Intercellular information transfer in the immunogenesis process. VIII. Analytical separation on agar and polyacrylamide gel of RNA fractions from the spleens of immunized and intact rats

RNA isolated from the spleens of intact rats and from rats with immunized sheep red cells was fractionated through three steps: 1 - extraction from phenol nuclei at 50-55 degrees and 65-75 degrees C, 2 - calcium-phosphate chromatography, 3 - agar electrophoresis. Eight agar fractions were obtained of the spleens of immunized rats, an increased RNA content was manifested in at least three agar fractions: the first (4 S), the third (21 S) and the eighth (26 S) ones. The first and the eighth immune RNA fractions, as it was shown earlier, induce the synthesis of antibodies in the rat transplantable lymphosarcoma cell. The first agar fraction of nuclear RNA from the spleens of immunized and intact rats were additionally separated using PAAG electrophoresis. The 4 S agar RNA fraction appears to be rather heterogeneous. It contains 4 S, 4.5 S, 5 S, 5.8 S, U1, U2 and 8 SII fractions, which are low-molecular nuclear RNAs, the 4 S subfraction prevailing. It is suggested that the 4 S PAAG subfraction is most active in the synthesis of antibodies induced by the heterogeneous agar 4 S RNA.     

Intercellular information flow in the process of immunogenesis. VII. The effect of low- and high-polymer immune nuclear RNA on antibody synthesis by the cells of rat transplantable]U

Rats were immunized with sheep red cells. From their spleen tissue 4S and 26S electrophoretically homogenous RNA fractions were extracted. Effects of these RNA fractions on the hemolysin synthesis were studied in cells of rat transplantable lymphosarcoma in the presense of actinomycin D (AD). The time intervals between the introduction if RNA into the lymphosarcoma cells suspension and the addition of AD were different. The duration of the period within which no synthesis of antibodies occurred was determined, this period being termed the AD-dependent period in induction of antibody synthesis. AD being introduced later (after the end of this period) failed to exert its inhibiting action. With highmolecular (26S) RNA, the AD-dependent period was somewhat shorter as compared with the lowmolecular (4S) RNA. This difference is suggested to be due, presumably, to different mechanism of action of both the RNA fractions on recipient cells.     

Intercellular information transfer in the process of immunogenesis. VI. The induction of antiphage antibody synthesis in the cells of rat transplantable lymphosarcoma under the influence of splenic RNA from rats and mice immunized with phage T2]

It has been proved that nuclear and cytoplasmic RNAs, isolated from spleens of T2 phage immunized rats and mice, can induce T2 phage antibodies in cells of the transplantable rat lymphosarcoma. With the nuclear RNA from rat spleens, the effect is persisting in a number of subsequent cell generations. The data presented are principally in accord with results of the authors' previous studies in which lymphosarcoma cells were treated with RNA extracted from spleens of rat immunized with sheep red cells. These results well compare with the authors' earlier advanced hypothesis suggesting a possible involvement of RNA in deblockation of genes responsible for the synthesis of the antibodies in question.     

Nuclear translocation of insulin receptor substrate-1 by oncogenes and Igf-I. Effect on ribosomal RNA synthesis

The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally localized in the cytosol/membrane fraction of the cell. We show here that a substantial fraction of IRS-1 is translocated to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1 occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized role in rRNA synthesis and/or processing.     

Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta.

A 4-8-fold increase in the rate of hepatic nuclear RNA synthesis occurred within 11 h after a single injection of oestradiol-17 beta to male Xenopus to induce egg-yolk protein synthesis. 2. By using a gentle procedure for fractionating nuclei into their major structurally different components [J. R. Tata& B. Baker (1974) Exp. Cell Res. 83. 111-124], it was found that the hormone-induced increase in the total amount of newly made RNA was associated with a 2-10-fold increase in the poly(A) content of nuclear RNA. 3. When the poly (A) content of nuclear RNA was determined by hybridization to poly[3H](U) or specific binding to oligo(dT)-cellulose, most of the increase (10-fold) in poly (A) content of newly synthesized RNA was associated with the euchromatin fractions, whereas the increase was less marked in the other subnuclear fractions. 4. Resolution of nuclear RNA into poly (A)-poor and poly(A)-rich RNA species by chromatography on oligo(dT)-cellulose, followed by polyacrylamide-gel electrophoresis with sodium dodecyl sulphate or in the pressence of 99% formamide, revealed that the hormone caused a preferential enhancement of high-molecular-weight (25S-60S) poly (A)-rich HnRNA (heterogeneous nuclear RNA,) much of which was associated with euchromatin and not with the nuclear sap. 5. Induction of vitellogenin in male frogs was in particular characterized by the appearance of a high-molecular-weight polyadenylated component exhibiting a peak at 35-36S, i.e. a molecular weight of approx. 2.05x10(6)+/-0.15x10(6). Although there is no evidence as yet that such a polyadenylated high-molecular-weight nuclear RNA species contains sequences corresponding to vitellogenin mRNA, it is possible that a high proportion of the most stable form of the putative nuclear precursor to vitellogenin mRNA induced by oestrogen in male Xenopus liver may be only marginally bigger than the cytoplasmic mRNA, and may at any one time be predominantly associated with the euchromatin fraction.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Shutoff of Host RNA Synthesis in Bacteriophage T4-Infected Escherichia coli in the Absence of Host DNA Degradation and Nuclear Disruption

In contrast to its effect on host DNA synthesis, nuclear disruption in phage T4-infected Escherichia coli B/5 cells has no effect on the shutoff of host RNA synthesis. Host RNA synthesis is shut off normally after infection with T4 multiple mutants that fail to induce both nuclear disruption and host DNA degradation.     

Immunohistochemical demonstration of nuclear S1 proteins in various cells

Summary S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.     

Immunohistochemical demonstration of nuclear S1 proteins in various cells

S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.     

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.