RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

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.     

RNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Increase in Rate of RNA Synthesis during G1

Cultures of mitotic Chinese hamster cells, prepared by mechanical selection, were pulse-labeled with methionine-methyl-¹⁴C or with uridine-³H at different stages in the life cycle. The rate of ¹⁴C incorporation into 18S RNA was measured, as was the rate of uridine-³H incorporation into total RNA for both monolayer and suspension cultures. The rate of incorporation increased continuously throughout interphase in a fashion inconsistent with a gene-dosage effect upon RNA synthesis.     

RNA SYNTHESIS IN CHINESE HAMSTER CELLS : I. Differential Synthetic Rate for Ribosomal RNA in Early and Late Interphase

The incorporation of methionine-methyl-¹⁴C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1–1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.     

EFFECTS OF ADDED SUBSTANCES ON RNA AND PROTEIN SYNTHESIS IN IMMATURE NEURONS

Abstract— A newly described method for the isolation of morphologically intact neurons from newborn rat brain was used to study the influence of inhibitors and neuroactive substances on RNA and protein synthesis in these cells in vitro . Incorporation of [¹⁴C]-uridine into RNA and [³H]leucine into protein proceeded rapidly and continued up to 3 h. When the incorporation mixture was chased at 20 min with an excess of nonradioactive uridine and leucine, hardly any degradation of labelled RNA was noted during the following 2 h 40 min. In contrast, the specific radioactivity of proteins decreased by 22 per cent indicating turnover of cellular proteins.
Incorporation of labelled leucine into protein was markedly inhibited in the presence of NaF and cycloheximide but not affected in the presence of chloramphenicol or pancreatic RNase. A mixture of ATP + GTP depressed the incorporation by 38 per cent. The responses to ATP + GTP and RNase indicated that the incorporation system was typically cellular. Acetylcholine, γ-aminobutyrate, noradrenaline and phenylalanine in the incubation medium depressed the incorporation of labelled uridine into RNA by 10–30 per cent and 5-hydroxytryptamine by 75 per cent. Acetylcholine, γ-aminobutyrate and noradrenaline had no effect on protein synthesis, while 5-hydroxytryptamine and phenylalanine inhibited the incorporation by 60–80 per cent. Testosterone and prednisolone depressed both RNA and protein synthesis while thyroxine caused slight but non-significant stimulation.     

INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G₁ nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G₁ or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.     

AUTORADIOGRAPHIC STUDIES OF NUCLEIC ACIDS AND PROTEINS DURING MEIOSIS IN LILIUM LONGIFLORUM

Taylor , J. Herbert (Columbia U., New York, N. Y.) Autoradiographic studies of nucleic acids and proteins during meiosis in Lilium longiflorum. Amer. Jour. Bot. 46(7): 477–484. Illus. 1959.—A study was made of the incorporation of glycine-C¹⁴, orotic acid-C¹⁴ and cytidine-H³ into nucleic acids and proteins of sporogenous and tapetal cells of lily anthers preceding and during meiosis. Methods for differential extraction of nucleic acids from tissue sections, which had been frozen, dehydrated by alcohol-substitution, and fixed in hot alcohol, were tested by chromatographic analysis of extracts. Both acid and enzyme hydrolysis were shown to be useful for quantitative or, at least, semi-quantitative work. DNA synthesis was shown to occur only during premeiotic interphase in sporogenous cells, but at two intervals in tapetal nuclei, once when the microsporocytes are in zygotene and again during pachytene. Each time the synthetic period was followed by a normal mitosis. Accumulation of RNA in microsporocytes occurred at stages up to late leptotene. After this period, labeled RNA accumulated almost exclusively in their nuclei and at a slower rate than in earlier stages. DNA synthesis, as measured by incorporation of glycine-C¹⁴ and orotic acid-C¹⁴, gave the same results and confirm earlier results with inorganic phosphate-P³². For RNA, glycine-C¹⁴ and orotic acid-C¹⁴ gave different results. When glycine-C¹⁴ was the source of label, incorporation of C¹⁴ in RNA stopped during DNA synthesis in sporogenous cells. Glycine-C¹⁴ was not utilized to a significant extent at any time by tapetal cells for RNA synthesis, but extensively for DNA and protein synthesis. Orotic acid-C¹⁴ was incorporated into RNA of both tapetum and sporogenous cells at various periods in development apparently including the interval of DNA synthesis. Protein synthesis as measured by incorporation of glycine is relatively rapid during premeiotic interphase and leptotene. It continues during the remainder of prophase, but at a much reduced rate. In tapetal cells the rate is rapid in the nuclei during periods of DNA synthesis, but even faster in both cytoplasm and nucleus after divisions are completed and the microsporocytes are in late prophase and division stages. This period of synthesis is perhaps necessary for the postmeiotic functioning of tapetum when it appears to secrete the wall materials for the microspores.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

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.     

Ultrastructural evidence of mitotic perichromosomal ribonucleoproteins in hamster cells

The mitotic chromosomes of cultivated hamster cells were studied at the ultrastructural level with cytochemical methods preferential for ribonucleoproteins. A perichromosomal layer of RNP was thus revealed throughout mitosis. Its cytochemical identification was ascertained by enzymatic digestions, whereas autoradiography after labelling with [³H]UdR demonstrated that its synthesis occurs at the end of the G2 phase of the cell cycle or at the beginning of prophase. The type of RNA involved is discussed.     

Nuclear and microtubular cycles in heterophasic multinuclearTriticum root-tip cells induced by caffeine

Summary Nuclear and microtubular cycles were studied in large heterophasic multinuclear cells induced in root tips ofTriticum turgidum by caffeine treatment. Multinuclear cells and cells with polyploid nuclei exhibited various configurations of multiple and complex preprophase microtubule (Mt) bands (PPBs), including helical ones. The developmental stages of PPBs in some heterophasic cells did not comply with the cell cycle stages of the associated nuclei, a fact indicating that these events are not directly controlled by the associated nuclei. The heterophasic cells exhibited asynchronous nuclei at different stages of mitosis. In cells displaying prophase and interphase nuclei, the prophase spindle was either absent or developed around both of them or developed around the prophase nuclei earlier than around the interphase ones. During prometaphase-metaphase of the advanced nuclei the lagging interphase nuclei were induced to form prematurely condensed chromosomes (PCCs) along with spindle formation around them. These observations suggest that the mitotic transition in heterophasic cells is delayed but is ultimately achieved due to the effect of the advanced nuclei, which induces a premature mitotic entry of the lagging nuclei. Although kinetochore Mt bundles were found associated with PCCs, their metaphase and anaphase spindles were abnormal resulting in abnormal or abortive anaphases. In some heterophasic cells, metaphase-anaphase transition did not take place simultaneously in different chromosome groups, signifying that the cells do not exit from the mitotic state after anaphase initiation of the advanced nuclei. Asynchronous pace of mitosis of different chromosome groups was also observed during anaphase and telophase. Implications of these observations in understanding plant cell cycle regulation are discussed.Abbreviations cdk cyclin dependent kinase - Mt microtubule - PCC prematurely condensed chromosome - PPB preprophase band     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

RNA synthesis in Chinese hamster cells. III. Non-coordinate increases during interphase in synthesis rates for informosomal, polysomal and heterogeneous nuclear RNAs.

Cultured Chinese hamster ovary cells were synchronized by mitotic selection. Relative synthesis rates for informosomal messenger-like RNA (mlRNA), polysomal messenger RNA (mRNA), and heterogeneous nuclear RNA (HnRNA) were estimated from the amount of labeled adenosine or uridine incorporated into these species in early and late interphase. The amounts of uridine incorporated into HnRNA, mRNA, and mlRNA during a pulse administered 9.75-10.75 h post-mitosis were 3.48 4.64, and 2.82 times the amounts incorporated 1.5-2.5 h post-mitosis. Adenosine incorporation values 9.5-11.0 h post-mitosis were 1.64 (HnRNA), 2.49 (mRNA), and 1.18 (mlRNA) times the 1.5-3.0 h values. The realitive incorporation into MRNA of large polysomes corresponded to incorporation into mRNA of smell polysomes. Thus, the synthesis rates of mRNA, mlRNA, and HnRNA increase during interphase in a noncoordinate fashion.     

Binding of E-MAP-115 to microtubules is regulated by cell cycle- dependent phosphorylation

Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15- fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition.     

Individual nuclei in polykaryons can control cyclin distribution and DNA synthesis.

Nuclear patterns of cyclin (PCNA) distribution that subdivide S-phase (determined using PCNA autoantibodies specific for this protein) as well as [3H]thymidine incorporation followed by autoradiography have been used to determine the S-phase synchrony of homophasic polykaryons produced by polyethylene glycol (PEG)-induced fusion of populations of mitotic transformed human amnion cells (AMA) exhibiting the following average distribution of phases: prophase, 9%, metaphase, 60% (including early and late prometaphase), anaphase, 3.8%, telophase, 26.2% and interphase, 1%. Both synchronous and asynchronous polykaryons were generated from these fusions; the latter being frequently observed only amongst populations of multinucleated cells having three or more nuclei. These results are taken to imply that individual nuclei in these polykaryons can control cyclin distribution and DNA synthesis in spite of the fact that they share a common cytoplasm.     

LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.