INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

Differential effects of ACTH or 8-Br-cAMP on growth and replication in a functional adrenal tumor cell line

The effects of ACTH and 8-Br-cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y-1) were investigated. ACTH and 8-Br-cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G₁; an additional point of arrest in G₂ occurred with 8-Br-cAMP. Cells whose growth was arrested in G₁ by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G₁ by serum deprivation. When ACTH or 8-Br-cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8-Br-cAMP had no effect on the serum-induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre-replicative period. Partial inhibition of the serum-induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH-arrested Y-1 cell resembles an in vivo hypertrophied adrenal cortical cell.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: The imposition of a G2 block

Through a receptor-mediated process glucocorticosteroids block cell division by 20–45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4′,6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G₁, as previously thought, but in G₂.     

Uridine uptake and its intracellular phosphorylation during the cell cycle

The rate of 5-³H uridine uptake into Chinese hamster V79 cells and the rate of its incorporation into RNA increase tenfold during the cell cycle. Both reactions exhibit the same apparent K_m(1.7 × 10^?5 M ). Chromatography of acid-soluble material from cells incubated with 5-³H uridine (0.25 μM) at different times of the cell cycle revealed that intracellular uridine was rapidly phosphorylated at all times, even though cells in late S and G₂ take up roughly ten times as much uridine as cells in G₁. Uridine kinase activity in synchronized cells increases about two and one-half-fold during the same time period, and in exponentially growing cells is not saturated until the external uridine concentration is raised above 200 μM. It is concluded that the change in uridine kinase activity during the cell cycle is not responsible for the tenfold increase in the rate of uridine transport, and that these two processes are independently regulated.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

INDUCTION OF PROPHASE IN INTERPHASE NUCLEI BY FUSION WITH METAPHASE CELLS

Fusion of an interphase cell with a metaphase cell results in profound changes in the interphase chromatin that have been called "chromosome pulverization" or "premature chromosome condensation" In addition to the usual light microscopy, the nature of the changes has been investigated in the present study with electron microscopy and biochemical techniques Metaphase and interphase cells were mixed and fused at 37°C by means of ultraviolet-inactivated Sendai virus. After cell fusion, morphological changes in interphase nuclei occurred only in binucleate cells which contained one intact set of metaphase chromosomes Irrespective of the nuclear stage at the time of cell fusion, the morphologic changes that occurred 5–20 min later simulated very closely a sequence of events that characterizes the normal G₂-prophase transition. Radioautography revealed that, late in the process, substantial amounts of RNA and probably protein were transferred from the interphase nucleus into the cytoplasm of fused cells. Thus, the findings indicate the existence in metaphase cells of factor(s) which are capable of initiating biochemical and morphological events in interphase nuclei intrinsic to the normal mitotic process.     

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

Developing erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May-Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non-mammalian vertebrates. Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t-test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post-injection. The ³H-TdR studies showed that stages I-IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell). Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post-injection. Grain counts showed that stages I-IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded. Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post-injection. Grain counts indicate that stages I-V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self-sustained for about 5 days.     

PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C¹⁴. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C¹⁴. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

The incorporation of 5-³H-uridine and 5-³H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-³H and cytidine-³H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G₁ in both the nucleus and the reconstituting nucleolus. Thereafter, through the G₁ and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G₁ or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

ANALYSIS OF GROWTH OF TETRAPLOID NUCLEI IN ROOTS OF VICIA FABA

Growth of nuclei of a marked population of cells was determined from G₁ to prophase in roots of Vicia faba. the cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G₁ and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. of the marked population of cells, about 65% had completed a cell cycle 14–15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

ON THE DIFFERENTIAL CYTOTOXICITY OF ACTINOMYCIN D

Actinomycin D (AMD) at concentrations that inhibit cellular RNA synthesis by 85% or more causes an acute phase of lethal cell degeneration in HeLa cultures beginning as early as 3 hr after drug exposure, resulting in the nearly complete loss of viable cells by 12 hr. The loss of cells during this acute phase of lethality is closely dose dependent. Vero, WI38, or L cells are not susceptible to this early acute cyto-intoxication by AMD, and may begin to die only after 1–2 days. Differential susceptibility to acute cyto-intoxication by AMD, or other inhibitors of RNA synthesis (daunomycin or nogalamycin), among different types of cultured cells is analogous to that observed in vivo in certain tissues and tumors, and cannot be accounted for by differences in the effect of AMD on RNA, DNA, or protein syntheses, or by the over-all loss of preformed RNA. Actinomycin D in a dose that inhibits RNA synthesis causes an equivalent loss of the prelabeled RNA in all the cell types studied. Inhibition of protein synthesis with streptovitacin A or of DNA synthesis with hydroxyurea does not cause acute lethal injury in HeLa cells as does inhibition of RNA synthesis. Furthermore, since Vero or L cells divide at about the same rate as HeLa cells, no correlation can be drawn between the rate of cell proliferation and susceptibility to the cytotoxicity of AMD. Susceptibile cells are most vulnerable to intoxication by AMD in the G₁-S interphase or early S phase. Inhibition of protein synthesis (which protects cells against damage by other agents affecting DNA) does not protect against AMD-induced injury. Although HeLa cells bind more AMD at a given dose than Vero or L cells, the latter cell types, given higher doses, can be made to bind proportionally more AMD without succumbing to acute cyto-intoxication. It is suggested that the differential susceptibility of these cell types to acute poisoning by AMD may reflect differences among various cells in the function or stability of certain RNA species not directly involved in translation whose presence is vital to cells. In HeLa cells, these critical species of RNA are presumed to have a short half-life.     

CONTRAST BETWEEN THE ENVIRONMENTAL pH DEPENDENCIES OF PROPHASING AND NUCLEAR MEMBRANE FORMATION IN INTERPHASE-METAPHASE CELLS

In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes. Only one of these phenomena occurred in a given interphase-metaphase (I–M) binucleate cell. At pH 7.4, there was about an equal probability that either event could occur amongst the population of I–M cells. The effect of pH changes in the medium containing the fused cells was examined. At pH 6.6, prophasing was the predominant event; at pH 8.0, membrane formation predominated. It was found that the rate of progression of a mononucleate cell from G₂ to metaphase was appreciably faster at pH 6.6 than at pH 8.0. Conversely, the progression from metaphase to G₁ was faster at pH 8.0 than at pH 6.6. These results with the mononucleate cells strengthen the hypothesis that structural changes in I–M cells are reflections of normal mitotic phenomena. Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium. The double membrane around the chromosomes of the I–M cell was indistinguishable from the normal NE. The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes.     

SYNTHESIS OF RNA IN MAMMALIAN CELLS DURING MITOSIS AND INTERPHASE

Chinese hamster cells in the mitotic and G₁ phases of the growth cycle were incubated for 30 or 60 min in suspension tissue culture and pulse-labeled with tritiated uridine. After appropriate chases, washes, and extractions, it was found that all incorporation into the nucleic acid may be accounted for by those cells in interphase. An average of 410 counts was found for incorporation into the cell population (approximately 2.0 x 10⁵ cells) of which over 80% of the cells was initially in mitosis. The increasing number of cells leaving mitosis and entering interphase during the 30 min incubation was theoretically able to account for 470 counts. In addition, short-pulse labeling experiments have shown a consistent linear relationship between the percentage of cells in division and the incorporation of the isotope, which strongly suggests that, if 100% of the cells were in mitosis, the counts would be essentially zero. Thus, the entire label may be attributed to those cells in interphase where portions of the chromosomal material are known to be already extended.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine