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.     

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.     

Der Zellzyklus in Fibroblastenkulturen vom Menschen

Summary Autoradiography using H³-Thymidin and Feulgen photometry of nuclear DNA-content were carried out on human euploid fibroblast cultures.The average durations of S- and G2-periods are about 9 hours and 5 hours respectively, with only minor variation. The duration of G1-period may vary from a few hours to more than 20 hours.A combined study of H³-thymidin uptake and Feulgen photometry on the same cell nuclei showed that all cells whose DNA-content places them into the S-period are labelled when they are fixed immediate after a H³-thymidin puls. After continuous H³-thymidin uptake for 5 hours, all S- and G2-nuclei are labelled, whereas the G1-nuclei remain unlabelled.The Feulgen histogramm as well as grain counts over labelled nuclei indicate a constant rate of DNA synthesis during S-period.     

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.     

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.     

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.     

Ultrastructure of Deoxyribonucleic Acid-Membrane Associations in Escherichia coli

Areas of contact between deoxyribonucleic acid (DNA) and intracytoplasmic membrane are frequently seen in the “extra” membrane-forming strain Escherichia coli 0111a₁. By examination of serial sections, it has been estimated that these DNA-membrane associations occur in at least 60% of the extra membrane-containing cells. Most of the DNA masses contained only one contact area. Several cells in which the DNA had been stretched revealed individual fibers connecting to the membrane, suggesting a firm attachment of DNA to membrane. The areas of membrane associated with DNA fibers were usually between 100 and 500 nm in diameter, although some smaller areas were seen. Electron microscopic autoradiography of cells in which the replication forks were labeled showed grains over 24% of the profiles containing a contact area, whereas there were grains over only 16% of the profiles without a contact area. Data from autoradiographs of cells in which the label was “chased” away from the replication fork showed the reverse labeling pattern. These data indicate that the areas of contact between DNA and intracytoplasmic membranes seen in electron micrographs contain the DNA replication forks.     

Reproduction of chicken chromosomes at the end of the DNA synthesis period]

Reduplication of chicken chromosomes at the end of S-period in the cells of bone marrow was studied, using the method of prolonged labelling with 3H-thymidine and autoradiography. It is shown that during the last hour of the S-period the DNA synthesis in macrochromosomes ends primarily in the centromere region and the regions adjacent to it. Sex chromosomes (ZZ) of cocks accomplish the reproduction simultaneously.     

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.     

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).     

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.     

Stimulation by insulin of DNA synthesis in cultured mouse fibroblasts]

With the aid of autoradiography, the effect of insulin on entering S- from G1-period of the mitotic cycle and on the rate of DNA synthesis of the mouse fibroblasts (L), was studied,--in the cells incubated for 24 hr in serum-free medium. In these conditions the cells were temporarily blocked in G1-period. Insulin (100 mcU/ml) increased by 1.5-fold the amount of cells in S-period as well as caused a marked stimulation of DNA synthesis.     

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.     

Dependence of the nucleolar structure on DNA and RNA synthesis

The dependence of nucleolar structure on DNA and RNA synthesis in synchronous cultures of the slime mold Physarum polycephalum was traced through the mitotic cycle. The blockage of RNA synthesis produces a characteristic abnormality of the nucleolar structure when imposed at any time during interphase. But differences in the function of the early and late replicating DNA molecules were observed. The blockage of DNA synthesis causes abnormality of nucleolar structure only when imposed during the early part of the S-period.     

Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis]

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.     

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Effects of cycloheximide on protein, RNA and DNA synthesis in cultures of CHO cells and human diploid fibroblasts]

In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go--an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long-term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

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.