Coding capacity of poly(A)+ mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells

Total poly(A)⁺ mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8 h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 per cent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

Coding capacity of poly(A)+mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells.

Total poly (A)+mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 percent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes.

A recombinant library has been constructed using the plasmid pAT153 and double stranded cDNA prepared from normal human lymphocyte poly(A)+ RNA. Transformation conditions were optimized to yield approximately 200,000 recombinants per microgram of double stranded cDNA. Statistical analysis as well as sequence complexity analysis of the inserted sequences indicates that the cDNA library is representative of > 99% of the poly(A)+ RNA present in the normal human lymphocyte.     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)⁺ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)⁺ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [³H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)⁺ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)⁺ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [³H] poly(U) hybridization experiment.     

Associations of small nuclear RNAs in human cells

Several observations have been made about the associations of small nuclear RNAs (snRNAs) in human cells. When nuclear RNA was extracted with phenol and chloroform under standard nondenaturing conditions, the proportion of the nuclear snRNA content that cosedimented with high molecular weight RNA was very low. These results do not support the proposal that it is a large percentage of the cellular snRNA content that is involved in relatively stable base-paired interactions with heterogeneous nuclear RNA at any given time. The various small nuclear ribonucleoprotein particles (snRNPs), in which the snRNAs are found in the cell, appear to differ substantially in their sedimentation rates under conditions of physiological ionic strength. Using anti-RNP and anti-Sm antibodies to analyze various subcellular fractions, we found that most, if not all, of the U1 snRNA cellular content is associated with the polypeptide(s) bearing the RNP determinant (in interphase and mitotic cells) and with the polypeptide(s) carrying the Sm determinant (in mitotic cells).     

Stability of poly(A)+RNA in nucleate and anucleate cells of Acetabularia

The stability of poly(A)⁺RNA was compared in nucleate and anucleate cells of Acetabularia. While in the absence of the nucleus poly(A)⁺RNA exhibits a pronounced stability, it is significantly less stable in the presence of the nucleus.     

Effects of exogenous poly(A)+ mRNA on antigenic properties and growth of Krebs-2 tumor cells]

The treatment of Krebs-2 ascitic tumor cells (TC) with total RNA from the liver of Wistar rats (2 mg/ml) altered their antigenicity. As a result, the growth of treated TC in contrast with untreated TC was inhibited when transplanted i. m. to mice preimmunized with rat liver homogenate. Investigations of poly(A+)mRNA, rRNA and tRNA isolated from the same tissue established that the alteration of antigenicity is due to mRNA (8-24 micrograms/ml). In the cytotoxicity assay with antisera against rat Wistar antigens, the expression of rat antigens in TC treated mRNA was observed in the next cell generations.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).     

Quantitation and characterisation of poly(A)-containing messenger RNAs from mouse neuroblastoma cells

Comparison of several isolation procedures for neuroblastoma poly(A)-containing mRNAs shows that the highest percentage recovery of undegraded and biologically active messenger RNAs is obtained using proteinase K prior to phenol extraction. The messenger RNAs thus isolated comprise approximately 1.5% of the total ribosomal RNAs and have negligible contamination with 18 and 28 S RNAs. On denaturing polyacrylamide gels they have an average molecular weight of 6.5-10(5) with a range from 2.2-10(5) to 1.53-10(6). The messenger RNAs have an average poly(A) content of 154 nucleotides. They are highly active in wheat germ in vitro protein synthesizing systems, giving as much as 4.3 pmol [35S]methionine incorporation into total protein per mol of mRNA. This is almost as active as a control globin mRNA preparation.