RNA metabolism in nuclei: selective transport of kappa exons from myeloma nuclei and adenoviral transcripts from infected HeLa nuclei.

Two independent systems and several analytical procedures have been used to establish that isolated mammalian nuclei selectively transport mature RNA polymerase I and II products. Murine myeloma nuclei retain physiologic restriction in our transport assay as assessed by the transport of the immunoglobulin kappa light chain mRNA and 18S and 28S rRNAs. Nearly 50% of the total kappa exons are transported as structurally intact mature mRNA molecules while less than 8% of either pulse-labeled or steady state kappa intron sequences are detected in the transported fraction. Ribosomal external transcribed spacer sequences also are absent in transported RNA. Release of cytoplasmic RNA from the outer nuclear membrane during the transport assay accounts for less than 10% of transported RNA. Nuclei isolated from adenovirus-infected HeLa cells at 20 hours post infection retain cellular actin mRNA and transport viral poly A+RNA. Ribosomal RNA is transported from infected nuclei although at a reduced rate compared to transport from mock-infected nuclei. Inhibition of transport of host mRNA is paralleled by the absence of pulse-labeled actin mRNA in the cytoplasm of infected cells. The implications of our transport data in relationship to intranuclear RNA trafficking are discussed.     

Cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells.

Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.     

Histone-specific acetyltransferase from the rat liver nuclei

Certain properties of histone-specific acetyltransferases A, B and C, obtained from the rat liver are determined. pH optimum for enzyme A is within the range of 7.5-8.5, for B--7.8 and for C--7.5. The maximal activity for enzymes A, B and C is observed with the 60 micrograms/ml concentration of the substrate. The activity is inhibited by N-maleimide, iodacetamide and chloromercuribenzoate. The results obtained show that a number of similar properties are typical of the above enzymes.     

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.     

Nucleolin from the multiple nucleoli of amphibian oocyte nuclei

When fixed preparations of newt germinal vesicle (GV) contents are treated with RNase and are then probed with radiolabeled single-stranded DNA in 0.1–2.0 × SSC, the extrachromosomal nucleoli bind the probe non-specifically. DNA/protein blot analysis of proteins from newt GVs shows that gv95, an acidic protein (pI = 5.0) of M_r = 95 000, is the most prominent nonspecific DNA-binding protein. Immunocytochemical analysis with affinity purified antibody directed against gv95 shows that it is located in the multiple nucleoli. We used an antibody directed against rat nucleolin to show that newt gv95 and two similarXenopus GV proteins are the amphibian versions of nucleolin, a nucleolar ribonucleoprotein originally identified in mammalian cells. We show that mAb 3A10, directed against newt histones H1 and H5, labels gv95 on protein immunoblots and the multiple nucleoli in cytological preparations. These results suggest that histone H1 and nucleolin share a cross-reacting epitope.     

Interpretation of the properties of chromatin extracts from mammalian nuclei

Chromatin from standard preparations of nuclei stabilized by magnesium ions at 0-4 degrees C was degraded during the nuclear isolation, and newly-synthesized chromatin was degraded slightly more slowly than the ;older' chromatin. The significance of this observation, and its relation to the interpretation of the properties of nucleoprotein extracts is discussed.     

RNA transport in isolated myeloma nuclei. Transport from membrane- denuded nuclei

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.     

Ribonuclease P from plant nuclei and photosynthetic organelles

RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date. Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate. No clearcut answer is yet possible regarding the order of processing events at the 5 or 3 end of tRNAs in the case of nuclear or chloroplast processing enzymes. RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from theCyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit. This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity. Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit. The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.Abbreviations RNase P Ribonuclease P - (pre-)tRNA transfer ribonucleic acid (precursor) - tRNA ^Ser (- ^Tyr , - ^Phe ) transfer ribonucleic acid specific for serine (tyrosine, phenylalanine) - CyRP RNA RNA component of cyanelle RNase P     

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.     

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.