Nucleic Acids Research, 1991, Vol. 19, No. 6 1325-1336
© 1991
MOLECULAR BIOLOGY |
Nucleoprotein hybridization: a method for isolating active and inactive genes as chromatin
Biophysics Research Division, University of Michigan 2200 Bonisteel Boulevard, Ann Arbor Ml, 48109-2099, USA
* To whom correspondence should be addressed
Received August 14, 1990. Revised February 9, 1991. Accepted February 9, 1991.
The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based only on the gene sequence and is independent of other physical properties such as protein composition and transcriptional activity, we were able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclel. Using T7 gene 6 exonuclease, the 3'termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix. DTT cleavage of the biotin linker yielded a chromatin fraction >700 fold enriched in SUEHGR. Overall yields were between 2% and 15%. The purity of the isolated material was independently measured to be >80%. The homogeneous native structure of the inactive genes was preserved as shown by electron microscopy and micrococcal nuclease digestion of the purified SUEHGR. Minor heterogeneity was observed for the purified active genes by micrococcal nuclease digestion but the main features of the active chiomatin were preserved during isolation. This isolation offers the first opportunity to study the structure of an RNA polymerase II gene at different stages of the cell cycle and development.
+Present address: Institute of Biochemistry, Warsaw University, Zwirki I Wigury 93, 02089 Warsaw, Poland
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