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Biomedical subjects

K R Fox

Publications and source records attributed to K R Fox.

At least 91 records · Page 5Linked to original sources
PubMed1995

Synthesis, DNA binding, footprinting and in vitro antitumour studies of a meta-hydroxy analogue of Hoechst 33258.

An analogue of Hoechst 33258, bearing a phenolic hydroxyl group in the meta rather than para position, was designed using molecular graphics to introduce hydrogen-bonding potentials between this OH group and the C = O group of cytosine-9 and the NH2 group of guanine-4', of the opposite strand of the B-DNA duplex, d(CGCGAATTCGCG)2. This derivative (meta-Hoechst) was synthesized in seven steps and characterized. Its binding to DNA was assessed by measurements of melting temperatures (Tm) and found to be similar in strength and AT preference to the parent Hoechst 33258 at this gross level. The AT preference of meta-Hoechst and Hoechst 33258 was probed further using hydroxyl radical footprinting on the tyrT DNA fragment, for which clear footprints were detected at AAT, AAA and ATAT runs, as for netropsin and distamycin. Hydroxyl radical footprinting was carried out on a trimer of CGCGAATTCGCG cloned into a longer DNA fragment, for which clear footprints for both Hoechst 33258 and meta-Hoechst were detected in regions with four or more contiguous AT base pairs. Three cell lines derived from haematological malignancies were more sensitive to both Hoechst 33258 and meta Hoechst than lines derived from solid tumours, but there was no significant difference between the activity of these two Hoechst derivatives.

Animals↗
PubMed1994

Effect of a triplex-binding ligand on parallel and antiparallel DNA triple helices using short unmodified and acridine-linked oligonucleotides.

We have used DNase I footprinting to investigate the effect of a triplex-binding ligand on the formation of intermolecular DNA triple helices at target sites that have been cloned into longer DNA fragments. In the presence of a triplex-binding ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine ), the concentrations of T5C5 and C5T5 required to generate DNase I footprints at the target sites A6G6.C6T6 and G6A6.T6C6, respectively, are reduced by at least 100-fold. Complexes with the acridine-linked oligonucleotides Acr-T5C5 and Acr-C5T5 are stabilized to a much lesser extent and produce footprints at concentrations similar to those of the unmodified oligonucleotides in the presence of the ligand. The stabilizing effects of acridine modification or the addition of a triplex-binding ligand are not additive. The position and length of the footprints produced by Acr-T5C5 and T5C5 at the target sequence A6G6.C6T6 are unaffected by the ligand. In contrast, footprints at the target site G6A6.T6C6 appear 3-4 bases shorter in the presence of the ligand, when viewed from the pyrimidine strand, and 1-2 bases longer on the purine strand. These results are explained by suggesting that the compound binds at T.AT triplets and prevents the transmission of any DNA structural changes into the flanking duplex. The compound has a smaller stabilizing effect on short antiparallel triplexes consisting of G.GC and T.AT triplets. Binding of Acr-G5T5 to A6G6.C6T6 is enhanced slightly by the compound, which increases the apparent footprinting site, probably by preventing fraying at the 3'-end of the third strand. The compound does not promote the binding of G5T5 to A6G6.C6T6 or that of Acr-T5G5 and T5G5 to G6A6.T6C6.

Acridines↗
PubMed1994

Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.

We have examined the formation of alternate strand triple-helices at the target sequence A11(TC)6.(GA)6T11 using the oligonucleotides T11(AG)6 and T11(TG)6, by DNase I footprinting. These third strands were designed so as to form parallel T.AT triplets together with antiparallel G.GC and A.AT or T.AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 microM) in the presence of manganese, only T11(TG)6 forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 microM). Examination of the interaction of (AG)6 and (TG)6 with half the target site confirmed that the complex containing A.AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)6 was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T.AT triplet is less stable that G.GC. We suggest that, within the context of G.GC triplets, the rank order of antiparallel triplet stability is A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.

Base Composition↗
PubMed1994

DNase I footprinting of triple helix formation at polypurine tracts by acridine-linked oligopyrimidines: stringency, structural changes and interaction with minor groove binding ligands.

We have investigated the binding of short (10 base) acridine-linked triplex-forming oligonucleotides to the target sequence A6G6.C6T6 by DNase I footprinting. Specific binding is detected at low pH (< 6.0) for 5'-Acr-T5C5 and 5'-Acr-5BrU5(5Me)C5. The sequence T5C5, lacking the acridine modification, binds less strongly, though specific binding is still evident. 5'-Acr-T5C5 produces footprints at slightly lower concentrations than 5'-Acr-5BrU5(5Me)C5. All three oligonucleotides produce enhanced DNase I digestion at the 3'-end of the target purine strand, suggesting that there is a DNA structural change at the triplex-duplex boundary. Target sequences AnG4A and TAC3Tn, containing one and two triplex mismatches, show no interaction with the acridine-free oligonucleotide, but bind the acridine-linked oligonucleotides. In these secondary binding modes the third strand is positioned so that the mismatches are located at the 3'-end of the oligonucleotide. Mithramycin and distamycin, binding in the minor groove to GC- and AT-rich sequences respectively, abolish triple helix formation.

Acridines↗
PubMed1994

A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply.

BACKGROUND: Early in the spring of 1993 there was a widespread outbreak of acute watery diarrhea among the residents of Milwaukee. METHODS: We investigated the two Milwaukee water-treatment plants, gathered data from clinical laboratories on the results of tests for enteric pathogens, and examined ice made during the time of the outbreak for cryptosporidium oocysts. We surveyed residents with confirmed cryptosporidium infection and a sample of those with acute watery diarrhea consistent with cryptosporidium infection. To estimate the magnitude of the outbreak, we also conducted a survey using randomly selected telephone numbers in Milwaukee and four surrounding counties. RESULTS: There were marked increases in the turbidity of treated water at the city's southern water-treatment plant from March 23 until April 9, when the plant was shut down. Cryptosporidium oocysts were identified in water from ice made in southern Milwaukee during these weeks. The rates of isolation of other enteric pathogens remained stable, but there was more than a 100-fold increase in the rate of isolation of cryptosporidium. The median duration of illness was 9 days (range, 1 to 55). The median maximal number of stools per day was 12 (range, 1 to 90). Among 285 people surveyed who had laboratory-confirmed cryptosporidiosis, the clinical manifestations included watery diarrhea (in 93 percent), abdominal cramps (in 84 percent), fever (in 57 percent), and vomiting (in 48 percent). We estimate that 403,000 people had watery diarrhea attributable to this outbreak. CONCLUSIONS: This massive outbreak of watery diarrhea was caused by cryptosporidium oocysts that passed through the filtration system of one of the city's water-treatment plants. Water-quality standards and the testing of patients for cryptosporidium were not adequate to detect this outbreak.

Adolescent↗
PubMed1994

Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.

Acridines↗
PubMed1994

Formation of DNA triple helices incorporating blocks of G.GC and T.AT triplets using short acridine-linked oligonucleotides.

We have used DNase I footprinting to assess triple helix formation at target sites containing the sequences A6G6.C6T6 and G6A6.T6C6. These sequences can be recognized by the acridine-linked oligopyrimidines Acr-T5C5 and Acr-C5T5 respectively at low pH, using well-characterised T.AT and C+.GC triplets. At pH 7.5 A6G6.C6T6 is specifically bound by Acr-G5T5, utilising G.GC and T.AT triplets in which the third strand runs antiparallel to the purine strand of the duplex. This interaction requires the presence of magnesium ions. No interaction was detected with Acr-T5G5, an oligonucleotide designed to form parallel G.GC and T.AT triplets. In contrast neither Acr-T5G5 nor Acr-G5T5 produced DNase I footprints with the target sequence G6A6.T6C6. These results suggest that, in an antiparallel R.RY triple helix, the T.AT triplet is weaker than the G.GC triplet. We find no evidence for the formation of structures containing parallel G.GC triplets.

Acridines↗
PubMed1994

Light-activated cleavage of DNA by cobalt-bleomycin.

We have studied the light-activated cleavage of DNA by cobalt-bleomycin using a series of synthetic DNA fragments containing (AT)n and (GC)n. This cleavage reaction requires high concentrations of the antibiotic and appears to be a stoichiometric process rather than a catalytic process. We find that, in common with the iron-complex, cobalt-bleomycin can cleave at ApT steps within regions of alternating AT residues; ApT steps within other sequences including (AAT)n. (ATP)n are not good substrates for cobalt-bleomycin cleavage. Some repetitive regions display an alternating pattern of cleavage products, revealing the preferred arrangement of ligand molecules along a saturated DNA lattice. A similar repetitive pattern is found for diethylpyrocarbonate modification and hydroxyl-radical cleavage. Although cleavage of ApT and GpC proceeds at equivalent rates, the data suggest that bleomycin binds more tightly to the latter. Adenine residues on the 3' side of both GpC-cleavage and ApT-cleavage sites are rendered more reactive to diethylpyrocarbonate, consistent with a ligand-induced alteration in local DNA structure. The cobalt-bleomycin-binding site consists of not more than four base pairs, and may be as small as three base pairs.

Base Sequence↗
PubMed1994

Interaction of mithramycin with isolated GC and CG sites.

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)n and An.Tn using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)n appear to be bound better than those surrounded by An.Tn. Although no footprints are produced at T9GCA9 and T15CGA15, DNase I cleavage is enhanced within the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)n.(CT)n.

Adenine↗
PubMed1993

Triple helix formation at A8XA8.T8YT8.

We have examined the formation of DNA triple helices between the oligonucleotides T8XT8 (X = A,C,G,T) and DNA fragments containing the target sequences A8XA8.T8YT8 (X = T,C,G; Y = A,G,C), by DNase I footprinting. We find that A8GA8.T8CT8 yields a footprint with T8CT8 and shows a weaker interaction with T17 and T8GT8. A8CA8.T8GT8 yields a footprint with T17, and shows weaker interaction with T8CT8. A8TA8.T8AT8 yields a footprint with T8GT8 and shows weaker interaction with T17. Each of the successful complexes is characterised by enhanced DNase I cleavage at the 3' end of the purine strand of the target, as well as protection at the 5' end. We have been unable to from triplexes with third strands of the type A8XA8.

DNA↗
PubMed1993

DNA-sequence binding preference of the GC-selective ligand mithramycin. Deoxyribonuclease-I/deoxyribonuclease-II and hydroxy-radical footprinting at CCCG, CCGC, CGGC, GCCC and GGGG flanked by (AT)n and An.Tn.

We have used hydroxy-radical and deoxyribonuclease-I footprinting to probe the interaction of mithramycin with DNA fragments containing the sequences (AT)10X(AT)10 (X = CCCG, CCGC or CGGC) and A14GCCCT15. As expected the drug produces clear footprints located around the central four GC base pairs. The exact position of the footprint is different for the four sequences; the footprint with CCCG is displayed by two base pairs in the 5' direction relative to GCCC. These variations are explained by suggesting that mithramycin avoids the dinucleotide CG and binds better to GG/CC than GC. Although there is little change in deoxyribonuclease-I cleavage of the surrounding blocks of (AT)n, cleavage by deoxyribonuclease II is markedly enhanced and certain thymines on the 5' side of the ligand-binding site become hyperreactive to hydroxy-radical attack. Adjacent regions of An.Tn show enhanced rates of deoxyribonuclease-I cleavage in the presence of the antibiotic.

DNA↗
PubMed1993

Interaction of mithramycin with DNA fragments complexed with nucleosome core particles: comparison with distamycin and echinomycin.

We have studied the sequence-specific interaction of mithramycin with nucleosome core particles which have been reconstituted with various DNA fragments. Mithramycin binds to these DNAs without disrupting the integrity of the nucleosome and produces clear DNase I footprints centered around GC-rich regions. In some instances, the footprints produced on free DNA are resolved into two or more smaller sites when the DNA is complexed with the nucleosome core. In a few cases, novel footprints are produced in sequences which did not bind the drug in free DNA samples. The results are explained by suggesting mithramycin binds to GC-rich regions in which the minor groove faces away from the protein core, and which possess a wider than normal narrow groove on account of their location. Hydroxyl radical footprinting and diethyl pyrocarbonate modification confirm that mithramycin does not affect the rotational positioning of the nucleosome-bound DNA. Although distamycin and echinomycin induce novel DNase I digestion products in nucleosomal DNA which are consistent with the proposed change in DNA positioning [Low, C. M. L., Drew, H. R., & Waring, M. J. (1986) Nucleic Acids Res. 14, 6785-6801], hydroxyl radical footprinting and diethyl pyrocarbonate modification suggest these ligands do not change the rotational positioning of the DNA on the nucleosome cores.

Base Sequence↗
PubMed1993

DNA structure influences sequence specific cleavage by bleomycin.

We have examined the cleavage of several synthetic DNA sequences by iron(II)-bleomycin. We find that, although bleomycin cuts mixed sequence DNAs with a preference for GC = GT > GA >> GG, it efficiently cleaves regions of (AT)n cutting exclusively at ApT, not TpA. Isolated ApT steps show very little cleavage while blocks of three or more contiguous ATs are cut as efficiently as GpT. This cleavage is specific for (AT)n, since sequences of the type (TAA)n.(TTA)n and (ATT)n.(AAT)n are hardly cut at all. No cleavage is observed at ApC or CpA within sequences of the type (AC)n.(GT)n; regions of An.Tn are also not cut. Although the cobalt-bleomycin complex (which binds to but does not cleave DNA) yields good DNase I footprints at GT and GC sites, no footprints are observed within (AT)n, suggesting that although the cleavage reaction is efficient, the binding affinity is relatively weak. We propose a model in which bleomycin cleavage is determined by local DNA structure, while strong binding requires the presence of a guanine residue.

Base Composition↗
PubMed1993

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.

Base Sequence↗
PubMed1993

Sequence-specific binding of [N-MeCys3,N-MeCys7]TANDEM to TpA.

The sequence selective binding of [N-MeCys3,N-MeCys7]TANDEM to DNA has been studied by footprinting experiments on DNA fragments containing the self-complementary sequences CGCGATATCGCG, CGCGTATACGCG, CGCGTTAACGCG and CGCGAATTCGCG. DNAase I and micrococcal nuclease reveal drug-induced footprints with the central sequences ATAT, TATA and TTAA, but not AATT, suggesting that the ligand binds to the dinucleotide TpA. The ligand renders certain adenines hyper-reactive to diethyl pyrocarbonate. These are observed with ATAT, TATA and TTAA, but not AATT, and are located both within, and distal to, the TpA-binding sites.

Base Sequence↗