A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.     

RNA synthesis in maturing avian erythrocyte nuclei

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)-sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.     

Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components

Linear double-stranded (ds)DNA, obtained by excision of the cloned A and B components of tomato golden mosaic virus (TGMV) from recombinant plasmids, was found to infect plants and to elicit symptoms identical to those obtained with TGMV or TGMV DNA. Progeny virus isolated from plants infected with cloned DNA was infective and indistinguishable from TGMV on the basis of (a) its circular single-stranded (ss)DNA genome, (b) its capsid polypeptide, (c) its particle morphology and (d) serological identity. Southern blot analysis of DNA extracted from cells infected with cloned DNA, or TGMV DNA, revealed the same intracellular ss and dsDNA species, represented in both A and B components, except for a subgenomic, possibly defective, DNA, which was not detected in infections with cloned DNA. Infection with cloned DNA was achieved when cloned A and B components were both present, but not with either cloned A or B components separately. TGMV is the first DNA virus for which unequivocal proof of a bipartite genome has been obtained.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.     

Qualitative and quantitative studies on DNA and RNA synthesis in Loxodes striatus nuclei.

In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei and differentiated morphologically into a clearly recoginzable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a 3H-labeled precursor ([3H]thymidine for DNA and [3H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [3H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [3H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macronucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components synthesis can occur in adult macronuclei. The possible explantion of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

Isolation and characterization of nuclei from rice embryos.

A method has been developed to isolate pure preparations of nuclei in high yield from commercially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol (without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of the isolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both whole nuclei and nuclear extracts obtained by SDS-PAGE, organellar marker enzyme activities, DNA and RNA analyses, and in vitro RNA synthesis, all indicate that the highly purified nuclei are isolated from rice embryos.     

Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

RNA synthesis in isolated nuclei of the dinoflagellate Crypthecodinium cohnii

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).     

Transcription of isolated nuclei and nucleoli by exogenous RNA polymerase A and B.

Both forms A and B RNA polymerases solubilised from rat liver nuclei transcribed templates within these organelles when added exogenously to freshly prepared nuclei. The enzymes initiated more efficiently in the presence of KCL than ammonium sulphate and required manganese rather than magnesium as the divalent cation. Form A enzyme initiated most successfully at 375 mM KC6, activity was proportional to the amount of template added and continued linearly for at least 30 min. Form B enzyme initiated with two ionic strength optima, 125 mM and 500 mM KCl. Activity in the latter case was critically dependent on the enzyme: nuclei ratio. In both instances incorporation of nucleotide precurors was linear for less than 20 min. Form A enzyme synthesised products with a size distribution mainly larger than 18 S; form B enzyme synthesised products of mainly less than 5 S at 125 mM KCl and about 10 S at 500 mM KCl. Subfractionation of nuclei indicated that exogenous RNA polymerase A activity and form B at 125 mM KCl were occurring in nucleoli; form B activity at 500 mM KCl was nucleoplasmic. Measurements of U : G ratios in the RNA products suggested that exogenous form A was synthesising species with similar base ratios to the ribosomal RNA precurosrs. Both enzymes formed rifamycin AF/0-13 resistant complexes with nucleolar templates. Size analyses of products showed that whereas form B enzyme synthesised very small RNA species, RNA polymerase A produced a range of species of similar sizes to the ribosomal RNA precurosors.     

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.     

Host DNA replication is induced by geminivirus infection of differentiated plant cells

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.     

Synthesis of histone mRNA sequences in isolated nuclei of cleavage stage sea urchin embryos

A qualitative assay for detection of histone mRNA sequences in nuclear RNA was developed using actinomycin D-CsCl gradients to separate histone DNA from bulk DNA by differences in buoyant density. A significant amount of RNA synthesized in vitro in isolated nuclei from early blastula stage sea urchin embryos hybridized coincident with the histone DNA satellite, and this hybridization was competed out by unlabeled “9S” polysomal RNA purified from embryos at the same stage of development. The biogenesis of these histone mRNA sequences appeared similar as observed during in vivo and in vitro synthesis. Nuclear RNA from embryos pulse labeled in vivo was found to lack histone sequences, suggesting a rapid exit time for these sequences from the nucleus. Attempts to study the exit of histone sequences from isolated nuclei labeled in vitro also suggested a rapid exit time for histone sequences. The histone sequences were synthesized to a much lesser extent in isolated nuclei from late blastula stage embryos, as anticipated from the much reduced amount of histone mRNA labeled on polysomes at this stage.