Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome.

We studied synthesis of viral and cellular RNA in the presence and absence of 5-fluorodeoxyuridine (FdU, an inhibitor of DNA synthesis) during lytic infection with polyoma virus in confluent, primary mouse kidney cell cultures. In the presence of FdU, synthesis of early 19S polyoma mRNA and of polyoma tumor (T)-antigen, i.e. expression of the early viral gene, is rapidly followed by a mitogenic reaction of the host cell; it leads to an increase of 30 +/- 5% in cellular, mainly 28S and 18S rRNA, followed by activation of the cellular DNA-synthesizing apparatus. Polyoma-induced cellular RNA synthesis is paralleled by increased production of early 19S mRNA and begin of expression of the late viral genes, leading to synthesis of small amounts of late 19S and 16S mRNAs. Changed expression of the viral genome occurs in the absence of detectable synthesis of polyoma DNA I. Infection in the absence of FdU induces the same sequence of events; it is followed, however, by duplication of the mouse cell chromatin (S-phase) and production of progeny virus.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.     

A modified nucleotide in the poly(A) tract of maize RNA

poly(A)+ RNA was isolated from maize by affinity chromatography on columns of oligo(dT)-cellulose. A modified nucleotide ('X') was detected in ribonuclease T2 digests of the RNA as part of a resistant dinucleotide. The dinucleotide was detected by means of the polynucleotide kinase-mediated transfer of a radioactive phosphate atom from adenosine triphosphate to the 5'-OH position of the dinucleotide. Intact poly(A) tracts were released from poly(A)+ RNA by digestion with ribonuclease T1 and A in a high salt buffer and were isolated by oligo(dT)-cellulose chromatography. The poly(A) preparation was found to consist of a series of polyadenylate fragments which varied in chain length from approximately 17 to greater than 70. The modified nucleotide was shown to occupy an internal position in these poly(A) tracts.     

Characteristics of rabbit globin mRNA purification by oligo(dT) cellulose chromatography

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [³H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN

A portion of rat brain RNA contains poly(A) sequences and binds to oligo(dT)-cellulose. Young rats have a greater amount of brain RNA which contains poly(A) than do adult animals. The length of the poly(A) sequence from the brain RNA of young animals was shown to be somewhat longer than that from the RNA of adults. The RNA which bound to the oligo(dT)-cellulose was found to be large and heterogeneous, and to be almost free of ribosomal or of small mol. wt. RNAs. When the polysomal RNA which bound to the oligo(dT)-cellulose columns and that which did not were used to prime a cell-free protein synthesizing system there was a noticeable difference in their‘messenger’activity; the RNA which bound to the oligo(dT)-cellulose was much more active than the unbound material. However, in the case of the nuclear RNA there was not as great a difference between the material which was bound and that which was not bound.     

Composition and in vitro cytotoxicity of cellular infiltrates in rejecting human kidney allografts.

Xenogeneic immune RNA (I-RNA) extracted from the spleens and lymph nodes of guinea pigs previously immunized with a murine fibrosarcoma was able to convert normal mouse lymphocytes to effector cells specifically cytolytic to the same murine tumor in vitro. This effect of I-RNA was dose-dependent, and destroyed by treatment with RNase, but not with DNase or pronase. I-RNA was fractionated by ultracentrifugation on a 5–20% sucrose density gradient and the fraction capable of transferring cell-mediated cytotoxicity (CMC) was shown to have a sedimentation coefficient of 8–16 S. I-RNA was also fractionated by oligo(dT)-cellulose affinity chromatography and the active fraction was found to possess polyadenylic acid (poly(A)) sequences thus resembling messenger RNA. The immunological activity of the poly (A)-containing RNA fraction was tumor-specific and RNase-sensitive. In further experiments, I-RNA fractionated by sucrose density gradient ultracentrifugation was subsequently chromatographed. on an oligo(dT)-cellulose column. CMC was transferred only by the fraction which sedimented at 8–16 S and also contained poly (A).     

Isolation of messenger RNA from polysomes by chromatography on oligo(dT)-cellulose.

A method for the isolation of messenger RNA (mRNA) from polysomes is described. Polysomes are dissolved in a solution containing 0.5 m NaCl and Na dodecyl sulphate and applied to an oligo(dT)-cellulose column. RNA species containing poly(A) sequences are retained by the column, whereas ribosomal proteins and other RNA species are washed off. The column is then eluted with a buffer not containing NaCl. mRNA from HeLa cells and from duck reticulocytes has been fractionated in this way. When fractionated on sucrose gradients, 10 s globin mRNA is obtained in addition to a 20 s component, which can be translated in a cell free system into duck globin. This 20 s RNA is an aggregate of mRNA, which can be disaggregated. Experiments with HeLa cells have shown that the only mRNA species which is not retained by oligo(dT)-cellulose is histone mRNA; this mRNA does not contain a poly(A) sequence.     

Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

The location of the globin mRNA sequence within its 16S precursor.

The coding sequence of globin mRNA has been located at or very close to the 3' end of its poly(A)-containing 16S precursor. The 16S RNA was annealed to globin cDNA and the hybrid digested with ribonuclease H. The undigested fragment did not bind to oligo(dT)-cellulose and its size was that expected for the intact 5' portion of the precursor.     

Partial purification and characterization of rat-liver messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP).

The mRNA coding for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was partially purified from the liver of cyclic-AMP-treated rats by a procedure involving multiple oligo(dT)-cellulose chromatographies and sucrose gradient fractionations. The purification was monitored by translational assay using a wheat germ extract. Relative to RNA bound once to oligo(dT)-cellulose, the final material was enriched 20-fold in template activity for phosphoenolpyruvate carboxykinase synthesis. With this RNA preparation, cell-free enzyme synthesis amounted to 5% of total mRNA-directed protein synthesis. The apparent sedimentation coefficient of phosphoenolpyruvate carboxykinase mRNA in sucrose gradients was between 20 and 22 S, corresponding to an average molecular weight of 0.93 X 10(6). By formamide/polyacrylamide gel electrophoresis the molecular weight of the enzyme mRNA was estimated at between 0.91 X 10(6) and 1.12 X 10(6). From these estimates, it was concluded that considerable non-coding sequence(s) are present in the mRNA. Approximately 20% of the enzyme mRNA in rat liver failed to bind to oligo(dT)-cellulose, presumably because of the absence of a poly(A) segment. The translation of phosphoenolpyruvate carboxykinase mRNA by the wheat germ extract was inhibited in the presence of 7-methylguanosine 5'-phosphate. The enzyme mRNA appears therefore to have a 'cap' at the 5' end.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)-cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)-cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one-half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)-containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA-precipitable material in a cell-free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.