Use of Sepharose 4B for Preparative Scale Fractionation of Eukaryotic Messenger RNA's

A new technique using Sepharose 4B column chromatography for the partial purification of the total messenger RNA population of several animal tissues is described. The column when eluted with 0.1M sodium acetate, pH 5.0, containing. 001M EDTA, resolves a total nucleic acid extract into four major peaks. DNA is eluted at the void volume, followed successively by peaks of 18S ribosomal RNA, 4S transfer RNA and 28S ribosomal RNA. Ribonucleic acid containing mRNA activities is eluted after the DNA peak but immediately before the 18S rRNA peak. Hence the column enables quantitative removal of DNA, 5S RNA, tRNA and 28S rRNA from the majority of total cellular mRNA's. Partial segregation of mRNA's in the column is also demonstrated. The method does not require the isolation of polysomes as the initial procedure in mRNA isolation and is readily adaptable to large scale preparations. One hundred mg of total nucleic acid extracted from whole tissues can be fractionated on a 5 × 100 cm Sepharose 4B column. Recovery of total mRNA activities ranges between 60–70% and purification with respect to the total cell extract is 7 to 8 fold.     

Adsorption chromatography of nucleic acids on silicone-coated porous glass

Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations. tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.     

Overestimates of the size of poly(A) segments.

Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Using a cationic carbocyanine dye to assess RNA loading in Northern gel analysis.

In Northern blotting, one must have a means of assessing the uniformity of RNA loaded into each lane of a gel. As an alternative to "common gene" controls and previously published nucleic acid dyes (ethidium bromide, acridine orange, methylene blue), we have utilized a cationic carbocyanine dye (Stains All) for the assessment of RNA gel loading uniformity over the range of 5-25 micrograms RNA/lane. The following protocol is suitable for messages of well-characterized mobility and utilizes xylene cyanol as a 4-kb marker; as such, it will migrate between 28S and 18S rRNA over a wide range of agarose concentrations. Optimally, it is best that the message(s) of interest should migrate either as a smaller species than 18S or as a larger species than 28S; this allows either the 28S or 18S ribosomal band to be separated from the message(s) of interest by severing the gel transversely at the xylene cyanol front. Severing the gel in such a manner makes it possible to simultaneously submit that portion of the gel containing either the 28S or 18S rRNA band to Stains All staining while immediately continuing with the transfer of that portion of the gel containing the mRNA of interest. We have found the dye to interact linearly with rRNA whether data were gathered by densitometrically scanning the gels themselves or photographs of the gels.     

Virus Interference by Cellular Double-Stranded Ribonucleic Acid

Ribonuclease-resistant ribonucleic acid (RNA) was isolated from uridine-labeled cultures of rabbit kidney, chicken embryo, and HeLa cells. This RNA, regardless of its source, was found to induce interference with virus growth in either rabbit kidney or chicken embryo cultures. Nuclease-treated cellular nucleic acids exhibited interference-inducing activity which eluted with a small fraction of RNA in the exclusion volume of a 6% agarose gel column. Besides resistance to ribonucleases, the interference inducer and RNA isolated from partially digested nucleic acids have in common two properties of double-stranded RNA: (i) similar sharp melting profiles were obtained for inducer and ribonuclease-resistant RNA, with T(m) dependent on NaCl concentration; (ii) ribonuclease-resistant inducer and RNA banded together in Cs(2)SO(4) density gradients at a density characteristic of known double-stranded RNA. After melting at low ionic strength, the labeled RNA shifted to a higher density and its capacity to inhibit virus replication was lost. Velocity sedimentation analysis of the cellular ribonuclease-resistant RNA indicated that the majority sedimented between 7 and 11S, but only RNA sedimenting at >==8 to 20S had a high specific activity of interference induction. Without prior ribonuclease treatment, the ribonuclease-resistant RNA can be precipitated with 2 m LiCl and thus appears to exist in purified cellular nucleic acids as part of molecular complexes with both single- and double-stranded regions of RNA. The biosynthesis of cellular double-stranded RNA is inhibited by actinomycin D.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

Purification of 5S RNA from Plant Leaves

A simple and rapid method for large scale preparation of 5S RNA from plant leaves is described. To begin, all nucleic acids were extracted from the leaves with a mixture of phenol-chloroform-n-butyl alcohol and extracting buffer. After precipitation of the high-molecular-weight nucleic acids from the crude extract with 2 M LiCl, the low, molecular-weight RNA in the supernatant (containing about 6% 5S RNA) could be separated by eleetrophoresis on denatured polyaerylamide gels. The band of 5S RNA was excised from the preparatory slab gel under UV light and then purified by eleetrophoretie elution. In our experiments, several mg pure 5S RNA was obtained from 100 g leaves in a single run which takes about 4 days. The purified final produet was pure as showing a single hand on denatured polyaerylamide gel and a typical UV absorption peak of nucleic acid. The sedimentation coefficient (S20w) of the product was 4.6 as determined by ultracentrifugation.     

Cytoplasmic RNA from hen reticulocytes, mouse sarcoma 180 ascites cells, rat liver and barley embryos. Their preparation and purification by a standard procedure and characterization by polyacrylamide gel electrophoresis.

1. This paper describes a standard procedure for the preparation and purification of RNA from the post-mitochondrial supernatants of a number of eukaryotes. 2. Cytoplasmic RNA was fractionated by NaCl precipitation. The 28S (26S), 18S and 5.8S rRNA, and 9S RNA, in the NaCl insoluble fraction were separated by a two-step sucrose gradient fractionation procedure. Poly(A)-containing mRNA in hen 9S RNA was purified by affinity chromatography. The 5S rRNA and tRNA in the NaCl-soluble fraction were fractionated by gel filtration. 3. Polyacrylamide gel electrophoresis showed that the above RNA species were remarkably stable and homogeneous. Differences were found in the 26-28S rRNA, 5.8S rRNA, and 9S RNA of different eukaryotes, but other cytoplasmic RNA species were identical.     

Isolation of rapidly labeled RNA from mouse L-cells using cupric sulfate.

Cultured mouse L-cells, pulse labeled for 5 min with 3H-uridine, were gently suspended in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate at 4 degrees C and treated with cold phenol. Only the RNA, containing less than 1% DNA, was extracted by this procedure. The rapidly labeled ribosomal RNA precursors (44S, 34S) and cytoplasmic 8S RNA showed specific activities higher than that of tRNA and were present in the RNA fraction insoluble in 2M NaCl.     

Application of high-performance ion-exchange chromatography to the analysis of cytosolic glucocorticoid receptor

The use of high-performance ion-exchange chromatography (HPIEC) on a Mono Q column was investigated for the analysis of glucocorticoid receptor. In the presence of 10 mM sodium molybdate, both liganded and unliganded glucocorticoid receptor were eluted as a single and sharp peak (0.32 M NaCl). In the absence of molybdate and after exposure to heat and salt, another peak of specifically bound radioactivity was eluted with 0.08 M NaCl. When HPIEC was performed in the absence of molybdate, two molecular forms of the liganded receptor were detected which eluted with 0.08 M NaCl (Stokes' radius Rs = 5.1 nm, s20,w = 4.6 S, calculated mol. wt Mr approximately 100,000) and 0.32 M NaCl (Rs = 7.3 nm, S20,w = 9.0 S, calculated Mr approximately 280,000). Analysis of both forms with mini-columns of DNA-Ultrogel, DEAE-Trisacryl and hydroxylapatite (HA-Ultrogel) confirmed the identity of the two peaks with transformed and non-transformed glucocorticoid-receptor complexes. These results suggest that HPIEC may provide a useful tool for the rapid resolution and quantification of receptor molecular forms.     

Proteoglycans synthesized and secreted by pancreatic islet beta-cells bind amylin

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.     

Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

The separation of ribonucleic acids from Escherichia coli on lysin-agarose.

Lysine-agarose provides a simple means of separating RNA species of different moleecular weight. When RNA from Escherichia coli is added to a small column of lysine-agarose and elution is carried out at neutral pH with a shallow linear gradient of NaCl the RNA species are eluted according to size; 4S and 5S RNA species are completely separated and after a delay the 16S and 23S species are eluted as separate peaks. The process is very reproducible and the different species are eluted at a fixed salt concentration even when changes are made in the gradient, provided other conditions, under which the column is run remain constant.     

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10?nM Ang II for 5?min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30?mM NaCl and 3?mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150?mM NaCl and 3?mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.     

Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)

Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in ³H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4–5 × 10⁵ dpm/μg. 4 + 5S, 18S and 25S RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18S and 25S RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.