Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

'Cap' structures in maize poly(A)-containing RNA.

Poly(A)-containing RNA was isolated from maize embryos by chromatography on columns of oligo(dT)-cellulose and exhaustively digested with ribonucleases T2, T1, and A. Fractionation of the digests by two-dimensional electrophoresis revealed the presence of three 7-methylguanosine-terminated 'cap structures' of the type m7GpppNp.     

Fractionation and characterization of rat liver poly(A)-containing RNA.

Three fractions of poly(A)-containing RNA were separated from total rat liver RNA using poly(U)-Sepharose 4B affinity chromatography. The poly(A)-containing RNA fractions were released by thermal elution. Fraction 1, eluted under the mildest conditions, and had poly(A) tracts of approx. 200 AMP units in length which appeared to be associated with poly(U) sequences of 20-50 UMP in length. Fraction 1 appeared to be present mainly in the nucleus and, its size distribution was similar to that of fractions 2 and 3. Fractions 2 and 3 eluted at higher temperatures and were associated mainly with polysomal and microsomal fractions. Poly(U) sequences were absent in fractions 2 and 3 while their poly(A) sequences had a size distribution characteristic of those reported in the mRNA of other organisms.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.     

Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.     

Altered fractionation profiles of higher plant poly(A)-containing RNA by selective formation of a complex with a hydrophobic impurity

Synthetic polyadenylate (poly(A)) which originally sedimented at 6–7 S in a sucrose density gradient did so at 15–19 S in the presence of phenol-extracted nucleic acids from higher plant tissues. After centrifugation, the input poly(A) was found to be insensitive to RNase T2 (EC 3.129.1), suggesting a newly-formed complex with a hydrophobic substance contaminating the nucleic acid preparation. The artifically-formed poly(A)-impurity complex migrated more slowly than unbound poly(A) during gel electrophoresis. The complex was retained rather effectively by an oligodeoxythymidylate (oligo-(dT))-cellulose column and a polyuridylate (poly(U))-glassfiber filter. However, a nitrocellulose membrane filter retained the poly(A)-complex to various extents depending on plant materials, suggesting different varieties of the poly(A)-binding impurity in the higher plant kingdom. Fractionation of mRNA by these ordinarily used procedures is suggested to be variously altered by complex formation with this hydrophobic impurity.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

Metabolic heterogeneity of nuclear poly (A)-containing RNA in mouse liver.

The analysis by the approach to equilibrium labeling method has shown that the poly(A)+ fraction of liver hnRNA is not a uniform class of molecules, but is comprised of two distinct subclasses with half-lives of 5 and 60 min, while the poly(A)- hnRNA was metabolically homogeneous and turned over with a rather uniform half-life of 30 min. The results suggest that (a) poly(A) synthesis and addition is not limiting for the rate of hnRNA processing, and (b) there is a correlation between the kinetics of mRNA appearance in the cytoplasm and kinetic behavior of their possible nuclear precursors.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Structure of poly (I).poly (A).poly (I)

The molecular structure of poly (I).poly (A).poly (I) has been determined and refined using the continuous intensity data on layer lines in the x-ray diffraction pattern obtained from an oriented fiber of this polymorphic RNA complex. The polymer forms a 12-fold right-handed triple-helix of pitch 39.7A and each base-triplet is stabilized by quasi Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in all the three strands have C3'-endo conformations. The final R-value for this best structure is 0.24 and the x-ray fit is significantly superior to all the alternative structures where the different chains might have different furanose conformations. This all-purine triple-helix, counter-intuitively, has a diameter roughly 3A shorter than that of DNA and RNA triple-helices containing a homopurine and two complementary homopyrimidine strands. Its compact, grooveless cylindrical shape is consistent with the lack of lateral organization.