Toxic effects of herbal medicines and food supplements.
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Foodborne Diseases↗
Biomedical subjects
V Murray
Publications and source records attributed to V Murray.
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↗
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↗
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↗
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↗
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↗
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↗
Toxicity of Chinese herbal remedies.
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Adult↗
Direct PCR sequencing of dystrophin polymorphic CACA alleles after purification to remove shadow bands.
A method is described that allows the sequencing of polymerase chain reaction (PCR) products containing CACA repeats. The method was tested using a DNA polymorphism that exists at the 3' end of the dystrophin gene. This polymorphism consists of a variation in the length of a CACA dinucleotide repeat. Four alleles from a total of 16 individuals were sequenced at this locus after the DNA sequence had been amplified by the PCR. Five examples of each of the common alleles were sequenced. For each allele all five sequences were the same. The only example of a rare allele was also sequenced. The PCR products of DNA sequences containing dinucleotide repeats consist of a number of bands differing by 2 bp below the most intense main band. Previously, direct sequencing of the PCR products lead to ambiguities and smearing at and above the CACA repeat. In this paper, the main PCR band was cut out of a sequencing gel and directly sequenced to give a clear DNA sequence. Our results indicate that for a particular allele, all individuals had exactly the same DNA sequence. This implies that with the appropriate choice of oligonucleotide primers, polymorphisms could be detected without electrophoresis.
Alleles↗
Hospital response to a chemical incident: report on casualties of an ethyldichlorosilane spill.
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Accidents, Traffic↗
Are transposons a cause of ageing?
A hypothesis for ageing is proposed based on the properties of transposons. During the process of transposition, one copy of the DNA sequence generally remains at the same position while the other copy moves to another location in the genome. In this manner the DNA sequence of the transposon is effectively duplicated. With time the number of transposons increases exponentially and since in their new location they can inactivate an essential gene, they will eventually kill a cell line or organism. Thus transposons could be a cause of ageing. This hypothesis is attractive because it can explain many of the properties of senescent cells. Other processes capable of DNA self-duplication (e.g., reverse transcription) could also contribute to the increase in transposable DNA sequences.
Aging↗
Radiation sensitization by an iodine-labelled DNA ligand.
An iodinated DNA ligand, iodo Hoechst 33258, which binds in the minor groove of DNA, enhances DNA strand breakage and cell killing by UV-A irradiation. The sites of UV-induced strand breaks reflect the known sequence specificity of the ligand.
Animals↗
Improved double-stranded DNA sequencing using the linear polymerase chain reaction.
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DNA↗
The degree of ultraviolet light damage to DNA containing iododeoxyuridine or bromodeoxyuridine is dependent on the DNA sequence.
The sequence selectivity of 300 nm ultraviolet light damage to DNA containing bromodeoxyuridine or iododeoxyuridine was examined on DNA sequencing gels. This was accomplished using a system where an M13 template was employed to direct synthesis of DNA in which thymidine was fully substituted with bromodeoxyuridine or iododeoxyuridine. The sites of damage corresponded to the positions of analogue incorporation. The extent of damage varied considerably at different sites of cleavage and ranged from the undetectable to over fifteen times the limit of detection (as assessed by laser densitometer scans). Strong damage sites had the "consensus" sequence CTT while sites of no detectable damage had the "consensus" sequence GTR. Bromodeoxyuridine and iododeoxyuridine had the same sites of damage although the extent of damage varied at different sites and bromodeoxyuridine damage was slightly greater than iododeoxyuridine. DNA containing thymidine was not damaged to any detectable level in this system with 300 nm ultraviolet light. The use of three closely related DNA sequences as targets for damage confirmed that (1) the sites of analogue incorporation are the cause of ultraviolet damage; and (2) that the neighbouring DNA sequence is an important parameter in determining the extent of damage. It is proposed that the microstructure of DNA--in particular the distance between the 5-carbon of the pyrimidine base (which is attached to the halogen) and hydrogen on the 2' carbon of the 5'-deoxyribose--ultimately determines the degree of cleavage with large distances giving a small degree of damage and smaller distances a large degree of damage.
Adenosine↗
Medical planning for toxic releases into the community: the example of chlorine gas.
Emergency planning for a major accidental release of chlorine gas from industrial installations into the community is outlined for emergency services and hospitals. Realistic planning has been made possible with the advent of computer models for gas dispersion which may be used to estimate the numbers of deaths and casualties, according to their severity. For most purposes sufficient accuracy may be obtained by using a small number of computer analyses for the most serious reasonably foreseeable events under typical day and night weather conditions, and allowing for the emergency response to be scaled up or down according to the size of an actual release. In highly populated areas triage should be preplanned to deal with a large number of victims; field stations will be needed for the treatment and observation of minor casualties. The management and treatment of casualties is summarised. The best protection against a gas cloud is afforded by buildings whose windows, doors, and ventilation systems have been closed. Hospitals in the vicinity of an installation should draw up plans to protect patients and staff. Coordination in a disaster will require toxicological and epidemiological expertise and hospital plans should allow for this.
Accidents, Occupational↗
The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions.
The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.
Base Sequence↗
Sequence specificity of 125I-labelled Hoechst 33258 damage in six closely related DNA sequences.
The sequence selectivity of [125I]Hoechst 33258 in six 340 base-pair DNA sequences has been investigated. [125I]Hoechst 33258, which is a bis-benzimidazole and binds to the minor groove of B-DNA, preferentially binds to A + T-rich regions of DNA. Six out of nine strong binding sites contained four or more consecutive A.T base-pairs, while the other three strong binding sites were AAGGATT, TATAGAAA (the peak of damage was in the run of 3 A residues) and AAA. One of the six weak binding sites had five consecutive A.T base-pairs, two of the weak binding sites had three, and three did not have any. In addition to genomic 340 base-pair alpha RI-DNA (which is a tandem repeat in human cells), five 340 base-pair alpha RI-DNA clones were generated that differed from the genomic "consensus" sequence by a number of random base alterations. The effect of these base changes on the sequence specificity of [125I]Hoechst 33258 damage indicated that of the base changes that interrupted 14 binding sites, six decreased and eight did not change the extent of damage, while two sites changed position. Of the base alterations that augmented 17 binding sites, five increased, two decreased and ten did not alter the degree of cleavage, while ten sites changed position. It was concluded from the data that, while runs of consecutive A.T base-pairs was the most important parameter that determines [125I]Hoechst 33258 binding, other factors including position in the DNA sequence, nearest neighbour and long-range interactions were also important.
Autoradiography↗
Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells.
Using polyacrylamide/urea DNA sequencing gels, the DNA sequence selectivity of 125I-labelled Hoechst 33258 damage has been determined in intact human cells to the exact base-pair. This was accomplished using a novel procedure with human alpha RI-DNA as the target DNA sequence. In this procedure, after size fractionation, the alpha RI-DNA is selectively purified by hybridization to a single-stranded M13 clone containing an alpha RI-DNA insert. The sequence specificity of [125I]Hoechst 33258 was indistinguishable in intact cells from purified high molecular weight DNA; and this is surprising considering the more complex environment of DNA in the nucleus where DNA is bound to nucleosomes and other DNA binding proteins. The ligand preferentially binds to DNA sequences which have four or more consecutive A.T base-pairs. The extent of damage was measured with a densitometer and, relative to the damage hotspot at base-pair 94, the extent of damage was similar in both purified high molecular weight DNA and intact cells. [125I]Hoechst 33258 causes only double-strand breaks, since single-strand breaks or base damage were not detected. These experiments represent the first occasion that the sequence specificity of a DNA damaging agent, which causes only double-strand breaks, has been determined to the exact base-pair in intact cells.
Autoradiography↗