Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Deoxyribo-oligonucleotide synthesis*

An enzyme, which is probably identical with polynucleotide phosphorylase, was prepared from Escherichiacoli B. In the presence of Mn(2+) it catalyzes the addition of one (and to a slight extent more) residue of deoxyribonucleotide residue from the diphosphate to an oligodeoxyribonucleotide primer. The shortest effective primers contained three phosphate residues. Ribodinucleotides were effective as primers and accepted two or three deoxyribonucleotide residues under these conditions. The application of the procedures to the convenient synthesis of certain defined oligodeoxyribonucleotides up to nine residues long is discussed.     

Single-step elongation of oligodeoxynucleotides using terminal deoxynucleotidyl transferase

Procedures for the stepwise addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleoside phosphate acceptor using commercially available terminal deoxynucleotidyl transferase is described. 2-80 nmol of acceptors with a chain length of four, five or nine monomer units were elongated with a single 2'-deoxyribonucleoside 5'-triphosphate in yields of 20-30%. The monomers carried no protecting groups and were used both radioactively labelled and unlabelled. The elongated oligodeoxynucleoside phosphates were isolated by reverse-phase (Nucleosil C18) high-performance liquid chromatography or paper chromatography. The isolated products were sequenced by the fingerprint method. Advantages and disadvantages of this new methodology for the enzymatic synthesis of defined oligodeoxynucleotides are discussed.     

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains.

It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.     

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that polyphosphates are primers and determination of the size of the synthesized polyphosphate

Polyphosphate kinase from Propionibacterium shermanii was purified to 70% homogeneity and shown to be a monomeric enzyme of molecular weight 83,000 +/- 3,000. It was demonstrated that short chains of polyphosphate serve as primers by using [32P]polyphosphate, 6-80 residues in length for synthesis of long-chain polyphosphate glucokinase, the radiolabel was found to be at the end of the polymer, proving that the mechanism of elongation of polyphosphate by polyphosphate kinase is strictly processive. Only 1 out of 3-8 of the polyphosphate chains contained the primer, indicating that there is a second unknown pathway of initiation which does not involve the polyphosphate primer. The termination of polyphosphate synthesis was investigated. With polyphosphate as a primer, the majority of the synthesized polyphosphate was 750 residues in length. With phosphate, in place of the polyphosphate primer, the major portion was about 2,000 residues in length but there was a large span of chain lengths down to 300. Termination is influenced by pH, temperature, and the concentration of the polyphosphate primer, with the chain length decreasing as either the temperature or the concentration of primer is increased.     

Vaccinia virus poly(A) polymerase. Specificity for nucleotides and nucleotide analogs

We have studied the nucleotide specificity of vaccinia virus poly(A) polymerase using a novel primer extension assay. Oligoribonucleotide primers labeled at the 5' end with 32P were elongated by the enzyme in the presence of ATP, leading to the 3' addition of greater than 1000 adenylate residues/primer molecule. In the presence of UTP, the enzyme catalyzed 3' polymerization of long poly(U) tails, albeit at a reduced rate of chain growth. In the presence of both ATP and UTP, 3' addition was selective for ATP. The transient accumulation of RNAs elongated by 10-16 residues suggested that polyadenylation (and polyuridylation) was a biphasic reaction. Quantitative 3' addition of GMP (from GTP) or CMP (from CTP) to the primer was also observed, although the rate of chain growth was so slow as to allow synthesis of only short oligo(G) or oligo(C) tails. The deoxynucleotides 3'-dATP (cordycepin triphosphate) and ddATP were markedly inhibitory to poly(A) polymerase. Primer elongation studies were consistent with inhibition due to 3' incorporation of inhibitor and chain termination. Incubation of enzyme with [alpha-32P] cordycepin triphosphate resulted in labeling of the Mr 57,000 enzyme subunit, apparently via formation of a covalent nucleotidyl-protein complex. These data are discussed in light of their implications for the catalytic mechanism of polyadenylation.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells.

The de novo biosynthesis of fatty acids of 12 to 18 carbons from precursors of 5 carbons or fewer has been demonstrated in Acholeplasma laidlawii B. Radiolabeling experiments indicated that the normal primers for the synthesis of the even- and odd-chain fatty acids are acetate and propionate or valerate, respectively. Saturated straight-chain monomethyl-branched fatty acids of up to five carbons were readily utilized as primers, wheras more highly branched species and those possessing halogen substituents or unsaturation were not utilized. At primer concentrations of 1 to 3 mM, up to 80% of the total cellular lipid fatty acids were derived from exogenous primer. The mean chain length of the exogenous primer-derived fatty acids rose with increasing primer incorporation for methyl-branched short-chain fatty acids but was invariant for propionate. The products of de novo biosynthesis varied only slightly with temperature or cholesterol supplementation, suggesting that de novo biosynthesis is not directly influenced by membrane fluidity. Cerulenin inhibited de novo biosynthesis in a fashion that suggests the presence of two beta-ketoacyl thioester synthetases, which differ in substrate chain length specificity and in susceptibility to inhibition by the antibiotic.     

Sequence confirmation of synthetic phosphorothioate oligodeoxynucleotides using Sanger sequencing reactions in combination with mass spectrometry

A protocol relying on Sanger sequencing reactions in combination with mass spectrometry (MS) for sequence confirmation of antisense phosphorothioate oligodeoxynucleotides is described. In this procedure, synthetic phosphorothioate oligodeoxynucleotides are used as reverse primers for extension of matched templates with enough length (approximately 150-300 bp) for well-established Sanger sequencing. Because the complementary strand of modified primer is used directly for sequencing primer extension, the base order shown in the sequencing result is reversely complementary to phosphorothioate oligodeoxynucleotide. This sequencing method can be applied not only to phosphorothioate oligodeoxynucleotides with different lengths (13-21 mer) and base composition but also to sequences with bases' switch, deletion, or insertion. In addition, modified primers incorporate the 5' end of polymerase chain reaction (PCR) products conveying the characters of phosphorothioate modification. The method requires only common reagents and instruments and so is better suited to routine sequence analysis in quality control of phosphorothioate antisense drugs.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis

The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P. polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.     

Fatty acid chain elongation by microsomal enzymes from the bovine meibomian gland

The composition of meibomian gland lipids suggested that fatty acid chain elongation might play a major role in the synthesis of such lipids. A fatty acid synthase preparation from the bovine meibomian gland catalyzed the formation of C16 acid and the enzyme was immunologically quite similar to that in the mammary gland. The microsomal fraction from the gland, on the other hand, catalyzed elongation of endogenous fatty acids in the presence of ATP and Mg2+ and of exogenous C18-CoA using malonyl-CoA and NADPH as the preferred reductant. The elongated products, ranging up to C28 in chain length, were found mainly as CoA esters and products derived from them. With C18-CoA as the exogenous primer, the elongation rate was linear with incubation time up to 20 min but the rate changed in a sigmoidal manner with increasing protein concentration. The elongation rate was maximal at a pH around 7.0. Typical Michaelis-Menten-type substrate saturation patterns were observed with both malonyl-CoA and NADPH. From linear double-reciprocal plots, the Km values for the two substrates were calculated to be 52 and 11 microM, respectively, with a V of about 340 pmol min-1 mg protein-1 with respect to malonyl-CoA. Exogenous CoA esters of C16 to C22 fatty acids were elongated to give products up to C28 without exhibiting any preference for the primer. The present elongation system could account for the formation of most of the very long chains found in meibomian lipids.     

A convenient and versatile method for the purification of CoA thiol esters

A simple and versatile chromatographic procedure has been described for the purification of fatty acyl-CoA thiol esters, ranging in chain length from C₂ to C₁₈. The method yields products that are over 90% pure, as judged by several criteria, in yields of 80% or greater. It may also be applicable to analytical procedures where the resolution and isolation of acylated CoA, CoASH, CoA-S-S-CoA and adenine nucleotide from a large number of reaction mixtures is required.     

Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides

The enzyme exonuclease I from Escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers. When the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated. The distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease I with homopolymers of deoxyadenylate, deoxythymidylate, or deoxycytidylate is a decamer. These results suggest a model in which exonuclease I possesses at least two nucleotide binding sites. When both sites are filled, with 11-mers and longer polymers, the enzyme does not dissociate from the polymer during hydrolysis. When, with smaller oligomers, only a single site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation. The kinetics of the reactions of exonuclease I with purified polydeoxyriboadenylates of defined size distributions have been investigated. The maximum rates of hydrolysis are nearly independent of polymer size while the apparent Michaelis constants are inversely proportional to the polymer size. A simple steady state model yields a kinetic equation that is consistent with our results. Competition experiments indicate that the rate at which exonuclease I associates with the 3'-terminus of a polydeoxyribonucleotide is independent of the polymer's chain length.     

Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater

The human-specific HF183 Bacteriodes 16S rRNA genetic marker can be used to detect human faecal pollution in water environments. However, there is currently no method to quantify the prevalence of this marker in environmental samples. We developed a real-time polymerase chain reaction (PCR) assay using SYBR Green I detection to quantify this marker in faecal and environmental samples. To decrease the amplicon length to a suitable size for real-time PCR detection, a new reverse primer was designed and validated on human and animal faecal samples. The use of the newly developed reverse primer in combination with the human-specific HF183 primer did not decrease the specificity of the real-time PCR assay but a melting curve analysis must always be included. This new assay was more sensitive than conventional PCR and highly reproducible with a coefficient of variation of less than 1% within an assay and 3% between assays. As the Bacteroides species that carries this human-specific marker has never been isolated, a bacteria real-time assay was used to determine the detection efficiency. The estimated detection efficiency in freshwater ranged from 78% to 91% of the true value with an average detection efficiency of 83+/-4% of the true value. Using a simple filtration method, the limit of quantification was 4.7+/-0.3x10(5) human-specific Bacteroides markers per litre of freshwater. The aerobic incubation of the human-specific Bacteroides marker in freshwater for up to 24 days at 4 and 12 degrees C, and up to 8 days at 28 degrees C, indicated that the marker persisted up to the end of the incubation period for all incubation temperatures.     

Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence

Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg²⁺, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.     

Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.     

Enzymatic synthesis of low molecular weight amyloses with modified terminal groups.

Low molecular weight amyloses with modified terminal groups were synthesized by cyclomaltohexaose (alpha-cyclodextrin) transfer using (1----4)-alpha-D-glucan: 4-alpha-D-(1----4)- alpha-D-glucopyranosyltransferase (cyclising) (EC 2.4.1.19) from Bacillus macerans. 4-Nitrophenyl alpha-malto-oligosaccharides d.p. 2-7 served as acceptors, and cyclomaltohexaose served as the donor. The reaction was optimized to obtain a majority of species of definite chain lengths in a range of d.p. 10-20, depending upon the chain length of the acceptor. The course of the coupling reactions, as well as the action of the enzyme in disproportionation, cyclisation, and hydrolysis of the products, were observed by h.p.l.c. analysis of the oligomer distributions. Using a 15-fold molar excess of cyclomaltohexaose and 0.5 units enzyme per mumol of acceptor at pH 5.2, the chromatograms revealed that the products of the coupling reaction were predominant during the first reaction period. By incubating the acceptors with the enzyme, but without the donor, the mechanism of disproportionation was elucidated as a transfer of malto-oligosaccharyl residues dependent upon the substrate chain length. The minimum chain length required for a direct cyclisation reaction was d.p. 7. The results were confirmed by separation and investigation of the products of hydrolysis and cyclisation, which were nonmodified alpha-malto-oligosaccharides and cyclomalto-oligosaccharides.     

Evaluation of deep eutectic solvents as new media for Candida antarctica B lipase catalyzed reactions

This study aimed at analyzing the advantages and limitations of several deep eutectic solvents (DESs) as ‘green solvents’ for biotransformation using immobilized Candida antarctica lipase B as catalyst. The transesterification of vinyl laurate was chosen as model reaction and the influence of substrate polarity was assessed using alcohols of various chain lengths. Results showed that grinding of immobilized lipase was essential parameters for good lipase activity. Moreover, in our model reaction some hydrogen-bond donor component from the DES can compete with the alcoholysis reaction. Indeed, side reactions were observed with DES based on dicarboxylic acid or ethylene glycol, leading to some limitations of their use. However, the results showed that other DESs such as choline chloride:urea and choline chloride:glycerol could exhibit high activity and selectivity making them promising solvents for lipase-catalyzed reactions. Finally, the best DES's specific activity – and stability up to five days incubation time – were analyzed and compared with conventional organic solvents. Experiments revealed that iCALB is less influenced by the chain length of alcohol in DES than organic solvents and it is preserves its activity with minimally destructive to protein structure.     

Unusual amino acid usage in the variable regions of mercury-binding antibodies

Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza-specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens.