The structure of partly decondensed metaphase chromosomes.

The technique of freeze-drying was applied to examine the submicroscopic organisation of metaphase chromosomes from Chinese hamster after removal of bivalent cations with EDTA and removal of histone HI with 0,6 M NaCl. Treated chromosomes increased in size, and nucleosomal filaments appeared at the periphery of the chromosomes. Removal of bivalent cations is accompanied with the appearance of regularly organized structures of the "beads-on-a-string" type. The regular organization of the fibers is damaged as soon as histone H1 is removed. After decondensation in a 0.6 M NaCl solution the metaphase chromosomes were treated with staphylococcal nuclease in situ on EM grids nd the residual structures analysed using electron microscopy. Nucleohistone fibers wer visible at the periphery of the chromosomes at the beginning of digestion. After complete elimination of the nucleohistone fibers in the course of digestion the remaining proteinaceous material was represented by aggregates of irregular shape and of varying size. These were either concentrated along the central axis of the chromatids or, at the final step of digestion, scattered evenly over the entire area that had been occupied by the chromosome. Presumably, in the chromosome prior to digestion, the material did not form an integral protein structure similar to a scaffold in dehistonised and spread chromosomes. An alternative interpretation for the fragmentation of protein material in the chromosome considers possible degradation of the protein scaffold in the course of digestion.

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes. III. Distribution of AT- and GC-nucleotide pairs along the length of chromosomes 1, 2, 3, and 16 in the two types of human cells.

Distributions of AT- and GC-base pairs along the length of chromosomes 1, 2, 3 and 16 in primary cultures of embryonic fibroblasts and of peripheral blood leucocytes were studied by autoradiography with: 1. 3H-thymidine and 3H-deoxycytidine; 2. 3H-deoxyadenosine and 3H-deoxyguanosine. It has been shown that the two types of cells differ in the DNA content and proportion of AT- and GC-nucleotide pairs in the centromeric heterochromatin of chromosome I: this region contains more DNA in fibroblasts than in leucocytes mainly due to AT-pairs. In both types of cells the telomeric region of the short arm of this chromosome contains more GC- than AT-pairs. Similar results were obtained for C-heterochromatin of chromosome 16: the frequencies of labelling of this region by 3H-deoxyadenosine and 3H-thymidine in fibroblast cultures were higher than in case of 3H-deoxycytidine and 3H-deoxygyanosine, and in leucocyte cultures these frequencies were almost equal. No differences in the distributions of base pairs along the length of chromosome 2 and 3 were established in the two types of cells. -- The nature of the established phenomenon may be connected with under-replication or loss in another way of part of the genetic material in the process of development and differentiation of cell systems.

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes. II. Replication of centromeric regions of chromosomes at the termination of the S period.

Replication of regions of chromosomes 1, 2, 3, 16, and group 4-5 was studied at the termination of the S period in primary cultures of embryonic fibroblasts (two embryos) and in cultures of peripheral blood (two women). Distinct differences were established in the pattern of late replication of the studied chromosomes in the cultures of the two types of cells. These differences consern first of all the centromeric and neighbouring regions of the chromosome. The content of late label in this region is 1.5-3 times higher in the cultures of fibroblasts than in the corresponding regions of leucocyte cultures. The difference is most pronounced in chromosomes 1, 3 and 16. It is suggested that the difference between cultures of these two types of cells in chromosome replication may be connected with the different genetic functioning of the centromeric and neighbouring regions in them. It is also possible that this difference is due to underreplication (or partial loss in an other way) of heterochromatin DNA of centromeric and neighbouring regions in leucocytes functioning for a long period without division.