Uptake of homologous single-stranded fragments by superhelical DNA. III. The product and its enzymic conversion to a recombinant molecule

When superhelical DNA (RFI)² of phages φX174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RFI is displaced, forming a displacement loop (D-loop). The length of the base-paired region was estimated from the fraction of the associated ³²P-labeled fragment that was resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonucleases that act on single-stranded DNA, the S₁ nuclease of Aspergillus oryzae and the recBC DNAase of Escherichia coli. Acting on complexes, both enzymes converted the form I [³H]DNA into form II DNA, and left some of the associated ³²P-labeled fragment undigested. The remaining ³²P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S₁ nuclease also produced the amount of acid-soluble ³H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining ³²P-labeled fragment to ³H-labeled strands, which proves that S₁ nuclease digested the D-loop.     

A rapid DEAE disk assay for photoreactivation of pyrimidine dimers in [3H]DNA.

We have developed a rapid, sensitive, and specific assay for photoreactivation of pyrimidine dimers in ³H-labeled DNA. It is based on the nuclease resistance of dimercontaining sequences in DNA, and the adsorption of these sequences to DEAE-substituted paper (DE-81). The method maintains the advantages of the rapid dimer assay of B. M. Sutherland and M. J. Chamberlin (1973, Anal. Biochem., 53, 168–176), while avoiding its major drawback, the frequent preparation of high specific activity, ³²P-labeled, bacteriophage DNA. In addition, the use of scintillation spectrometry in our assay should allow more widespread use of this method.     

Improved two-step method for the assay of adenylate and guanylate cyclase.

A two-step assay for adenylate and guanylate cyclase is described utilizing α-³²P-labeled ATP or GTP as substrate and involving purification of the resulting ³²P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mm HCl for cGMP and 10 mm HClO₄ for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the ³²P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.     

Characterization of bacteriophage T4 and D RNA, a low-molecular-weight RNA of unknown function

The nucleotide sequence of T4 band D RNA, a stable RNA species encoded by bacteriophage T4, has been deduced from analysis of the ³²P-labeled RNA and comparison with the DNA sequence of the T4 genome in the region encoding the RNA. The sequence is: pA-U-G-A-G-A-A-A-C-C-G-G-G-U-C-G-C-U-A-C-C-G-G-U-A-A-G-U-C-G-U-C-G-G-A-C-U-G-A-U-G-G-U-U-C-C-C-U-G-A-G-U-A-A-G-G-A-A-U-U-G-C-G-U-U-A-A-U-A-A -U-C-U-U-U-G-C-G-U-U-U-A-U-U-G-A-U-G-C-C-C-U-C-U-U-A-C-A-U-C-A-C-A-G-C-A-G-A-A-A-C-G-G-C-G-C-A-C-C-A_OH. Band D RNA is 120 nucleotides long, and contains no modified nucleotides. The sequence can be arranged in a secondary structure consistent with the results of limited digestion with nuclease S1, but shows no striking similarities to tRNAs. While a biological function for band D RNA is unknown, similar molecules are encoded by bacteriophages T2 and T6, indicating that the molecule has been preserved during evolution. This retention may reflect a significant function for the RNA.     

Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase.

Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]_o > K_m indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [³²P]phosphate ion. Incubation of wheat germ acid phosphatase with ³²P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of ³²P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A ³²P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme.     

Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle

A method is described for purification of (N⁺, K⁺)-ATPase which yields approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 μmol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600–700 pmol of [³H]ouabain/mg protein when incubated in the presence of either Mg²⁺ plus P_i or Mg²⁺ plus ATP plus Na⁺, and incorporated more than 600 pmol ³²P/mg protein when incubated with γ-³²P-labeled ATP in the presence of Mg²⁺ and Na⁺. The preparation is approximately 35% pure.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Zonal rate centrifugation of proteoglycans in sucrose gradients

A simple reverse-phase chromatographic system for separating deoxyribonucleoside monophosphates is described. Using isocratic elution at room temperature, clear separation of seven of the deoxyribonucleoside monophosphates that occur in either procaryotic or eucaryotic DNAs can be achieved in less than 2 h. Thus, this method allows a sensitive and rapid analysis of submicrogram quantities of ³²P-labeled deoxyribonucleoside monophosphates derived from DNA labeled in vivo with ³²P_i or from DNA labeled enzymatically in vitro at the 5′ or 3′ ends. The suitability of the method for studying methylation of mammalian DNAs is illustrated by presenting examples of its application to (a) quantitation of major and minor nucleotides in newly synthesized DNA, (b) determination of the specificity of in vitro methylation of DNA, and (c) quantitation of the extent to which specific restriction endonuclease sites are methylated in vivo.     

Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex.

The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.     

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.     

Purification of globin messenger RNA from dimethylsulfoxide-induced friend cells and detection of a putative globin messenger RNA precursor

Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate ³²P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. ³²P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified ³²P-labeled globin mRNA by RNAase T₁ digestion. The partial sequences for nine oligonucleotides corresponded to those predicted from the amino acid sequences of α and β globin; the other oligonucleotides were presumably derived from non-translated regions.In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [³²P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XII. Evidence for interaction of the acyl phosphate at the active site with neighboring groups

The sodium-potassium adenosinetriphosphatase (NaK ATPase), partially purified from beef brain, has been phosphorylated with [γ-³²P]ATP in the presence of Na and Mg and digested with pronase. A single ³²P-labeled peptide spot has been identified on paper electrophoresis, accounting for 60% of the radioactivity in the ³²P-labeled enzyme, the remainder of the radioactivity being [³²P]-orthophosphate resulting from breakdown of the highly labile acyl phosphate during pronase digestion. The ³²P in the pronase peptide was released as [³²P]-orthophosphate by N-propylhydroxylamine—as to be expected of an acyl phosphate compound. The pH stability of the acyl phosphate in the denatured phosphorylated NaK ATPase, in the pronase peptide and in acetyl phosphate were quite different. The phosphorylated protein had the lowest stability of higher pHs, acetyl phosphate had the highest stability, and the pronase peptide had an intermediate stability. These results indicate that the neighboring groups in the polypeptide chain containing the acyl phosphate residue influence the stability of the acyl phosphate bond.