A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-[α-³²P]triphosphate ([α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-[α-³²P]triphosphate ([α-³²P]KTP) as a means to 3′-end label DNA. The [α-³²P]ddATP was found to be three to five times more efficient than [α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with [α-³²P]ddATP using terminal transferase than were the 5′-ends with [γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared [α-³²P]ddATP- and [α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.     

Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein

The 5′ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M_r protein, linked to a small [³²P]oligonucleotide, was isolated after DNase digestion of uniformly ³²P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [³²P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5′ ends of Ad5 DNA); (3) prior treatment of the [³²P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of O-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mm-NaOH at 70 °C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad5 DNA under these conditions we were able to label the 5′ termini with ³²P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5′ mononucleotides yielded [³²P]dCMP. We conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the β-OH of a serine residue of the protein and the 5′-OH of the terminal deoxycytidine residue of the DNA.     

Base compositional analysis of nanogram quantities of unlabeled nucleic acids.

After conversion of unlabeled DNA and RNA to 3′-mononucleotides accurate base compositional analysis can be performed on as little as 10 ng of the hydrolysate. The 3′-mononucleotides are first quantitatively postlabeled with [γ-³²P]ATP by T4 polynucleotide kinase and are then separated as mononucleoside diphosphates on Whatman DE-81 ion-exchange paper at pH 3.5 after hydrolysis of surplus [γ-³²P]ATP to ³²P₁. The locations of the four labeled nucleoside diphosphates are determined by autoradiography and the ratio of radioactivity in the four spots gives the base ratio of the sample.     

Enzymatic Preparation of 32P-Labeled β-L-2′, 3′-dd-5′ ATP and Its Use as a High-Affinity,Conformation-Specific Ligand for Labeling Adenylyl Cyclases

Abstract

An enzymatic method was developed for the preparation of unlabeled and [β-³²P]-labeled β-L-2′,3′-dd-5′ATP from the monophosphate with near quantitative yields. β-L-2′,3′-dd-5′ATP was a competitive and potent inhibitor of adenylyl cyclases (IC₅ ～ 30 nM). Upon uvirradiation β-L-2′,3′-dd-[β-³²P]-5′ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state, noncompetitive inhibitor.     

Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-³²P]ATP. After selective degradation of the DNA portions, [5′-³²P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.     

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-<Superscript>32</Superscript>P]d/rNTP

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d₂CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k _m and k _cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α-³²P]rNTP and [α-³²P]dNTP.     

In vitro polyoma DNA synthesis: Involvement of RNA in discontinuous chain growth

Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-³²P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-³²P-labeled deoxynucleoside triphosphates showed ³²P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived.     

Preparation of (beta-32P)ribonucleoside-5'-triposphates using permeable cells of Escherichia coli

A rapid method for the preparation of [β-³²P]ribonucleoside-5′-triphosphates is described. The method involves the incubation of a ribonucleoside triphosphate with ³²P_i and E. coli cells made permeable to nucleotides. The labeled triphosphates can be isolated by preparative thin layer chromatography on poly(ethylene)imine cellulose plates. Labeled GTP, CTP, and UTP obtained by this method are more than 99% pure [β-³²P]compounds. Labeled ATP contains about equal amounts of label in the β- and γ-phosphate position. Pure [β-³²P]ATP can be obtained from this preparation by exchanging the γ-³²P against unlabeled P_i and reisolating the labeled ATP by charcoal adsorption and elution.     

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 …     

The 5'-terminal sequence of Q 6S RNA

[γ-³²P]GTP-Labeled Qβ 6S RNA yielded only one major radioactive oligonucleotide after digestion with pancreatic ribonuclease. Nearest neighbor analysis of this 5′-oligonucleotide demonstrated that approximately 95% of the molecules terminate with the same sequence, pppGpGpCp. This sequence is the complement of the only major 3′-sequence found in this RNA. Both strands of 6S RNA therefore appear to have identical 3′- and 5′-terminal trinucleotide sequences.     

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques

The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of ³H-labeling procedures previously published and a new method for the characterization of 2′-O-methylated nucleosides based on enzymatic ³²P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails ³²P labeling by [γ-³²P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T₂-stable 2′-O-methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the ³²P-labeled 2′-O-methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.     

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.     

A radiotracer enzyme assay for phosphofructokinase using ( , , -32P) adenosine triphosphate

A radiotracer enzyme assay for phosphofructokinase using adenosine 5′-triphosphate[α,β,γ-³²P] is described in this paper. Here the rates of appearance of both [1-³²P]d-fructose 1,6-diphosphate and [α,β-³²P] adenosine 5′-diphosphate were followed to establish enzyme activity. The unique advantages of multiple rate determinations in a single reaction sequence which accrue from the use of a readily available multiply labeled cosubstrate are discussed. By an extension of this approach other labeled(¹) nucleotides of the type, N(¹P)_n, and enzymes in the Enzyme Commision categories, EC 2.7(phosphotransferases) and EC 6.1–6.4(ligases) are equally amenable to radionuclide assay.     

Human erythrocyte spectrin: Phosphorylation in intact cells and purification of the 32P-labeled protein in a non-aggregated state

[³²P]spectrin (0.5 Ci/mMole) has been isolated from human erythrocytes incubated with ³²Pi and purified to homogeneity by preparative rate zonal sedimentation on linear sucrose gradients. ³²P-label, localized in band 2, co-elutes with spectrin from ghosts with a similar dependence on ionic strength and Mg⁺⁺ ion, and has the same sedimentation coefficient and an identical effective Stokes radius. [³²P]spectrin reassociates in a specific manner with spectrin-depleted membranes. Bands 1 and 2 bind in equal ratios, and the ³²P-label is distributed with band 2. Purified [³²P]spectrin is not aggregated since this protein migrates as a symmetrical peak on Sepharose(C1)4B at about 1.6 Vo and sediments at 8S_20,w on sucrose gradients.     

Cytidylate cyclase: the product isolated by the method of Cech and Ignarro is not cytidine 3',5'-monophosphate.

When mouse liver homogenates were incubated with [α-³²P]-CTP according to the method of Cech and Ignarro (4,5), a [³²P] product (100–120 pmoles/mg protein/minute) was isolated by chromatography on alumina column, in the same fraction as [³H]-cyclic CMP.¹ However this product did not behave as cyclic CMP in various chromatographic systems. Moreover the amount of this [³²P] product isolated was markedly reduced by removal of protein prior to chromatography on alumina. Chromatography of the enzymatic reaction product either before or after alumina purification on Dowex 1 — formate column indicated that 5′-CMP and CDP are the major products, with no formation of cyclic CMP. These results indicate that the [³²P] labeled material isolated by Cech and Ignarro is mainly if not entirely 5′-CMP and CDP.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.