Rate of DNA replication in the DNA synthetic period of the barley chromosomes

The rate of DNA replication, as judged by H³-thymidine incorporation, at the specific time of the S-period in chromosomes of barley (Hakata No. 2) is studied by means of autoradiography.In the barley chromosomes, two different DNA units with respect to replication-time are distinguishable. The early replicating DNA is replicated at least within 1 hour ab init. of the S-period, and the late replicating DNA within 1/2 to 1 hour before the end of the S-period. The replication scarcely occurs in the middle of the S-period. These evidences suggest that the replication of chromosomal DNA in the present material does, therefore, not proceed in a continuous time sequence. Topographically, the early replicating DNA is almost confined exclusively to the distal regions of the chromosomes 1 and 5, and this situation seems applicable to other chromosomes as well, whereas the late replicating DNA is close to the centromere on its both sides. Hence, the replication of chromosomal DNA does not proceed uniformly in a longitudinal sequence along the chromosomes. The interrelationships among chromosome structure in its cytological expression, replication -pattern and -time of chromosomes, and regulating mechanisms of DNA replication are discussed.     

Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.     

Telomeric fusion of the chromosomes in cells with micronuclei during the subsequent incorporation into the DNA of thymidine halogenated analogs

As was shown elsewhere among halogenated analogs of thymidine there exist strong (5-chlorodeoxyuridine, 5-ChldU) and weak (5-iododeoxyuridine, 5-IdU) inducers of dicentric chromosomes in cells with micronuclei. Since from colcemid administration to the moment of fixation two cycles of replication are passed, a study was made of the pattern of induction of dicentric chromosomes under subsequent administration of 5-ChldU and 5-IdU, and the other way round. It was shown that the pattern of induction of dicentric chromosomes was strong or weak, and depended on the fact which analog was administered in the first S-period.     

Dynamics of 3H-thymidine and 3H-deoxycytidine incorporation into the nuclei of a rabbit kidney cell culture during the S period

Using a combined cytophotometric-autoradiographic method a study was made of 3H-thymidine and 3H-deoxycytidine incorporation rates into the interphase nuclei of rabbit kidney cell culture during the S-period. The rate of 3H-deoxycytidine (10(-4) M--10(-6) M) incorporation into nuclei increases throughout the first part of the S-period and decreases from its middle to the end. The patterns of variations of 3H-thymidine and 3H-deoxycytidine incorporation rates into the nuclei of cultured rabbit kidney cells during the S-period were identical.     

Periodicity of the changes in DNA and RNA levels in Penicillium chrysogenum conidia at the stage preceding the S-period

It is ascertained, that conidia enter S-period after 11 hours of incubation on Capek-Dock medium and after 7 hours of incubation on Reistrick medium. Five peaks of DNA and RNA synthesises preceding the S-period were revealed. Moreover, DNA decay to a certain degree depending on the peak number is characteristic for every DNA synthesis peak. By the moment the cell enters into S-period the amount of DNA remains at the initial level. It is noted, that the RNA synthesis curve corresponds to the character of the DNA synthesis curve. A hypothesis is offered to explain the data obtained, which pressuposes the non-specific duplication of the greater part of the genome and its selective degradation.     

DNA and histone synthesis rate change during the S-period in Ehrlich ascites tumor cells

Data from flow-cytometric analysis of DNA of Ehrlich ascites tumor cells were fitted using non-linear least squares curve fitting routines. Analysis of rates of synthesis from the derived S-period profiles revealed a pattern of changing rates of DNA synthesis during the S-period. Three main peaks are seen whose trough to through periods range from 0 to 16%, 16 to 65%, 65 to 100% of the DNA synthesized during S. The differences between the peak rates and rates in the intervening troughs are small, about 10% of the maximum, but these occur reproducibly. Some differences in the DNA distribution profiles, hence rate profiles, can be seen among samples taken at different times during the day. These are thought to reflect the effects of circadian rhythms, but they are not large enough to obscure the general pattern of rate shifts that occur during the S-period. Analyses of radioactivity of ³H-thymidine pulse labelled cells, sorted across the S-period, were in accord with the results obtained from the DNA distributions. A parallel analysis of DNA and histones showed a correspondence in the timing and direction of shifts in rate for both during the middle part of the S-period.     

An analysis of the impulse character of cellular proliferation in the livers of sexually immature mice]

The quantitative ratio between labelled pre-and postmitotic nuclei of hepatocytes was studied 8 hr after H-3-thymidine administration in the 12-15 g mice. The karyoautoradiographic analysis showed that DNA-synthetizing cells are concentrated at a certain stage of S-period. This indicates the complete synchronization of DNA synthesis in the liver parenchyma. The times during which all the hepatocytes enter the S-period did not exceed 4 hr.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

DNA synthesis in the mono- and binuclear cells of regenerating rat liver after x-ray irradiation

The effect of X-irradiation on the dynamics of DNA synthesis during the S-period in bi- and mononucleated of regenerating rat liver was studied autoradiographically and microphotometrically. Rats were treated with X-rays at doses 3.84 X 10(-2), 15.48 X 10(-2), and 30.96 X 10(-2) Kl/kg 23 hours after a partial hepatectomy, and were sacrificed one hour after irradiation. In the control liver the rate of DNA synthesis was the lowest at the beginning of the S-period and the highest at the last quarter of this period in both mono- and binucleated cells. The irradiation results in the inhibition of DNA synthesis mainly at the end of the S-period depending on doses employed. This inhibition was the same in bi- and mononucleated cells. In addition, the increase of correlation of the 3H-thymidine incorporation rate and DNA content was found between nuclei of binucleated cells after irradiation.     

Differential spiralization along mammalian mitotic chromosomes

The morphology of human metaphase chromosomes of peripheral blood lymphocytes taken from normal persons of both sexes and cultured at the final stages of the S-period in the presence of 5-bromodeoxyuridine (BUdR), or 5-bromodeoxycytidine (BCdR) was studied. It was observed that the chromosomes of the complement were capable of responsing to the treatment with analogs by the appearance of extended segments along their length. The pattern of segmentation was constant and specific for a given chromosome, serving as a basis for its identification, and appeared to be similar for both analogs. — Autoradiography of such chromosomes performed with ³H-thymidine (³H-TdR), ³H-deoxycytidine (³H-CdR), and ³H-BUdR showed that the extended chromosomal segments are late replicating. In accordance with this correlation, the most regular and distinctive segmentation was observed in chromosomes having large late replicating regions, such as Nos. 4, 6, 9, 13, 16, X, and Y. — A comparative analysis of the BUdR-induced differential spiralization pattern and banding pattern obtained with the G-staining technique was carried out. A good correspondence between the extended segments and Giemsa-positive bands was found. The data are discussed in relation to the mechanism of differential staining of metaphase chromosomes.     

The effect of cycloheximide on the entry into the S period of the nuclei in heterodikaryons]

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts were fused with serum-stimulated (10%) proliferating cells to elucidate mechanisms of entering into S-period operating in the nuclei of the heterokaryons under the effect of cycloheximide--an inhibitor of protein synthesis. Using radioautography DNA synthesis was investigated in mono-, homo- and heterodikaryons. After short (0.5-3.0 h) depressing of protein synthesis, the nuclei of stimulated cells in heterokaryons were found to enter into S-period. Under these conditions no induction of DNA synthesis was found in the nuclei of resting cells in heterodikaryons. In other experiments, resting cells were under the effect of cycloheximide during 2-4 h before the fusion, that led to a great induction of DNA synthesis in the nuclei of these cells in heterodikaryons. The data obtained are consistent with the idea of fibroblast transition to the rest under the action of labile proteins-repressors.     

Effect of the alkylating agent dipin on the induced proliferation of hepatocytes. I. The kinetics of the cell population]

The alkylating drug dipin was injected to mice 2 hours before a partial hepatectomy. Liver regeneration was characterized by a decrease of the intensity of 3H-thymidine label, an increase of the labeled cell index, absence of mitoses, constant number of binuclear cells. The analysis of these data has shown that dipin causes a sharp (more than by 2 times) increase of the S-period and prolonged (up to 6--20 days) blocking of cells in the G2-period. No phenomenon of unbalanced growth was recorded. No changes in duration of prereplicative period, or in the volume of proliferative pool were recorded. The increase of mitotic cycle periods resulted in the cell population synchronization: by the end of the second ay more than a half of hepatocytes were in S-period, by the end of the third day about 80% of cells passed to G2-period.     

Pre-Meiotic DNA Synthesis and Recombination in CHLAMYDOMONAS REINHARDI

The time of the pre-meiotic S-period was determined by ³²P incorporation in synchronously germinating zygospores of Chlamydomonas reinhardi at six and one-half to seven hours after the beginning of germination. Phenethyl alcohol treatment caused death of zygospores at a period one hour before the S-period, and also during meiotic prophase. Recombination between arg-1 and arg-2 was increased by treatment with phenethyl alcohol or mitomycin C at a time between the first sensitive period to phenethyl alcohol and the S-period. Actinomycin D caused an increase in recombination at the time of this sensitive period. FUdR, nalidixic acid and hydroxurea all cause a decrease in recombination when applied during S-period, and have no effect earlier. These results are explained by postulating (1) that the units of delayed premeiotic replication are whole replicons, and (2) that the amount of recombination is proportional to the number of replicons in which synthesis is delayed. It is suggested that the control of DNA replication controls the distribution of recombination events.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

Localization by Q-banding of mitotic chiasmata in cases of Bloom's syndrome

In this paper methodology is described which yields three-way Giemsa differentiation (light-medium-dark) in human metaphase chromosomes exposed to 5-bromode-oxyuridine (BrdU) for 3 DNA synthetic periods (or exposed for 2 DNA synthetic periods and removed from exposure for the third) by means of which all of the sister chromatid exchanges (SCEs) occurring during (or shortly after) S1, S2 and S3 can be accurately counted and distinguished from one another. Using these methods it has been demonstrated that approximately twice as many SCEs occur during the first S-period in the presence of the drug (labeling= B₁T₀×T₀B₁)¹ as occur during the second S-period (labeling=B₂B₁× T₀B₂)¹. The three-way differentiation pattern is thought to result from a stepwise decrease in the amount of BrdU incorporated during the first, second and third DNA synthetic periods. These methods can also be used to differentiate between unlabeled (T₂T₀) and unifilarly labeled (B₁T₂) sister chromatids and are potentially useful in the detection of sub-chromatid exchanges (none were detected).     

Effect of intratracheal gamma-globulin administration on the mitotic activity and proliferative pool of lymphoid cells of the respiratory tract

After injection of gamma-globulin into respiratory tracts of rats a proliferative pool of lymphoid cells in the lungs increases and remains unchanged in the spleen. The increase in the proliferative pool was accompanied by a decrease in a mitotic cycle in the S-period. Immunization was followed by intensification of division processes in a group of medium lymphocytes and processes of blast cells differentiation.     

Changes in the chondriome during the cell cycle of a tissue culture of embryonic pig kidney

The regular cyclic changes of number, size, shape and ultrastructure of cells mitochondria during G1, S, G2-periods and mitosis have been shown by morphometric methods on nonsynchronized culture of PEK tissue. Number of mitochondria is equal in G1 and S cells. Middle size and unbranched mitochondria prevail in G1-period, middle size and large organellae of complicated shape are characteristic for S-period. In G2-period number of mitochondria increases in 1,5 times. Simultaneously the portion of middle and small unbranched mitochondria increases and number of large organellae of complicated shape decreases. Number of mitochondria in mitotic cells in comparison with cells of G2-period does not change distinctly. Most mitochondria are middle and small size, usually unbranched in this period. Considerable increase of mitochondria number in G2-period is probably due to division of the branched mitochondria characteristic for the previous S-period. The mitochondria ultrastructure does not undergo marked changes during the interphase of the cell-cycle and characterizes by prevailing of the orthodox forms of mitochondria: in late G2-period and in the process of mitosis the most mitochondria become condensed.     

Meiosis in the male mouse. An autoradiographic investigation

Meiosis in the male mouse has been studied autoradiographically in air-dried preparations. Information has been obtained on the relative rates of DNA synthesis and the lengths of the S-periods in spermatogonia and spermatocytes. The average rate of synthesis in the spermatocyte is lower, and the S-period is of longer duration than the preceding spermatogonial generations. The labelling pattern of the sex-chromosomes and autosomes observed at diakinesis and metaphase II in cells labelled at the spermatocyte S-period appears to be similar to that found in cells labelled during the spermatogonial S-periods. Replication in the autosomes commences before the sex-chromosomes. Late replicating autosomal centric regions show a marked degree of asynchrony in labelling both between and within bivalents. The Y chromosome starts and finishes replication later than the X. There is a short, late-replicating, segment of the X in the vicinity of the centromere. There is a short, early-replicating segment of the Y in the vicinity of the centromere which may represent the euchromatic short arm. The X and Y appear to associate at diakinesis by the distal ends of their long arms.     

Replication in Drosophila chromosomes

The temporal order of replication of specific sites in polytene chromosomes from salivary glands and gastric caeca of Drosophila nasuta larvae was compared using ³H-thymidine autoradiography. Labelling of different cytological regions in segments of chromosome 2R (section 47 A to 49 C) and chromosome 3 (section 80 A to 82 C) was examined in detail in nuclei showing late S-period labelling (2 D and 1D types) in both cell types. The different labelling sites (22 on the 2R segment and 38 on the chromosome 3 segment) are cytologically similar in the two cell types. However, there are profound differences in the labelling frequencies of certain sites in polytene nuclei from salivary glands and gastric caeca during the late S-phase. This suggests that even though a comparable number of chromosomal replicating units operates in the two polytene cell types, the temporal order of completion of replication differs.