Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin.
We describe a new technique designed to isolate specific eukaryotic genes as native oligonucleosome fragments. The isolation method consists of hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes, followed by isolation of the hybrids by sulfhydryl-Sepharose chromatography. SV40 minichromosomes were used to test the effectiveness of the technique. About 80% of KpnI- or BamHI-restricted and lambda exonuclease treated SV40 minichromosomes hybridized to an appropriate DNA probe after a 12-h hybridization reaction under mild conditions (0.1 M aqueous salt, 37 degrees C, pH 8). When the restricted minichromosomes were mixed with a 15-fold excess of "background" chromatin from sea urchin embryos, nucleoprotein hybridization was able to reisolate the SV40 chromatin to 88% purity with a 63% yield. This represented a 115-fold enrichment of specific genes as chromatin. Results of electron microscopy and polyacrylamide gel electrophoresis indicate that the hybridized SV40 chromatin has not lost the major chromosomal proteins characteristic of SV40 nor acquired significant amounts of protein due to exchange with background chromatin. Our experimental results show that it is currently possible to isolate repeated genes from higher eukaryotes for structural and biochemical study of the proteins involved with gene regulation.
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000302:isolation & purification*; D002875:Chromosomes / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D005260:Female; D005796:Genes; D007202:Indicators and Reagents; D007700:Kinetics; D008722:Methods; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins / Q000235:genetics*; D010957:Plasmids; D012617:Sea Urchins / Q000196:embryology; D013539:Simian virus 40 / Q000235:genetics; D014779:Virus Replication
D002843:Chromatin; D007202:Indicators and Reagents; D009698:Nucleoproteins; D004247:DNA; D004262:DNA Restriction Enzymes