[Show abstract][Hide abstract] ABSTRACT: Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.
[Show abstract][Hide abstract] ABSTRACT: The variability of genome size was studied in animal populations and in cell populations of different animal species by means of DNA flow cytometry with the precision level of several tenths of percent. For populations of frog Rana esculenta and laboratory mouse lines C57B1 and CBA the analysis was made with cells of different tissues: erythrocytes for R. esculenta, splenocytes for mice and haploid cells of testes for both species. The results of DNA cytometry, obtained with cells of different species, were shown to be correlated, which indicates the objectivity of individual intrapopulation differences in the genome size recorded with DNA flow cytometry. The level of variability (expressed as CV) was 0.3-0.4% for the population of frogs, and 0.2-0.3% for mouse lines. The analysis of genome size variability in cell populations of different animal species revealed a relationship between the variability level and the genome size: the coefficient of variance of the peak of DNA histogram was inversely related to the square root of genome size. The latter effect may be explained by fluctuations of a measured multicomponent object, but it is still unclear whether these fluctuations could be related to the genome size or to pecularities of chromatin structure. It is concluded that the method of flow DNA cytometry may be effective for studying individual differences in the genome size.
[Show abstract][Hide abstract] ABSTRACT: Each chromosome of eukaryotic cells contains multiple units of DNA replication that are activated during S-phase of cell cycle according to a definite program. It is considered at present that the main independent units of replication in mammalian cells represent groups of 20-25 adjacent synchronously activated small (with the average size 100 kbp) replicons. After labelling of nascent DNA with nonradioactive DNA precursors and immunofluorescent staining of incorporated label, discrete replication domains (RDs) are detected in S-phase nuclei. It is assumed that each RD is formed by a single group of synchronously activated small replicons. Since the average rate of replication fork movement is 2 kbp/min, a group of small replicons should finish DNA synthesis within 25 min, and only during this time one RD should incorporate the replicative label. We have studied the duration of DNA synthesis in individual RDs in S-phase human cells using double replicative labelling that can be detected in the nucleus by specific reagents. Our results indicate that in the main fraction of RDs DNA synthesis lasts more than 90 min, that contradicts the generally accepted model of organization of replication units in mammalian cells (Hand, 1978), but is in agreement with an alternative model, according to which the main replication units are single or clustered big replicons more than 300 kbp in size (Liapunova, 1994).