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B R Shaw

Publications and source records attributed to B R Shaw.

At least 19 recordsLinked to original sources
PubMed1997

Direct PCR sequencing with boronated nucleotides.

A method is described to simultaneously amplify and sequence DNA using a new class of nucleotides containing boron. During the polymerase chain reaction, boron-modified nucleotides, i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]-triphosphates, are incorporated into the product DNA. The boranophosphate linkages are resistant to nucleases and thus the positions of the boranophosphates can be revealed by exonuclease digestion, thereby generating a set of fragments that defines the DNA sequence. The boranophosphate method offers an alternative to current PCR sequencing methods. Single-sided primer extension with dideoxynucleotide chain terminators is avoided, with the consequence that the sequencing fragments are derived directly from the original PCR products. Boranophosphate sequencing is demonstrated with the Pharmacia and the Applied Biosystems 373A automatic sequencers, producing data that is comparable with cycle sequencing.

Bacteriophage T7↗
PubMed1996

Accelerated deamination of cytosine residues in UV-induced cyclobutane pyrimidine dimers leads to CC-->TT transitions.

The rate of UV-induced deamination of cytosine to uracil at a specific site in double-stranded (ds) DNA was monitored using a genetic reversion assay. M13mp2C141 ds DNA was exposed to 160 J/m2 UV (254 nm), incubated at 37 degrees C, pH 7.4, for various time intervals to allow for deamination, and treated with Escherichia coli photolyase in the presence of 365 nm light to reverse cyclobutane-type pyrimidine dimers. Upon transfection into uracil-glycosylase deficient (ung-) E. coli cells, the mutation (i.e., reversion) frequencies in the CCCC target sequence increased greatly with post-UV time of incubation at 37 degrees C, nearly doubling every day that the DNA had been held at 37 degrees C. After 8 days, the reversion frequencies had increased by two orders of magnitude upon transfection into ung- cells, relative to isogenic ung+ cells, indicating that most of the mutations arising in UV/photolyase-treated ds DNA were C-->T mutations mediated by a uracil intermediate. Sequencing of the revertants revealed that all mutations were single C-->T or tandem double CC-->TT mutations. An increasing percentage of tandem double CC-->TT mutations was found with longer post-UV incubation times, yet none occurred if the post-UV delay time step was omitted before photoreversal. After a 4-day delay between UV and photoreversal at 37 degrees C, greater than 84% of the total revertants had tandem double CC-->TT mutations. Thus, the generation of a tandem double mutation is a time-dependent process that arises in DNA after the initial UV exposure. The rate of appearance (with a pseudo-first-order rate constant ca. 10(-6) s-1) of tandem double mutations during incubation of UV-irradiated DNA is inconsistent with two random, independently occurring mutational events and suggests a concerted deamination of both residues in a tandem cytosine pyrimidine (C < > C) dimer. Considering that deamination in a C < > C dimer occurred here with a half-life of ca. 5 days, in contrast to the measured half-life of ca. 20,000 years for spontaneous (non-UV-treated) cytosine deamination for the same target, these studies show that the formation of pyrimidine dimers in DNA increases the rate of deamination by six orders of magnitude, leading to the accelerated formation of single C-->T and tandem double CC-->TT mutations.

Bacteriophage M13↗
PubMed1996

Base-boronated dinucleotides: synthesis and effect of N7-cyanoborane substitution on the base protons.

Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N7-cyanoborane-2'-deoxyguanosine ((7b)dG) at the 3', 5' or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry. Fmoc was used as the 5'-protecting group because of incompatibility of the cyanoborane moiety with 5'-DMT cations generated during the deprotection step. The presence of the cyanoborane group was confirmed on the basis of Fab-MS and 1H NMR spectroscopy. The H-8 proton of (7b)dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D20 exchange kinetics of the H-8 proton at 60 degrees C showed that H-8 of (7b)dG is very labile relative to unmodified dG, indicating that the N7-cyanoborane modification increases the acidity of the H-8 proton of (7b)dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N7-guanine to block Hoogsteen pairing in the DNA major groove.

Amides↗
PubMed1995

Boron-containing oligodeoxyribonucleotide 14mer duplexes: enzymatic synthesis and melting studies.

A set of three 14mer oligodeoxyribonucleotides of sequence d(5'-CTATGGCCTCAG*CT-3'/3'-GATACCGGAGTCGA-5') containing G* variants either as 2'-deoxyguanosine phosphate (unmodified), N7-cyanoborane 2'-deoxyguanosine phosphate (base-modified) or 2'-deoxyguanosine boranophosphate (backbone-modified) were synthesized by template-directed primer extension. Both the N7-cyanoborane 2'-deoxyguanosine triphosphate and 2'-deoxyguanosine alpha-boranotriphosphate nucleotides are good substrates for Sequenase. We infer that a single Sp boranophosphate linkage (which has a stereochemistry equivalent to the corresponding Rp thiophosphate analog) is formed in the backbone-modified 14mer. Thermally induced helix-coil transitions were monitored for the hybridized duplexes using UV and circular dichroism (CD) spectroscopy. The CD spectra of the two types of boron-modified hybrids closely resemble the unmodified parent duplex, forming B-type helices in 150 mM NaCl, 1 mM EDTA, 10 mM phosphate, pH 7.4, buffer. UV melting results indicate that both hybrids have stabilities comparable with the parent duplex as measured by Tm or delta G degree 25. These studies indicate that singly modified base- or backbone-boronated DNA are good analogs of normal DNA.

Base Sequence↗
PubMed1995

N7-cyanoborane-2'-deoxyguanosine 5'-triphosphate is a good substrate for DNA polymerase.

The 5'-triphosphate of the boronated nucleoside analog N7-cyanoborane-2'-deoxyguanosine (7bdGTP) was synthesized, and a series of experiments was initiated to assess the potential of the compound to serve as a substrate for DNA polymerases. We show here that 7bdGTP can be incorporated into DNA by Sequenase. The resulting hemiboronated extension products are resistant to cleavage by treatment with either DMS and heat or a number of restriction enzymes. Further, in the polymerase chain reaction, 7bdGTP can be utilized as a substrate for Taq polymerase. Finally, by kinetic analysis, we have found that 7bdGTP is a more efficient substrate for exonuclease-free Klenow than normal dGTP. Thus, the introduction of a cyanoborane moiety to the N7 position of dGTP results in a nucleotide that is accepted in lieu of normal dGTP by a number of DNA polymerases.

Base Sequence↗
PubMed1994

Bisulfite induces tandem double CC-->TT mutations in double-stranded DNA. 2. Kinetics of cytosine deamination.

Deamination of cytosine to uracil in double-stranded DNA (ds DNA) by sodium bisulfite has been monitored with a sensitive genetic assay. In this system, reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 C141 was detected by employing an ung- bacterial strain defective in the enzyme uracil glycosylase. Within the 4-base target, it is possible to measure the rates of induction of C-->T, C-->A, C-->G, and CC-->TT mutations in DNA that has been incubated at physiological temperature and pH and then transfected into ung+ and ung- E. coli cells, respectively, for amplification and detection of the mutation. For concentrations of bisulfite from 1 to 50 mM, the reversion frequency in ung- cells increased linearly with time of incubation. The most interesting features of the bisulfite reaction were as follow: (1) Mutations were reduced 5-fold in ung+ cells, indicating ung is involved in repair of bisulfite-treated transforming DNA. (2) Sequencing of 157 revertants revealed that C-->T and tandem CC-->TT transition mutations comprised 100% of the mutations scored. (3) A unique finding was that, at the highest concentrations and longest incubation times, almost every mutant obtained in ds DNA exposed to bisulfite was found to be a CC-->TT tandem double mutation. (4) The high frequency of tandem double mutants is inconsistent with two random, independent mutational events and, coupled with the observed ung dependence, lends support to the concept of catalytic deamination, wherein bisulfite induces deamination in contiguous cytosines by a concerted mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1994

Anti-inflammatory and anti-osteoporotic activities of base-boronated nucleosides and phosphate-boronated nucleotides in rodents.

The 2'-deoxyribonucleoside cyanoboranes were effective anti-inflammatory agents in rodents at 2-8 mg/kg; they blocked induced edema, septic shock, and pleurisy. Overall compounds 3',5'-O-(bis- (triisopropylsilyl)-2'-deoxyinosine (1), 3',5'-O-bis(triisopropylsilyl)-2'-deoxycytidine (10), N3-(cyanoboryl)-2'-deoxycytidine (11), N7-(cyanoboryl)-N2-isobutyryl- 3',5'-O-bis(triisopropylsilyl)-2'-deoxyguanosine (20), and N7-(cyanoboryl)-N2- isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(triisopropylsilyl)-2' -deoxyguanosine (22) were the most active when all the anti-inflammatory screens are considered. The agents also blocked both local and central pain caused by inflammation. These nucleosides blocked calcium resorption but were less effective compared to other amine carboxyboranes. The inflammation process appeared blocked by these compounds because of their effectiveness in reducing both hydrolytic lysosomal enzyme and proteolytic enzyme activities. The agents were also dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities in leukocytes and macrophages. These agents at 10(-4) M demonstrated no specific organ toxicity to ileum mucosa cells grown in tissue culture.

Animals↗
PubMed1993

Cytosine deamination in mismatched base pairs.

The rate of deamination of cytosine in mismatched base pairs has been determined. Incubation of M13mp2 nicked heteroduplex DNA molecules containing T.C or C.C mispairs in the lacZ alpha-complementation gene results in deamination of cytosine to uracil, producing T.U or C.U mispairs. Strands which have undergone deamination at the target site to produce uracil will yield dark blue plaque revertants, while all other strands yield faint blue or colorless plaque phenotypes upon transfection of an ung- alpha-complementation Escherichia coli host strain. Rate constants were calculated from the reversion frequencies for several different heteroduplexes incubated at either 60 or 37 degrees C. For the 60 degrees C incubations, the hydrolytic deamination rate constants for mispairs in three different local sequence environments ranged from 8 x 10(-10) to 40 x 10(-10) s-1. For incubations at 37 degrees C, the rate constants were between 0.4 x 10(-10) and 1.3 x 10(-10) sec-1. At both temperatures and for all mispairs, these rate constants are significantly greater than deamination rate constants in properly matched Watson-Crick G.C base pairs and are similar to those constants determined for cytosine deamination in single-stranded DNA. Since deamination most likely occurs via a single-stranded intermediate, the data suggest that, at 37 degrees C, the T.C and C.C mispairs exhibit from 20% to 100% single-stranded character. We conclude that cytosine residues involved in a mispair in DNA are 1-2 orders of magnitude more prone to deaminate to uracil than are cytosines in double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1993

Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA.

The rate of bisulfite-induced deamination of cytosine to uracil in single-stranded (ss) DNA at physiological temperature and pH was monitored by a sensitive genetic assay. The assay is based on reversion of a mutation in the lacZ alpha gene of bacteriophage M13mp2 and employs ung- (NR9404) and ung+ (MC1061) bacterial strains which are isogenic except for uracil glycosylase activity. For ss DNA incubated with 1-50 mM bisulfite and transfected into an ung- cell strain, the reversion frequency increased linearly with time of incubation and with concentration of bisulfite. Of 54 revertants sequenced, all were C-->T transitions. Reduction in reversion frequency upon transfecting ss DNA into ung+ cells indicated that the majority of mutations were occurring via a uracil intermediate. Assuming that all revertants arose via uracil, the pseudo-first-order rate constant for deamination in 10 mM sodium bisulfite and 10 mM Hepes-NaOH, pH 7.4, at 37 degrees C as measured by transfecting into an ung- cell strain was 3.5 x 10(-10) s-1, as compared to a spontaneous background rate constant of 0.6 x 10(-10) s-1 in buffer alone.

Bacteriophages↗
PubMed1993

Echinomycin, a bis-intercalating agent, induces C-->T mutations via cytosine deamination.

Echinomycin, a bis-intercalating, antitumor drug, has been studied for its ability to induce the deamination of cytosine to uracil (C-->U) in double-stranded DNA. We have employed a sensitive lacZ alpha-complementation reversion assay to detect G.C-->A.T mutations at a number of sites in M13mp2 DNA to determine the extent to which distortions of DNA structure induced by echinomycin may affect C-->U rates. When double-stranded M13mp2 DNA with a 12-base target containing a CpG site was incubated at 37 degrees C, the reversion frequency of the echinomycin-treated DNA increased linearly over time, with a rate constant 3-fold greater than DNA incubated without echinomycin. Of the 11 ways that blue pseudo-revertants can occur in the target, 96% of the observed revertants arose from C-->T and tandem CC-->TT transitions, with 78% attributable to single-base C-->T changes at three sites. Transfection into ung+ cells decreased the reversion frequencies by 85% to near background levels, indicating that the increase in C-->T mutations was due to deamination of C to U. The cytosine deamination rate constants for the entire target at pH 6.0 and 37 degrees C were 1.2 x 10(-11) sec-1 for untreated DNA and 3.5 x 10(-11) sec-1 for echinomycin-treated DNA. The increase in C-->T mutation rates occurred at cytosines both proximal and distal to a CpG echinomycin-binding site. We hypothesize that this increase in deamination rate is due to a more open or single-stranded DNA structure caused by the echinomycin: DNA interaction.

Base Sequence↗
PubMed1993

Hypolipidemic activity of boronated nucleosides and nucleotides in rodents.

Base-boronated nucleoside and phosphate-boronated nucleotides were potent hypolipidemic agents in rodents, lowering both serum cholesterol and triglyceride levels. Rat VLDL and LDL cholesterol levels were generally reduced and HDL cholesterol levels were significantly elevated after 14 days dosing at 8 mg/kg/day. Tissue cholesterol, triglyceride and phospholipid levels were reduced by selected derivatives. Increased fecal excretion of lipids did not appear to be a mechanism by which these derivatives lowered serum lipids in rodents. Rather, the agents suppressed appetite and reduced the activities of rate-limiting enzymes for de novo lipid synthesis, specifically cytoplasmic acetyl CoA synthetase, squalene synthetase, and phosphatidylate phosphohydrolase with IC50 values of approximately 10(-5) m.

Animals↗
PubMed1992

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.

Base Composition↗
PubMed1992

The synthesis and anti-neoplastic activity of N2-isobutyryl-2'-deoxyguanosine-N7-cyanoborane derivatives.

N2-Isobutyryl-2'-deoxyguanosine-N7-cyanoborane derivatives were observed to be potent antineoplastic agents and to be active against a number of human tissue culture tumor cells, e.g. Tmolt3 leukemia, HeLa-S3 uterine carcinoma. Selective agents were active against colon adenocarcinoma, osteosarcoma and glioma growth. These agents preferentially inhibited both DNA and RNA synthesis of L1210 cells. De novo synthesis of purines was significantly inhibited at the regulatory sites of PRPP amido transferase and IMP dehydrogenase. Other sites of inhibition were thymidylate synthetase, OMP decarboxylase and thymidine kinases. The agents also significantly reduced deoxyribonucleotide levels and caused DNA strand scission.

Animals↗
PubMed1990

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.

Bacteriophages↗
PubMed1989

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA.

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

Chemical Phenomena↗
PubMed1987

Base stacking and molecular polarizability: effect of a methyl group in the 5-position of pyrimidines.

Substitution of a methyl group in the 5-position of pyrimidines increases melting temperatures and modifies biological properties of DNA. Increased DNA stability is often attributed to hydrophobic interactions between water and the methyl group. However, we present evidence that the major effect of methyl substitution is to increase the molecular polarizability of the pyrimidine, thereby increasing the base stacking. Experimentally determined base stacking interaction constants for free bases in water are shown to correlate well with calculated molecular polarizability and DNA melting temperatures.

Adenine↗