Demonstration, in leukemia L-1210 cells, of a phosphodiesterase acting on 3':5'-cyclic CMP but not on 3':5'-cyclic AMP or 3':5'-cyclic GMP.

cCMP-specific phosphodiesterase activity was demonstrated in the 80 to 100% ammonium sulfate fraction obtained from disrupted leukemia L-1210 cells. The activity was linear with time (up to 60 min), was a function of protein concentration, and was markedly stimulated by Mg2+ and by ammonium sulfate. Under identical assay conditions, no significant hydrolysis of cAMP or cGMP was observed, although these cyclic nucleotides served as substrates for phosphodiesterase(s) present in all the fractions obtained by less than 80% ammonium sulfate saturation. This is the first demonstration of a cCMP-specific phosphodiesterase.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

A rapid direct assay of 3'-5' cyclic nucleotide phosphodiesterase activity using chromatography on immobilized acriflavin

A chromatographic method using immobilized acriflavin has been developed for the separation of unreacted cyclic nucleotides from their corresponding 5'-nucleotides, in a direct assay of 3'-5' cyclic nucleotide phosphodiesterase using [3H]-cyclic nucleotides as substrate. The method based on the so-called charge-transfer overlap recognition between flavin and indol rings, provides a rapid (15-20 min) and sensitive elution of [3H]-5'nucleotides with high recovery (up to 98%) and low blanks, while [3H]-cyclic nucleotides are retarded on the column. By this method, the formation of some secondary products by purine metabolizing enzymes such as 5'-nucleotidases, nucleosidases and/or deaminases is taken into account, using [14C]-5'-AMP thus allowing an accurate determination of phosphodiesterase activity in any preparations.     

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.     

Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase.

Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.     

A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography.

A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.     

A polymorphism in the bovine gamma-S-crystallin gene revealed by allele-specific amplification

Summary
A polymorphism was detected in the 3' untranslated region of the bovine gamma-S-crystallin gene by direct sequencing of polymerase chain reaction (PCR) products from genomic DNA of an N'Dama bull and a Boran cow. A set of three PCR primers was designed to detect this difference and thus give allele-specific amplification. The two allele-specific primers differ in length by 20 nucleotides so that the allelic products may be distinguished by simple agarose gel electrophoresis following a single PCR reaction. This provides a simple and rapid assay for this polymorphism.     

Characterization of the 3':5' ratio for reliable determination of RNA quality

Determination of RNA quality is a critical first step in obtaining meaningful gene expression data. The PCR-based 3′:5′ assay is an RNA quality assessment tool. This assay is a simple, fast, and low-cost method of selecting samples for further analysis. However, its practical applications are unexploited primarily because of the absence of an experimental threshold. We show that, by anchoring the 5′ assay a specific distance from the 3′ end of the sequence and by spacing the 3′ at a distance of a number of nucleotides, a cutoff determines whether a sample is suitable for downstream quantification studies.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

An assay for the endonucleolytic clevage of RNA to large oligonucleotides

A rapid assay for endonuclease activity which cleaves high-molecular-weight RNA to acid-precipitable fragments has been developed. RNA is covalently coupled to beaded agarose under conditions that produce relatively few coupling sites. The immobilized RNA can be used qualitatively or semiquantitatively in an assay for endonuclease activity by determining the release of acid-precipitable RNA from the complex. This assay is compared to one employing separation of degraded RNA by gel electrophoresis.     

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA.

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.     

An isotopic assay of cyclic 3',5'-nucleotide phosphodiesterase with aluminum oxide columns

A method is described for separating purine bases and nucleosides from cyclic 3′,5′-nucleotides on aluminum oxide columns. Purine bases and nucleosides were found to pass through columns equilibrated with ammonium acetate buffer at pH 4.0 while the cyclic nucleotides were retarded enough to permit separation. Optimal conditions and factors affecting separation are described. The method was shown to be applicable in the isotopic assay of cyclic 3′,5′-nucleotide phosphodiesterase activity over a broad range of substrate concentrations. The advantages of this method and its possible use in a simultaneous cyclase-phosphodiesterase assay are described.     

A rapid histidine decarboxylase assay

Histidine decarboxylase (EC 4.1.1.22) catalyzes the conversion of histidine to histamine. Because current assays for enzyme activity are time consuming and require additional enzymes or large amounts of tissue, a rapid radioisotopic assay was devised. Using commercially available radioactive histidine (without additional purification), the enzyme mediates the formation of histamine. The product is resolved from precursor by paper electrophoresis in a formic acid-acetic acid solution for 15 min. After drying and ninhydrin staining, radioactive histamine is measured by liquid seintillation spectrometry. This assay procedure is sensitive enough to measure decarboxylase activity in milligram quantities of rat brain.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

RNA cleaving '10-23' DNAzymes with enhanced stability and activity

‘10-23’ DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5′-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3′-3′-inverted thymidine, phosphorothioate linkages, 2′-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.