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.     

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 α-amanitin was used to determine the relative and absolute levels of RNA synthesis by 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 monitored by hybridization to viral DNA, and of viral 5.5S RNA, 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 or from functional modifications of the RNA polymerases or from a combination of these effects.     

SYNTHESIS OF NUCLEAR RNA IN NERVE AND GLIAL CELLS

—Tritium-labelled RNA precursors were injected at 30 min intervals into the fourth ventricle of rats or rabbits. After 4 h the nuclei from neurones, astrocytes, and other glial cells were isolated and RNA extracted. Investigations were performed in order to establish optimum conditions for RNA extraction from this particular material. The sedimentation patterns obtained in sucrose gradients were similar to those of nuclear RNA from other mammalian tissues and showed the presence of RNA species with high specific activities in the region of the gradient between 10S and 16S and above 28S. All three types of nuclei contained a 45S and a 38S RNA. Moreover, a 32S component could be identified in astrocytic nuclei, a 35S fraction in neuronal nuclei, and both a 32S and 35S RNA in nuclei from glial cells. The nuclei from the various cell types also differ with respect to the rate of incorporation of the label into the nuclear RNA, being four times higher in astrocytic and neuronal nuclei than in those derived from the other glial cells.     

Migration of newly synthesized RNA during mitosis : III. Nuclear RNA in the cytoplasm of metaphase cells

It was shown by autoradiography in previous papers that RNA which is synthesized before mitosis and located in the nuclei, enters the cytoplasm at the onset of mitosis and returns to the nuclei of the daughter cells after mitosis. In order to study thenature of this migrating RNA we performed a sedimentation analysis of RNA isolated from the cytoplasm and chromosomes (nuclei) of metaphase and interphase cells in the synchronized culture of the Chinese hamster. Whereas the cytoplasm of interphase cells is found to contain RNA with sedimentation constants not higher than 28S, the cytoplasm of metaphase cells includes precursors of ribosomal and messenger RNA with sedimentation constants 32S, 45S and even higher. This means that RNA migrating from nuclei to cytoplasm during cell division retains its nuclear character. It is suggested that this property provides for the return of RNA synthesized before mitosis to the nuclei of the daughter cells.     

In vitro synthesis and stability of RNA in isolated nuclei from bovine lymphocytes

Nuclei from Concanavalin A-stimulated lymphocytes (30 hr after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14–20% of the $\text{in vitro}$ labeled RNA is polyadenylated. Poly(A) (?)RNA from both types of nuclei sediments from 4–5S up to more than 30S on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18S than nuclei from resting cells. The same holds for poly(A) (+)RNA. Poly(A) (?) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions poly(A) (+)RNA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells.     

Radioautographic comparison of RNA synthesis patterns in the epithelial cells of mouse pyloric antrum following 3H-uridine and 3H-orotic acid injections

RNA synthesis was examined in the epithelial cells of the mouse pyloric antrum using radioautography 20 min after injection of either 3H-uridine or 3H-orotic acid. The epithelium of the mouse antrum was known to invaginate into blind tubular units composed of mucous cells arranged from base to top into a gland, an isthmus, and a pit. These were subdivided into segments and, after radioautography, silver grains were counted over cell nuclei in each segment. Following 3H-uridine injection, silver grains were present over all nuclei but were more abundant over those of the isthmus than of the gland or the pit. When nuclei were examined in the electron microscope, nucleoplasmic as well as nucleolar silver grains were more numerous in the isthmus than in the pit or gland. Following 3H-orotic acid injection, silver grains were again present over all nuclei; but maximal incorporation appeared to be in pit cell nuclei where, by electron microscopy, it was mainly assigned to the nucleoplasm. When the incorporation was calculated per whole nucleus, however, it was less in pit cell than in isthmal cell nuclei. Even so, the proportion of label in pit cell nuclei was much greater than after 3H-uridine injection. The interpretation of these findings is based on the fact that isthmal cells are immature, whereas cells migrating from the isthmus to become gland or pit cells show increasing differentiation. The immature cells of the isthmus incorporate both uridine and orotic acid more effectively than do the differentiated cells of pit and gland. Since silver grain counts over nuclei provide an index of the rate of RNA synthesis, this synthesis proceeds more actively in the isthmus than in the pit or gland. This is true of ribosomal RNA synthesis, as shown by nucleolar grain counts, and of other RNA's synthesis, as shown by nucleoplasmic grain counts. It seems, however, that while uridine is involved in the synthesis of all types of RNA, orotic acid is mainly implicated in the synthesis of the heterogeneous RNA from which the messenger RNA arises.     

On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

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.     

Thermosensitivity of Nuclear RNA Polymerase during the in vitro Senescence of Mouse Fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro.
The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture.
Preheating nuclei increases the inhibition of their total RNA polymerase activity by or-α-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the α-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells.
It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.     

In Vitro RNA Synthesis by Rat Liver and I Leukemic Nuclei. Isolation of Undegraded RNA

Incubation of nuclei from rat liver or human leukemic cells in the presence of ³H-UTP² and other factors results in th incorporation of label into a material precipitable by acid, alcohol or ether. This materials is isolated by phenolsds extraction, is sensititve to ribonuclease digestion and presumed to be RNA.

The addition of Cu++ to the incubation system is necessary to inhibit RNA breakdown and allows the isolation of undegraded RNA without interefering with th incorporation of radiosactivity. The time patterns of labl incorporation by the two nuclei preparations are different. Whereas label incorporation by th two nuclei preparations are different. Whereas labelincorporation by liver nuclei continues to increase up to 60 minutes, incorporation by th leukemic nuclei is high during the first 10 minutes and continues at a slower rate up to 45 minutes of incubation. further, th two nuclei preparations also synthesize diferent RNA species. While liver nuclei synthesize RNA sedimenting at 4.5S and 7S to 13S, leukemic nuclei synthesize a heterogeneous, polydisperse type of RNA.     

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.