation event could prevent or alter the presumed default packaging of large stretches of DNA into inactive chromatin or, alternatively, how an active chromatin structure could propagate over a large distance.…”

Restriction fragment length polymorphism of hsp70 gene, localized in the RT1 complex, is associated with hypertension in spontaneously hypertensive rats.

“…The hybridization process has been described previously. 21 Autoradiography was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).…”

“…21 These DNA fragments were labeled with [a-32 P]dCTP and DNA polymerase (Klenow fragment) by random-primer extension. 22 Mouse /wp6S-containing plasmid was provided by Dr. L.A. Moran.…”