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)
We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.
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