TY - JOUR
T1 - Nick-translation of metaphase chromosomes
T2 - In vitro labeling of nuclease-hypersensitive regions in chromosomes
AU - Kuo, M. T.
AU - Plunkett, W.
PY - 1985
Y1 - 1985
N2 - Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNse 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 DNse 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.
AB - Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNse 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 DNse 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.
UR - http://www.scopus.com/inward/record.url?scp=0021996156&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021996156&partnerID=8YFLogxK
U2 - 10.1073/pnas.82.3.854
DO - 10.1073/pnas.82.3.854
M3 - Article
C2 - 3856236
AN - SCOPUS:0021996156
SN - 0027-8424
VL - 82
SP - 854
EP - 858
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 3
ER -