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Biomedical subjects

V Murray

Publications and source records attributed to V Murray.

At least 37 records · Page 2Linked to original sources
PubMed1994

Comparison of the sequence specificity of cis-diamminedichloroplatinum (II) damage in guanine- and 7-deazaguanine-containing DNA.

The N7 of guanine is thought to be the primary target for adduct and crosslink formation between cisplatin and DNA. However, reactive sites in DNA other than the N7 of guanine may also participate in the formation of adducts with cisplatin. The possibility that these interactions arise and form DNA polymerase blocking lesions was investigated by primer extension reactions with Taq DNA polymerase. To differentiate between damage produced at relatively weak sites from those formed at the N7 of guanine, a modified DNA template was synthesised with the N7 of guanine replaced with a carbon atom. This was achieved in a PCR designed to incorporate 7-deazaguanine instead of normal guanine. The sequence specificity of cisplatin damage in the modified and unmodified DNA substrates was compared (after linear amplification) by DNA sequencing gel analysis. For concentrations of cisplatin (1 to 5 microM) that induce blocking lesions in normal DNA, no significant damage was observed in the modified DNA. This confirmed that the N7 of guanine is the major site of adduct formation in normal DNA. At higher concentrations of cisplatin (50 microM and 100 microM), lesions were found at AA dinucleotides and other novel sites in the modified DNA. These results indicate that the N7 of guanine is not required in the formation of some cisplatin adducts.

Base Sequence↗
PubMed1994

Ultraviolet light-induced cleavage of DNA in the presence of iodoHoechst 33258: the sequence specificity of the reaction.

IodoHoechst 33258 sensitizes DNA to cleavage by near ultraviolet light (UV-A). Following an earlier study which showed that the UV-induced cleavage is at discrete locations corresponding to the ligand binding sites, this paper reports a more extensive analysis of the sequence specificity of cleavage. The experiments involved use of double-stranded DNA synthesised on primed M13 templates. Analysis of cleavage in a 280bp sequence in M13mp18 and a 310bp sequence in three M13mp9 clones ('alpha-32', 'alpha-82' and 'alpha-22') derived from human alpha-DNA, showed that for all of the twenty-nine strong and very strong damage sites, cleavage was at the 3' end of a run of three or more consecutive AT base pairs. The extent of cleavage was higher for sites with consecutive Ts than for consecutive As, and greatest for the sequence cTTTTca. Comparison of three closely-related alpha-DNA clones enabled assessment of single bp changes and essentially confirmed the results of detailed analysis of binding cleavage sites in mp18 and alpha-32. Decreasing the input ratio of iodoHoechst/per bp DNA from 0.13 to 0.013 altered the sequence specificity, and sites possessing only three consecutive AT bps were generally not cleaved. The contributions of both the strength of ligand binding and the efficiency of photolytic cleavage to the overall extent of cleavage by UV/iodoHoechst are discussed, in view of the potential utility of the ligand as a probe of DNA conformation.

Bacteriophage M13↗
PubMed1994

Specificity in splicing.

Considerable information about the process of pre-mRNA splicing has accumulated, but the mechanism by which highly accurate splicing is achieved is unresolved. Fifteen years ago we proposed that accuracy in splicing might depend on small RNA molecules (splicer RNAs) which hybridise across adjacent exon termini, or intron termini. Gene expression, including alternative splicing, could be controlled by the transcription of specific splicer RNA genes. We re-assess our model here, in the light of subsequent developments.

Alternative Splicing↗
PubMed1994

An outbreak of illness among schoolchildren in London: toxic poisoning not mass hysteria.

STUDY OBJECTIVE: To determine the cause of an outbreak of acute gastrointestinal illness that occurred shortly after lunch in children attending a school in London, UK. DESIGN: A questionnaire survey of children at the affected school was carried out on the day after the incident. Microbiological, environmental, and toxicological investigations were also undertaken. SETTING: A school in London, UK. PARTICIPANTS: Altogether 374/468 (80%) of the children who had eaten lunch at the school on the day of the incident completed a questionnaire. MAIN RESULTS: There was a significant association between illness and the consumption of raw cucumber (relative risk = 6.1; 95% confidence interval 2.2, 16). Microbiological investigation of the foods served at lunch did not show any pathogens and toxicological investigations suggested that the cucumbers were contaminated by a pesticide. CONCLUSIONS: Although the outbreak displayed several typical features of mass psychogenic illness, the most probable cause was a toxic chemical present in cucumber served at lunch. Those responsible for investigating outbreaks of illness should be aware of the possible toxicological causes and the appropriate modes of investigation. They should be wary of too readily attributing a psychogenic cause to unusual outbreaks of acute illness in schoolchildren.

Acute Disease↗
PubMed1994

Toxicological problems resulting from exposure to traditional remedies and food supplements.

The National Poisons Unit, London, carried out a pilot survey to investigate the frequency and severity of adverse effects/toxicity from exposure to traditional medicines and food supplements reported to the Unit. Enquiries related to suspected poisoning events were reviewed retrospectively from January 1983 to March 1989, and prospectively in 1991. Further information about cases identified by the prospective review was obtained, when appropriate, by follow-up questionnaire, clinical consultation by a consultant toxicologist, toxicological analyses of samples from patients and from products, and botanical identification of dried plant material. In total, 5536 enquiries were identified. Symptoms were reported in 657 (12%) of these. There was a large number of reports of accidental ingestion of vitamin preparations by children under 5 years. Appropriate assessment was possible in only relatively few cases, due to insufficient documentation, and poor labelling of certain products. A probable link between exposure and adverse effects was identified in 42 cases, and was highly probable in two. Heavy metal poisoning resulting from use of contaminated traditional remedies was confirmed in 5 cases. There was evidence that some patients took excessive doses of food supplements, without realising that this might result in toxic effects. The results of this pilot study suggest that there is a need for further surveillance to provide an appropriate risk assessment of food supplements and herbal remedies, improved quality control and labelling of these products, and increased awareness of their potential hazard.

Adolescent↗
PubMed1994

Dideoxy genomic sequencing of a single-copy mammalian gene using more than two hundred cycles of linear amplification.

We explored the possibility of using a large number of reaction cycles to achieve genomic DNA sequencing in single-copy mammalian genes. A section of the beta-globin promoter was sequenced directly from a sample of human white blood cell DNA. The sequencing fragments were extended from a single, 5'-terminal-labeled oligonucleotide primer by Taq DNA Polymerase in the presence of dideoxyribonucleotides and more than 200 thermal cycles of denaturation, annealing and extension. The labeled sequencing fragments produced in this linear amplification were detected after electrophoresis on a DNA-sequencing gel. We propose that this scheme could be adopted in some instances as an alternative to conventional sequencing.

Base Sequence↗
PubMed1994

A polymerase chain reaction on human alphoid DNA produces a characteristic ladder of bands.

A polymerase chain reaction (PCR) was performed on the tandemly repeated human alphoid DNA sequence. A characteristic ladder of bands was produced which differed by 171 (or 340) base pairs. This laddering phenomenon occurs because the two oligonucleotides can bind within the same monomer or in neighbouring monomers. In the latter case a larger PCR product is produced which is 171 bp or a multiple of 171 base pairs greater in size. The alphoid DNA PCR is very sensitive because a repeated sequence is amplified. Hence it could have applications in archaeology or forensic science.

Base Composition↗
PubMed1993

The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR.

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.

Base Sequence↗
PubMed1993

Detection of polymorphisms using thermal cycling with a single oligonucleotide on a DNA sequencing gel.

A method is described for the detection of restriction fragment length polymorphisms (RFLPs) in single copy genes in mammalian cells using one 5'-labelled oligonucleotide. This linear amplification (LA) method employs a single oligonucleotide as primer, which is extended by Taq DNA polymerase up to a restriction enzyme cleavage site. The products are arithmetically amplified by thermal cycling. The size of the products are determined by the sequence of the oligonucleotide and the position of the restriction enzyme cleavage site. Hence, an RFLP can be observed by measuring the size of the products. Polymorphisms which differ in size by a small number of base pairs, as are found in (CA)n repeats, are especially suitable for analysis by the LA procedure since the products are run on DNA sequencing gels. A number of genes were examined by the procedure and all produced a satisfactory signal including GC-rich template. It is proposed that the LA method would be suitable for large-scale genetic linkage analysis. The LA procedure has many advantages including the ability to multiplex signals under the same conditions, and lower cost since only one primer is needed.

Evaluation Studies as Topic↗
PubMed1993

A multidisciplinary investigation of a cluster of deaths on a paediatric intensive care unit.

During late December 1989 and early January 1990, a cluster of six unexplained deaths occurred on a paediatric intensive care unit (PICU) among children with congenital heart disease who had undergone cardiac surgical procedures. The children were all aged three years or less. In each case death was preceded by an unexpected increase in ventilatory pressure requirement followed by the development of a similar pulmonary shadowing on chest radiography. The radiological abnormality was felt to be consistent with a pneumonitis associated with some small airway disease. The clustering of these deaths, occurring in a similar unusual manner, was felt to constitute an outbreak warranting investigation. An Incident Committee was established to plan and manage a large multidisciplinary investigation during which the unit was temporarily closed. Following extensive investigation no bacterium, virus, fungus or other pathogen, toxic agent, or any other explanation for the cluster of deaths could be found. The possibility that the cluster occurred by chance remains although this was felt to be unlikely.

Cause of Death↗
PubMed1993

Sequence-specific DNA damage using iodine-125-labeled antisense oligonucleotides.

A procedure is described that cleaves single-stranded DNA with sequence specificity. This process involves attaching a DNA damaging agent to an oligonucleotide. This oligonucleotide delivers the DNA damaging agent, iodine-125, to a specific DNA sequence by complementary hybridization. 5-[125I]Iodo-2-deoxycytidine 5'-triphosphate was enzymatically incorporated into an oligonucleotide that was designed to hybridize to a single-stranded DNA target. 125I decays by electron capture and causes breaks in the target DNA. These breaks were observed on a DNA sequencing gel. After 22 days of exposure to the 125I-labeled oligonucleotide, significant damage was observed within 1 to 2 bases of the expected site of hybridization. Densitometry showed that after 48 days the amount of damage had approximately doubled. This method facilitates easy design and testing of oligonucleotides that could potentially be used to inactivate gene expression in a wide variety of organisms.

Autoradiography↗
PubMed1992

An investigation of the sequence-specific interaction of cis-diamminedichloroplatinum(II) and four analogues, including two acridine-tethered complexes, with DNA inside human cells.

The sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II) (cisplatin) and four analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification system. The primer extension is inhibited by the drug-DNA adducts, and hence the sites of these lesions can be analyzed on DNA sequencing gels. The repetitive alphoid DNA was used as the target DNA in human cells. A comparison was made between adduct formation in human cells and in purified DNA. The sequence-specific position and relative intensity of damage was similar in both systems for cisplatin, dichloro(ethylenediammine)platinum(II) (PtenCl2), and N-[3-N-(ethylenediamino)propyl]acridine-4-carboxamidedichloropl atinum(II) (4AcC3PtenCl2). However, no DNA damage could be detected in cells for trans-diamminedichloroplatinum(II) (transPt) or N-[3-N-(ethylenediamino)propyl]acridine-2-carboxamide-dichloroplat inum(II) (2AcC3PtenCl2) despite the ability of these latter analogues to damage purified DNA. Cisplatin, PtenCl2, and 4AcC3PtenCl2, which significantly damaged DNA inside cells, also show antitumor activity in mouse models. However, transPt and 2AcC3PtenCl2, which did not detectably damage DNA inside cells, did not show such antitumor activity. This correlation between intracellular DNA damaging ability and in vivo antitumor activity indicates the potential use of the human cells/Taq DNA polymerase/linear amplification technique as a convenient method for screening new cisplatin analogues for useful chemotherapeutic activity.

Acridines↗
PubMed1992

The use of Taq DNA polymerase to determine the sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II), acridine-tethered platinum(II) diammine complexes or two analogues.

cis-Diamminedichloroplatinum(II) (cisplatin) forms adducts with DNA. The sequence specificity of formation of cisplatin adducts with plasmid DNA was investigated using Taq DNA polymerase. This procedure involved the extension of an oligonucleotide primer by Taq DNA polymerase up to the cisplatin adduct. Using thermal cycling, this process is repeated many times in order to amplify the signal. The products of this linear amplification can then be examined on DNA sequencing gels, and the sequence specificity of cisplatin adduct formation can be determined to the exact base pair. In the pUC8 plasmid, the sequences that produced the most intense damage sites (as determined by densitometry) were runs of two or more Gs. Adducts could also be detected at GA, AG, and GC dinucleotides. Four other cisplatin analogues were also tested in the system. Two of these analogues contained an attached intercalating chromophore, and the strong damage with these compounds was similar to that found for cisplatin, but the medium and weak damage tended to be different. Weak damage was also detected with trans-diamminedichloroplatinum(II). With this compound, a large number of the damage sites were at the CG dinucleotide. This technique represents a simple, accurate, and quick method for determining the sequence specificity of damage for a cisplatin analogue in any DNA sequence.

Autoradiography↗