Enrichment of selected active human gene sequences in the placental deoxyribonucleic acid fraction associated with tightly bound nonhistone chromosomal proteins.
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.
D001483:Base Sequence; D002843:Chromatin / Q000302:isolation & purification; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification; D004247:DNA / Q000302:isolation & purification; D005260:Female; D005796:Genes; D006801:Humans; D008970:Molecular Weight; D009695:Nucleic Acid Renaturation; D010920:Placenta / Q000032:analysis*; D010928:Placental Lactogen / Q000235:genetics; D011061:Poly A / Q000302:isolation & purification; D011247:Pregnancy; D011485:Protein Binding; D012333:RNA, Messenger / Q000302:isolation & purification
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D010928:Placental Lactogen