Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.
An amplification of tandem CAG trinucleotide sequences in DNA due to errors in DNA replication is involved in at least four hereditary neurodegenerative diseases. The CAG triplet repeats when translated into protein give rise to tracts of glutamine residues, which are a prominent feature of many transcription factors, including the TATA-binding protein of transcription factor TFIID. We have used a biotin-labeled, complementary peptide nucleic acid (PNA) to invade the CAG repeats in intact chromatin and then employed a method for the selective isolation of transcriptionally active chromatin restriction fragments containing the PNA.DNA hybrids. The PNA-containing chromatin fragments were captured on streptavidin-agarose magnetic beads and shown to contain all the CAG.PNA hybrids of the active chromatin fraction. DNA hybridization experiments using a DNA probe specific for unique sequences downstream of the CAG-tandem repeats confirmed that the PNA.DNA hybrids contained the transcribed gene for the TATA-binding protein. In contrast, no hybridization signal was detected with a DNA probe specific for the c-myc protooncogene, which is amplified and transcriptionally active in COLO 320DM cells but lacks CAG tandem repeats.
Boffa
L C
LC
Istituto Nazionale per la Ricerca sul Cancro IST, Genoa, Italy.
Carpaneto
E M
EM
Allfrey
V G
VG
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D003110:Colonic Neoplasms; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004276:DNA, Satellite / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010455:Peptides; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004273:DNA, Neoplasm; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; D010455:Peptides; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors