EDTA- and puromycin-derived duck- and rabbit globin-messenger ribonucleoprotein complexes isolated by oligo(dT)-cellulose chromatography

Duck- and rabbit globin messenger ribonucleoprotein complexes isolated by oligo(dT) cellulose chromatography reveal an identical protein pattern—two main proteins of molecular weights of 73000 and 49000 daltons and minor components—whether the complexes have been liberated from polyribosomes with the EDTA-or the puromycin-high-salt method. In the globin messenger ribonucleoprotein particles of both species predominantly the protein with a molecular weight of 73000 daltons is attached to poly(A)-containing regions of the messenger RNAs.     

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.     

Messenger ribonucleoprotein complexes isolated by oligodeoxythymidylate-cellulose chromatography from Neurospora crassa polysomes.

Messenger ribonucleoprotein (mRNP) complexes were isolated from ethylenediaminetetraacetic acid-dissociated polysomes of Neurospora crassa. Approximately 15% of the [3H]uridine incorporated into polysomal ribonucleic acid (RNA) during a 15-min pulse was eluted from oligodeoxythymidylate-cellulose as an mRNP complex. The isolated mRNP complexes exhibited sedimentation coefficients ranging from 15S to greater than 60S. RNA isolated from these mRNP complexes sedimented in sucrose gradients between 4S and 40S, with broad peaks at 15S and 24S. The buoyant density of mRNP complexes eluted with 25% formamide was 1.42 to 1.44 g/cm3, whereas for mRNP complexes eluted with 50% formamide it was 1.48 to 1.50 g/cm3. Six polypeptides, with molecular weights of 14,000, 19,000, 24,000, 31,000, 44,000, and 66,000, were associated with mRNP complexes eluted with 25% formamide. The mRNP complexes eluted with 50% formamide had one associated polypeptide, of molecular weight 27,000.     

Isolation of mouse reticulocyte globin messenger ribonucleoprotein by affinity chromatography using oligo(T)-cellulose.

Mouse globin messenger ribonucleoprotein (mRNP) has been isolated from reticulocyte polysomes by affinity chromatography to oligo(T)-cellulose, using a procedure modified from that of Lindberg and Sundquist. The messenger RNA and protein moieties fo the mRNP are indistinguishable from those isolated by less rapid techniques, such as zonal ultracentrifugation.     

Uterine oestrogen-receptor binding to oligo(dT)-cellulose. An inhibitor from hypothalamic cytosol.

The inactivation of thrombin and factor Xa by antithrombin was determined in the presence of heparin fractions of different molecular weights and with high affinity for antithrombin. The ability to potentiate the inactivation of either coagulation factor increased with increasing length of the polysaccharide chain.     

Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography.

A study has been made of the developmental changes that occur in the RNA and protein moieties of mRNA-protein particles isolated from newborn and adult rat forebrain free polyribosomes. mRNA-protein particles were isolated by oligo(dT)-cellulose chromatography from salt-washed polyribosomes dissociated by puromycin/0.5 M-KCl treatment as two fractions (E1 and E2) by using Tris/HCl/NaCl eluting buffers containing respectively 25 and 50% (v/v) formamide. Isopycnic centrifugation on CsCl gradients showed that the newborn-derived fractions E1 and E2 has buoyant densities of 1.48--1.50 and 1.41--1.43 g/cm3. Adult-derived E1 and E2 fractions had corresponding values of 1.47 and 1.42 g/cm3. The pooled mRNA-protein particles from the E1 and E2 fractions after deproteinization with proteinase K sedimented with a mean size of approx. 18 S on a sucrose gradient containing 85% formamide with little differences between mRNA molecules from newborn and adult. The mean lengths of the poly(A) segments were similar, being about 130 nucleotides long. Distinct changes were found in the protein composition of the mRNA-protein particles. Fractions E1 and E2 from the newborn contained two major proteins of mol.wts. 74 000 and 52 000 with differences in the relative proportions in each fraction. In contrast, adult fractions E1 and E2 contained predominantly the larger protein. However, the adult fraction E2 contained a more heterogeneous population of minor bands of proteins, including that of mol.wt. 52 000. The findings are discussed briefly in relation to other changes in the developing brain.     

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.     

Synthesis of nascent prothrombin and albumin in a heterologous system using rat liver messenger RNA purified in oligo (dT)-cellulose.

Highly active m-RNA was prepared by phenol extraction of rat liver polysomes followed by oligo (dT)-cellulose chromatography. This m-RNA preparation stimulated total protein synthesis in rabbit reticulocyte lysates and in wheat germ extracts. Nascent prothrombin and albumin synthesized in the reticulocyte system programmed with this m-RNA were precipitated with specific antibodies and identified by their electrophoretic mobilities on SDS-acrylamide gels.     

Duplex structure formation between oligo(dA)''s and oligo(dT)''s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.     

Hybridization of biotinylated oligo(dT) for Eukaryotic mRNA quantitation

Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using a biotinylated oligo(dT) probe, which hybridizes directly to the 3′-polyadenylated sequence of eukaryotic mRNAs. The method provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern analysis.     

Dimethyl suberimidate cross-linking of oligo(dT) to DNA-binding proteins

Dimethyl suberimidate is a bifunctional reagent that is used for cross-linking the protein components of oligomeric macromolecules. In this report, dimethyl suberimidate is shown to specifically cross-link oligo(dT) of varying lengths to the DNA-binding subunits of a multimeric helicase-primase encoded by herpes simplex virus type 1. This result indicates that dimethyl suberimidate and other imidoester cross-linking reagents may be useful for characterizing the interaction of oligo(dT) with proteins that bind single-stranded DNA.