Isolated mammalian metaphase chromosomes: II. Fractionated chromosomes of mouse and Chinese hamster cells

Abstract

Isolated metaphase chromosomes of mouse L-cells, mouse L-5178Y lymphoblast tumor cells and Chinese hamster cells have been separated into several distinct size classes by velocity sedimentation in steep sucrose gradients. Fractions showing relatively homogeneous chromosome size distributions were readily obtained, but none of the fractions was composed entirely of chromosomes of one specific type. Fractions of the smallest chromosomes were the most homogeneous in size, and the heterogeneity of the fractions increased progressively with increasing average chromosome length. Mouse satellite DNA was not restricted to chromosomes of a limited size class or type but was distributed throughout all fractions of the isolated chromosomes in nearly the same proportion of the total DNA. These results are consistent with the interpretation that the amount of satellite DNA in each chromosome is proportional to the total DNA of the chromosome and that it is not limited to a fixed amount per chromosome. Approximately 70% of the total chromosomal DNA remained in the supernatant or sodium chloride-soluble fraction after extracting the isolated L-cell chromosomes twice in 2 m-NaCl and centrifuging after each extraction. The sodium chloride-soluble DNA was main-band DNA, whereas satellite DNA remained associated with the insoluble chromosome pellet fraction. Isolated chromosomes of the Chinese hamster were also separated into several size classes by centrifugation in sucrose gradients. The DNA's from the various fractions showed no significant differences either in densities or band profiles in cesium chloride density-gradient centrifugation. The DNA's from two fractions showing highly restricted chromosome size-distributions also failed to renature after heating and annealing and their behavior in this respect resembled that of the total DNA of mammalian cells.