Nick-translation of metaphase chromosomes: in vitro labeling of nuclease-hypersensitive regions in chromosomes.
Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNase I followed by incubation with DNA polymerase I and radioactively labeled nucleotides. The labeled DNA was located on nuclease-hypersensitive regions of the chromosomes, as suggested by the following observations. (i) The labeled DNA was hypersensitive to the subsequent DNase I digestion. (ii) The labeled DNA contained no nucleosomes. DNA reassociation kinetic analysis suggested that the labeled DNA was enriched in repetitive DNA sequences. Base composition analyses showed that the labeled DNA was highly enriched in guanine and adenine residues, suggesting that the nick-translation reaction was asymmetrical and the strand enriched in purine was preferentially translated. Autoradiographic analysis revealed that the label was distributed on every chromosome, but there was a lower grain density on the Y chromosome, which is heterochromatic and exhibits a relatively low level of gene activity. The locations of silver grains on the Y chromosomes were generally consistent with that revealed by the in situ hybridization using [3H]cDNA synthesized from the total Chinese hamster messenger RNA. These observations suggest that a specific subset of genomic DNA on active chromatin is the preferred site of the nick-translation.
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002460:Cell Line; D002843:Chromatin / Q000032:analysis; D002875:Chromosomes / Q000032:analysis*; D006224:Cricetinae; D003412:Cricetulus; D004256:DNA Polymerase I / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005260:Female; D008297:Male; D014176:Protein Biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014998:Y Chromosome
D002843:Chromatin; D004256:DNA Polymerase I; D003850:Deoxyribonuclease I