We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of chromosomes.
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We would lrke to express our gratttude to Dr. David Ward who provided valuable information which helped us get the Bio-dUTP assay on its feet. We thank Cato Nisselson and David Goitein for their unending efforts to produce high quality photographs for this paper and Tsippi Israeli and Mrriam Horowitz for their help in preparing this manuscript. This research was supported by grants from the Deutsche Forschungsgemeinschaft SP-144/g-2, the National institutes of Health, and the Israel Academy of Sciences and Humanities.