Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation

Summary Cortex cells of the root meristem of Cucurbita pepo (0.0–0.5 mm from the cap junction), in the 3–4, 5–6 and 7–8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7–8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5–6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (³H thymidine uptake), template activity of DNA (³H actinomycin D(³H AMD)-binding), RNA synthesis (³H uridine incorporation), and protein synthesis (³H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3–4 mm segment reach 4C ploidy state, but in the 7–8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of ³H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5–6 and 7–8 mm segments, while in 3–4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, ³H uridine uptake, ³H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3–4 and 5–6 mm segments). ³H leucine is still incorporated intensely into 7–8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of ³H thymidine uptake into nuclear DNA with ³H AMD binding and ³H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3–4 and 5–6 mm segments; in the 7–8 mm segment binding of ³H AMD slightly decreases, while ³H uridine incorporation is considerably reduced. Divergence between the ploidy state, ³H AMD binding and ³H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3–4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.This work was partly supported by Polish Academy of Sciences, Botanical Committee, Grant 217/II     

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that >80% of those cells' DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,000–200,000 daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3′-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells' soluble cytoplasm. The second polymerase, comprising 1.5–3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfhydryl-blocking reagents and has an apparent molecular weight of 49,000. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.     

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).     

Vesicular stomatitis virus infection reduces the number of active DNA-dependent RNA polymerases in myeloma cells.

Infection of mouse myeloma (MPC-11) cells with vesicular stomatitis virus resulted in rapid loss in activity of cellular RNA polymerases associated with nuclear chromatin. No RNA polymerase inhibitor could be detected in extracts of infected cell nuclei. Reconstitution experiments with solubilized RNA polymerases dissociated from chromatin of infected and uninfected cells demonstrated that vesicular stomatitis viral infection did not affect the ability of the polymerases to function on endogenous or exogenous templates; nor did infection alter the template capability of the chromatin. Measurement of the number of actively growing RNA chains revealed that infected cell nuclei contained fewer active polymerase units; however, the rates of RNA chain elongation were the same in nuclei from infected and uninfected cells. Quantitation of the number of polymerase units active in nuclear chromatin revealed that the alpha-amantin-sensitive polymerase II was more severely reduced by viral infection than were polymerases I and III.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Summary Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1–7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G₂ phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G₂ phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.This work was supported by the Polish Academy of Sciences as a part of project no 09.7.1.4.5     

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

   

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.     

The effect of calcium on DNA synthesis in pea (Pisus sativus L.) roots after treatment with heavy metals.

Cortex cells of the meristematic (1 mm) and differentiated (7 mm) zones of Pisus sativus L. roots after 144 h culture in distilled water (control), Ca2+ (10(-3) M) and/or Cd2+, Cr3+, Pb2+ solutions (10(-4) M, each) were subject of the present studies. Reductions in the number of nuclei incorporating 3H thymidine was observed in meristem in the presence of Cd2+ and in differentiated zone in Pb2+ treated roots. Intensity of DNA synthesis diminished after Pb2+ treatment but mostly in Cd2+ treated roots, mainly in meristem. Addition of calcium to the metal solutions caused an increase in the number of nuclei witch uptook the radioactive material in both studied root zones. Positive effect of calcium alone was visible also in the differentiated zone. Presence of calcium in the metal solutions caused a marked increase in 3H thymidine incorporation into nuclear DNA in meristem, although neutralizing calcium effect in this root zone was visible only in roots treated with Cr2+ and Pb2+. In the differentiated zone of roots growing in Cd2+ solution, calcium addition stimulated DNA synthesis but the intensity of this process was lower than in control (water).     

Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAE-Sephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24 micrograms/ml) but not by Rifampicin (200 micrograms/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred single-stranded DNA. 5. RNA polymerase II was inhibited by a low concentration of alpha-amanitin (0.02 micrograms/ml). RNA polymerase I was also inhibited by the relatively high concentration of alpha-amanitin (IC50 = 100 micrograms/ml and IC70 = 750 micrograms/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.     

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.