Ribonucleic acid (RNA) may be hybridized to denatured deoxyribonucleic acid (DNA) at temperatures of 0-24°rather than the usual ca. 66°, provided that the hybridization reaction mixture contains an adequate concentration of formamide. The formamide concentration which yields maximum hybridization of RNA to DNA depends upon temperature and salt concentration. Maximum hybridization was achieved at 0°in a salt concentration of 0.16 m in 30 vol. % formamide, and at 24°in 0.32 m salt in the same formamide concentration. Under these two conditions the have found that RNA may be hybridized to denatured DNA at low temperature in the presence of appropriate concentrations of formamide. The procedure was originally developed during search for conditions under which RNA and protein might simultaneously be annealed to DNA. It has now been found, however, that the method has advantages over the use of heat for routine hybridization. We describe below the method and its merits.The general procedures are based upon those of Gillespie and Spiegelman (1965) in which RNA in solution is hybridized to denatured DNA which is immobilized on nitrocellulose filters. Deproteinized pea

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Chromatin from chicken reticulocytes and mouse Ehrlich ascites tumor cells has been extracted with 2 M NaCl, leaving a portion of the DNA still complexed with a fraction of nonhistones (designated M3, since it can be dissociated from DNA in solutions of 3 M NaCl containing 5 M urea). The DNA complexed with M3, separated from the bulk DNA by centrifugation, was found to contain sequences poorly represented in bulk DNA. Specifically we found that DNA--M3 complexes isolated from chicken reticulocyte chromatin were enriched in globin gene sequences by 20-fold relative to unfractionated DNA and by over 1000-fold relative to DNA rendered free of protein following the extraction of chromatin with 2 M NaCl. We have therefore isolated DNA fractions complexed with M3 which are enriched in specific sequences as may be determined by M3.

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A DNA fraction which is highly enriched in active gene sequences and tightly associated with a subset of nonhistone chromosomal proteins has been isolated from human placenta. After extraction with 2 M NaCl, placental chromatin was separated into two distinct components by centrifugation. Of the total DNA, approximately 96% (DNA-S) is protein free, while the remaining 4% (DNA-P) is tightly complexed with nonhistone chromosomal proteins. Reassociation studies revealed that the DNA-P fraction was enriched 22-fold in actively transcribed human placental lactogen gene sequences, while the DNA-S fraction was correspondingly depleted 22-fold in these sequences. Approximately 45% of the sequences present in DNA-P (equivalent to 1.8% of the genome) were not present in the DNA-S fraction. Reassociation of nick-translated DNA-P to DNA from a partial digest of DNase I treated nuclei indicated that 27% of the DNA-P sequences were DNAase I sensitive, suggesting they may represent actively transcribed gene sequences. Analysis of the overall sequence organization of DNA-P showed that relative to unfractionated DNA and DNA-S, DNA-P was enriched in single-copy sequences, slightly enriched in the class of middle repetitive sequences from C0t 0.01 to 100 M.s, devoid of the more highly repetitive sequences (C0t less than or equal to 0.01). The distribution of total active placental genes between DNA-P and DNA-S was measured by hybridization with a complementary DNA probe transcribed from total polysomal poly(A+) messenger RNA. We found that 57% of this cDNA probe reassociated to DNA-P and 58% to DNA-S, while 95% reassociated to DNA-P mixed with DNA-S at the observed ratio of 4 to 96, suggesting that the DNA-P fraction contained a different population of active gene sequences than DNA-S. From these results we estimate that approximately 85% of the transcribed sequences appear to be distinctly distributed and equally proportioned between DNA-P and DNA-S, while approximately 15% of the transcribed sequences are common to both fractions. We suggest that the strong affinity of the tightly bound nonhistone chromosomal proteins for the DNA-P fraction indicates a likely role for these proteins in the regulation of gene expression.