Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-chloroform extraction step during 5′-end labeling with polynucleotide kinase and [γ-³²P]ATP; (b) ZnSO₄ inactivation of RNase T₁ results in a highly efficient procedure for 3′-end labeling with T4 ligase and [5′-³²P]pCp; and (c) a rapid 4-min procedure for variable quantity range of ¹²⁵I and RNA results in a qualitative and quantitative sample for high-molecular weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.     

Efficient extraction and quantitative determination of nanogram amounts of cellular RNA

An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-³²P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-³²P]ATP. Radiolabeled products are separated from remaining [γ-³²P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.     

Isolating and sequencing the infrequent 3'-ends of a specific mRNA

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.     

Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Isoproterenol-induced selective phosphorylation invivo of the 214,000 dalton subunit of rat C6 glioma cell RNA polymerase II

The phosphorylation $\text{in}\text{vivo}$ of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na₂H³²PO₄ and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [³²P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [³²P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II.     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Ontogeny of ppp(A2′p)nA-binding protein in mouse brain

Mouse brain tissue extracts at various stages of development show a drastic change in the specific activity of pp(A2′p)₂A-[³²P]pCp binding protein. Identification of the ppp(A2′p)₃A-[³²P]pCp binding protein was established by (i) binding to the specific ligand ppp(A2′p)₃A-[³²P]pCp, (ii) displacement of binding by nanomolar concentration of pppA(pA)₃, and (iii) affinity labeling techniques in which periodate oxidized ppp(A2′p)₃A-[³²P]pC was specifically cross-linked to a protein with a molecular weight of 86 000. These data suggest that the ppp(A2′p)₃A-[³²P]pCp protein is closely associated with the process of cellular proliferation and differentiation.     

Detection of avocado sunblotch viroid (ASBV) by dot-spot self-hybridization with a [32P]-labelled ASBV-RNA

RNA was extracted from plants infected with avocado sunblotch viroid (ASBV) and was analyzed by electrophoresis in polyacrylamide gel. The ASBV related fraction was eluted from the gel, labelled with [³²P] using polynucleotide kinase and used as a probe for hybridization with a purified ASBV-RNA preparation dot spotted on nitrocellulose paper. Positive self-hybridization indicated a high degree of internal complementarity. Dot spots of whole cell RNA and of leaf sap from ASBV infected plants were shown to hybridize with the labelled probe. This hybridization procedure proved to be 16–64 times more sensitive in diagnosing ASBV when compared with polyacrylamide gel analysis.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

A covalently linked DNA-RNA molecule from human leukemia cells

An enzyme complex was prepared from the cytoplasm of a continuous line of monocytic human leukemia cells isotopically labeled in culture. Such preparations carry out RNA dependent DNA synthesis using endogenous primers and templates and contain radioactive RNA and DNA. The endogenous [³H]-thymidine labeled DNA in these preparations was characterized by sedimentation in Cs₂SO₄ and neutral sucrose density gradients in conjunction with heat and alkali treatments and digestion with RNase. The resulting data support a view that a portion of the DNA is covalently linked to a larger piece of RNA in a molecule with a sedimentation coefficient of approximately 24S. This in turn may be hydrogen bonded to additional DNA in the native state.