Preparation of immobilized phosphodiesterase from calf spleen and its use in oligonucleotide analysis

The phosphodiesterase from calf spleen (EC 3.1.4.18) was immobilized on several supports. Some properties of the most suitable enzyme support system--calf spleen phosphodiesterase bound to agarose-Concanavalin A--were investigated, e.g., pH dependence, influence of ionic strength of the buffer medium, and Zn2+-ion inhibition. The immobilized spleen phosphodiesterase showed about 60% of the activity of the free enzyme; the activity toward several oligonucleotide test substrates was unchanged for two months.     

Synthesis of glucosylceramide by purified calf spleen beta-glucosidase

Radioactive glucocerebroside was formed when purified calf spleen β-glucosidase was incubated in the presence of 4-methylumbelliferyl-β-$\text{D}$-glucoside and (¹⁴C) labeled ceramide. The product was identified by cochromatography on thin layer plates and carrier dilution and crystallization to constant specific activity. The radioactive glucocerebroside was also a substrate for hydrolysis by this same β-glucosidase preparation employed for its synthesis resulting in the liberation of labeled ceramide. Neither methyl-β-$\text{D}$-glucoside nor free D-glucose were effective in producing radioactive glucocerebroside when incubated with labeled ceramide in this system.     

Solid phase ligation of synthetic DNA fragments

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.     

Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli.

Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents. The inducible AlkA DNA glycosylase (3-methyladenine [m3A] DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A. In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal. In such DNA alkylated with [3H]dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag. This was further confirmed using both N-[3H]methyl-N-nitrosourea- and [3H]dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA. These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.     

Solid phase DNA sequencing using the biotin-avidin system.

A novel method for solid-phase DNA sequencing is described. A plasmid vector, pRIT27, has been designed to allow directional immobilization of double stranded plasmid to avidin agarose. The strategy involves enzymatic incorporation of 11-bio-dUTP into the plasmid and strand specific elution using alkali. The immobilized single stranded DNA is used as template for sequencing reactions and the resulting labelled oligonucleotides are eluted by alkali. The affinity gel containing the immobilized template is consecutively used for the four different dideoxy-nucleotide reactions. The solid-phase technique can be used for both primer specific or extension specific labelling. The possibility to use the system in automated DNA sequencing is discussed.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.     

Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen.

Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C. The fluorescence of enzyme-bound guanine was similar to that of anionic guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme, whereas inosine and guanosine were not bound to free enzyme in the absence of phosphate. Thus, synthesis proceeded by a random mechanism, and phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data for the phosphorolysis of 100 microM guanosine were fitted to a bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1. The dissociation rate constants for guanine from the enzyme-guanine complex at high and low phosphate concentrations were similar to the catalytic rate constants. Fluorescence changes of the enzyme during phosphorolysis suggested that ribose 1-phosphate dissociated from the enzyme ribose 1-phosphate-guanine complex rapidly and that guanine dissociated from the enzyme-guanine complex slowly. The association and dissociation rate constants for acyclovir diphosphate, a potent inhibitor of the enzyme (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069), were also dependent on phosphate concentration. The effects of phosphate are discussed in terms of a dual functional binding site for phosphate.     

A method of solid phase immunoenzyme analysis using antigen molecules immobilized on membranes

The enzyme-linked immunoassay modification has been worked out. The method combines advantages of membrane technology of antigen immobilization which is used in the enzyme immunosensory technique and of conventional enzyme-linked immunosorbent assay. The nitrocellulose and polypropylene membranes are used as a solid-phase. The purified rabbit immunoglobulin G is immobilized on the surface of membranes as the first layer. The competitive immunoassay is employed. The immunoglobulin G concentration range is 1-1000 ng/ml. The membranes with the immobilized antigen can be repeatedly used after incubation in 0.1 M glycine buffer, pH 2.5. The dry membrane with the immobilized antigen can be used after keeping for 6 months in refrigerator at 4 degrees C without changing the concentration range measured.     

Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule.     

Parentage assessment of an IVF calf from Bubalus bubalis by DNA fingerprinting

We used two synthetic oligodeoxyribonucleotide probes, OAT18 and OMS1 comprising (TGG)₆ and (GGTA)₄ repeat motifs, respectively, in combination with HinfI enzyme for DNA fingerprinting of a calf born from embryo transfer technology, its surrogate mother (SM) and the sperm donor (bull). Of the two polymorphic probes, OAT18 was found to be more informative for correctly establishing the parentage status of the in-vitro fertilized (IVF) calf and demonstrated the genetic relationship of the SM with the bull. Two types of cluster analysis, SAHN clustering and neighbour joining tree, performed with similarity indices of these individuals produced by OAT18 probe were found to be different. Using the SAHN cluster method, SM was found to be genetically closer to the bull than to IVF calf, whereas using the neighbour joining method, IVF was closer to bull than SM. A similar result was obtained by SAHN clustering with OMS1 probe. The relevance of this approach for parentage control in the context of animal biotechnology is described.     

The elongation of mismatched primers by DNA polymerase alpha from calf thymus

The ability of the 9S and 5.7S DNA polymerase alpha subspecies from calf thymus in elongating a mismatched primer terminus has been investigated. With poly(dA) as template, the elongation rate for (dT)8dG, (dT)8dC and (dT)10dGdT is 20-fold lower for the 9S enzyme and 5-fold lower for the 5.7S enzyme as compared to (dT)10. The presence of a second mismatch at the primer terminus reduces the elongation rate further by a factor of two. Exonucleolytic excision of the mismatches can be excluded. With (dT)8dG (dT)n as primer we show, that at least five T-residues have to follow the mismatch in order to establish the elongation rate of a perfectly paired primer. The KM value for (dT)10 dG as primer is 400 nM as compared to 10 nM for (dT)10. Addition of Mn2+ increases the relative efficiency of elongation of the mismatched primers.     

Stabilization of double-stranded DNA molecule by nonhistone peptidic effector from calf thymus

A peptidic effector from calf thymus causes a strong stabilization of DNA doublestranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (<5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.     

Processivity of the DNA polymerase alpha-primase complex from calf thymus

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.