Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.     

Epiplastome variation of the number of chloroplasts in stomata guard cells of sugar beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations, which is typical of inbred plants. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage ("cell hereditary memory") and successive sporophyte generations.     

Increase of uv-induced DNA repair synthesis during blast transformation of human lymphocytes.

UV-induced DNA repair synthesis, measured as unscheduled DNA synthesis, was studied in human peripheral lymphocytes in various phases of the cell cycle. Mitogen transformation of the lymphocytes was effected with phytohemagglutinin (PHA), and the stage in the cell cycle was determined by measuring the Feulgen DNA content and the dry mass in individual cells by cytophotometry. The initial rate of repair was determined by autoradiography after UV-light irradiation (19.2 J/m²) and incubation of the cells for 30 min with [³H]thymidine. When the cells progressed from the G0 to the G1 phase there was a 3-fold increase in the grain count. The correlation between the grain count and the dry mass indicated an increase in the initial rate of repair during the progression of cells from G0 to G2 phase. G2 cells were more heavily labelled than those in G1, but there did not seem to be any difference between these two phases as regards the relationship between grain count and DNA content. The results indicate that the initial rate of UV-induced DNA repair may differ in various phases of the lymphocyte cell cycle.     

Epiplastome Variation of the Number of Chloroplasts in Stomata Guard Cells of Sugar Beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage (cell hereditary memory) and successive sporophyte generations.     

The effect of chalone on the cell cycle in the epidermis during wound healing

A cut was made on the ear conch of mouse and an extract containing epidermal chalone was injected subcutaneously 2 days later. The time changes after the chalone administration in the number of cells labeled with ³H-thymidine, in the number of grains on labeled cells and in the number of mitoses within the regenerating epidermis surrounding the wound were investigated by means of autoradiography (ARG). Grain counts decreased temporarily in early phase (0–2 h) after chalone injection. This decrease in grain count resulted in a decrease in the number of labeled cells on the ARG of a short exposure but not in that on the ARG of a long exposure. A decrease in the number of labeled cells on the ARG of a long exposure was evident at 6 h when the grain counts reverted to a level similar to the control without chalone. The number of mitoses reached a minimum at 2 h and then recovered quickly, indicating a rapid disappearance of the inhibition of cells in G 2 from entering M phase. Mitoses decreased again thereafter, presumably as a result caused by inhibition of cells in the preceding S phase from completing DNA synthesis. The extract made similarly from liver or kidney affected neither the mitotic nor the DNA synthetic activities.These results indicate that the epidermal chalone or chalones inhibit the epidermal cell proliferation in, at least, 3 different processes of the cell cycle; the DNA synthesis in S phase, the transition from G 1 to S phase and the transition from G 2 to M phase.     

Variation in tritiated thymidine uptake during DNA synthesis in the adrenal cortex

Summary The interphase grain counts of adrenocortical cells labelled with tritiated thymidine (³H) Tdr,do not conform to a Poisson distribution, and therefore are not the result of a random disintegration process. The rate of (³H) Tdr incorporation during interphase DNA synthesis (the S phase) was studied by metaphase grain count analysis. Maximum rates of incorporation were found towards the middle of the S phase. The interphase grain count of adrenocortical cells is considered to be largely dependent on the position of the cell in the S phase.     

Gangliosides stimulate astroglial cell proliferation in the absence of serum

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.     

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.     

Selective inhibition of DNA synthesis in macronuclear fragments in Paramecium aurelia exconjugants and its reversal during macronuclear regeneration

In Paramecium exconjugants very rapid DNA synthesis takes place in the developing macronuclear anlagen, while DNA synthesis is suppressed in macronuclear fragments. The rate of DNA synthesis in fragments (as a percentage of the rate in anlagen or macronuclei in the same cells) decreases by about 40% during each successive cell cycle over at least the first five cell cycles after conjugation, even though macronuclear anlagen are fully mature by the end of the second cell cycle. — Suppression of DNA synthesis in macronuclear fragments is reversible. If macronuclear anlagen are removed at fission, a very high rate of DNA synthesis resumes in macronuclear fragments after a two-hour lag. The total rate of synthesis in the ensemble of macronuclear fragments in cells without anlagen is greater than that in anlagen in control cells. Thus, suppression of DNA synthesis in macronuclear fragments is not the result of any stable differentiation or irreversible change in the fragments but is the result of, and dependent on, the presence of macronuclear anlagen. — The results of injection of cytoplasm from vegetative cells into normal exeonjugants suggest that normal macronuclei produce an inhibitor which selectively suppresses DNA synthesis in macronuclear fragments. In control cells the relative rate of DNA synthesis in fragments ranged from 40 to 70% of that in anlagen in the same cells, while in injected cells the relative rate of incorporation of DNA precursors was suppressed to as little as 7%. The mean level of incorporation into fragments in injected cells was significantly lower than that in controls, suggesting that the injected cytoplasm contained an inhibitor.Contribution 822, Zoology Department, Indiana University. Supported in part by contract COO-235-66 of the USAEC and by grant No. Gm 15410-05 of the USPHS to T. M. Sonneborn.This paper is a portion of a dissertation submitted in partial fulfillment of the equirements for the degree of Doctor of Philosophy.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Autoradiographic studies of the synthesis of RNA and protein as a function of cell volume in Streptococcus faecium

Mid-exponential-phase cultures were either labeled continuously with tritiated leucine and uracil or pulse-labeled with tritiated leucine. The amount of leucine and uracil incorporated into protein or RNA per cell was determined by grain counts of autoradiographs of cells seen in electron micrographs; the volume of each cell was determined by three-dimensional reconstruction. The average number of autoradiographic grains around cells continuously labeled with uracil and leucine increased linearly with cell volume. In contrast, while the average grain count around cells pulse-labeled with leucine increased in a near-linear fashion over most of the volume classes, less than the expected number of grains were seen around cells in large- and small-size classes. The distribution of grains around cells from both the continuously and pulse-labeled populations could be fit at the 5% confidence level with a Poisson distribution modified to take into consideration the volume distribution of each population of cells analyzed. These findings suggested that large changes in the density of RNA and protein do not occur in most cells as they increase in size; however, there may be decreases in the rate of protein synthesis in some large and small cells. The decrease in the rate of protein synthesis appears consistent with the hypothesis that new sites of envelope growth must be introduced into cells that are close to the division event to restore rapid growth.     

Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

Purkinje cell maturation in the light of RNA synthesis

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.     

Computer-assisted imaging cytometry of nuclear chromatin reveals bone tumor virus infection and neoplastic transformation of adherent osteoblast-like cells

Established osteoblast-like (OB) cells infected with the bone tumor-inducing C-type retrovirus OA MuLV remained nontumorigenic over 104 cell culture passages. DNA histograms revealed a new cell population with a stem line peak at 5c. A second OA MuLV-infected OB cell line underwent neoplastic transformation with increasing passage level. These cells showed diffuse aneuploidy. Stepwise linear discriminant analysis of the chromatin structure of control, OA MuLV-infected, and FBR osteosarcoma virus-transformed cell lines resulted in various levels of discrimination ranging between 79.6% for control cells versus nontumorigenic OA MuLV-infected cells, and 96.6% for nontumorigenic OA MuLV-infected cells versus FBR osteosarcoma virus-transformed cells. OA MuLV-infected tumorigenic cells and FBR osteosarcoma virus-transformed cells were discriminated at a 93.6% level.     

Regulation of photosynthetic carbon metabolism in synchronously growing Chlorella pyrenoidosa

The physical properties and photosynthetic metabolism of synchronizedcells of Chlorella pyrenoidosa are described. Cells, synchronizedby successive periods of light and dark, photosynthesized understeady-state conditions for 30 min with ¹⁴CO₂. Pool sizes ofmetabolic intermediate compounds, and rates of flow of carbonthrough these pools, were determined. Cell properties and metabolismwere studied for cells just divided, at three periods duringthe growing stage, at the time of maximum DNA synthesis, justprior to division (after continuous light, and after 5 hr darkness),and following division after continuous light for 37 hr. Changes in pool sizes and flow rates are correlated with relativeshifts between amino acid and protein synthesis, which is greatestduring the growing period and DNA-synthesizing stage, and sucrosesynthesis, which is greatest in the divided cells (after darkness)and pre-division cells (after darkness). The effects of thestage of cell growth and of a prior period of darkness can beseparated to some extent by these studies, and in some respectsare additive. Specific sites of metabolic regulation discerned in these experimentsinclude the following: 1) Ribulose diphosphate carboxylase,2) fructose diphosphatase and sedoheptulose diphosphatase, 3)the synthesis of sucrose, probably at the reaction between fructose-6-phosphateand uridine diphosphoglucose to give sucrose phosphate and uridinediphosphate, 4) amino acid synthesis, at the level of nitratereduction, and 5) amino acid synthesis, at the level of carbonflow from the photosynthetic carbon reduction cycle to aminoacid carbon skeletons. (Received October 31, 1969; )     

NUCLEAR INCORPORATION OF RADIOACTIVE DNA PRECURSORS AND PROGRESSION OF CELLS THROUGH S

Bone marrow blast cells of nine children with untreated acute leukaemia (five lymphoid, three myeloid, one monocytic), myeloid precursor cells of a haema-tologically normal child and thoracic duct lymph cells of a patient with sclerodermia were pulse-labelled in vitro with tritiated thymidine and/or tritiated cytidine. Combined radioautographic and cytophotometric techniques were used for the determination of the median nuclear size and the median grain count of labelled cells in different segments of S. It was found that the median grain count reached maximum values in the second or third quarter of S in all cell populations studied, and that the variation, during S, of the median grain count appeared to be independent of the median DNA synthesis times of the cell types investigated. In six cell populations a clear-cut inverse relation existed between the number and the median grain count of labelled cells in different segments of S. In three populations this relation was less apparent and in two it was not found at all.     

Syntheses of nucleic acids during spermatogenesis in Nereis diversicolor (annelida polychaeta): a quantitative autoradiographic study

The development of DNA and RNA synthesis in the germ cell population was studied after a 3H-thymidine or 3H-uridine pulse at each stage of spermatogenesis. The autoradiographic results show that the first sign (after 3 days in vitro) of cellular changes is an increase in RNA synthesis which reaches a maximum at day 5. DNA replication (premeiotic S phase) occurred at day 7, then cells entered meiotic prophase (day 9). Meiotic divisions and spermiogenesis occurred after 11 days. Silver grain counts permit the conclusion that RNA synthesis is clearly higher during premeiotic interphase (days 3-7) than during spermatogonial proliferation (day 0). It appears therefore that male meiotic differentiation in Nereidae is accompanied by increased RNA synthesis.     

Influence of cellular differentiation on repair of ultraviolet-induced DNA damage in murine proadipocytes

The effects of cellular differentiation on the repair of DNA damage induced by uv radiation were investigated in the murine 3T3-T proadipocyte cell culture system. Upon exposure to human plasma, actively cycling 3T3-T cells (stem cells) undergo growth arrest, which is followed by terminal differentiation into lipid-laden adipocytes. In response to uv irradiation, the level of unscheduled DNA synthesis is significantly lower in adipocytes as compared to stem cells. The alkaline elution assay was used to monitor the appearance of repair-induced strand breaks in 3T3-T cells after uv irradiation. DNA strand breaks were detected in stem cells by 4 min post-uv with essentially no further increase after 8 min. When terminally differentiated adipocytes were irradiated and allowed to repair, however, more strand breaks were present at 4 min and, in marked contrast to stem cells, continued to accumulate in adipocytes for at least 16 min post-uv. Inhibition of repair-replication with hydroxyurea and cytosine arabinoside significantly increased accumulation of repair-induced strand breaks in stem cells, yet had little effect on this accumulation in adipocytes. For stem cells and adipocytes, incision activity was linear out to at least 10 Jm-2 without saturation. These data suggested that 3T3-T cell differentiation is accompanied by a defect in some postincision process of the excision-repair pathway.     

Effects of mitomycin C on the rate of DNA synthesis in normal and Fanconi anaemia cells

The effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patients with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This effect was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.