Biomedical subjects

M J Cairns

Publications and source records attributed to M J Cairns.

16 recordsLinked to original sources

PubMed2002

Catalytic DNA: a novel tool for gene suppression.

RNA, as an intermediate in the production of every gene encoded protein and the genetic material of many pathogenic viruses, presents an attractive target for both biological and therapeutic manipulation. Despite its extensive involvement in living systems, its chemical diversity based on four units is relatively low compared with protein. This provides the opportunity for a generic approach to targeting with specificity based on primary structure rather than complex higher order structures. This form of recognition occurs naturally in complementary nucleic acids, due to an ability to bind their single stranded target through Watson-Crick interactions. The most established nucleic acid based approach to gene suppression at the RNA level is through antisense oligodeoxynucleotides (ODNs). These compounds form heteroduplex with target RNA which are thought to either block its function or mediate its destruction by activation of RNase H. Alternatively, RNA can be targeted by catalytic RNA such as the hammerhead ribozyme. Ribozymes have the advantage of being equipped with their own RNA cleavage apparatus and are therefore independent of host nuclear protein activity. At present, the utility of ribozyme oligonucleotides is restricted by the relative difficulty synthesising active molecules with sufficient resistance to nuclease degradation. Recently the power of in vitro selection has been used to evolve catalytic DNA sequences with RNA cleavage specificity and activity rivalling the very best ribozymes, while maintaining the more robust chemistry of an ODN. These deoxyribozymes or DNAzymes have tremendous potential as gene suppression agents for both target validation and therapeutic applications. A number of studies evaluating the biological activity of these compounds have shown promising results. However, as with other oligonucleotide based strategies, future exploitation of this approach may depend on accessory technology to assist with the accessibility of a target which is folded by its own secondary structure and hidden within the intracellular compartment.

Animals↗

PubMed2000

Nucleic acid mutation analysis using catalytic DNA.

The sequence specificity of the '10-23' RNA-cleaving DNA enzyme (deoxyribozyme) was utilised to discriminate between subtle differences in nucleic acid sequence in a relatively conserved segment of the L1 gene from a number of different human papilloma virus (HPV) genotypes. DNA enzymes specific for the different HPV types were found to cleave their respective target oligoribonucleotide substrates with high efficiency compared with their unmatched counterparts, which were usually not cleaved or cleaved with very low efficiency. This specificity was achieved despite the existence of only very small differences in the sequence of one binding arm. As an example of how this methodology may be applied to mutation analysis of tissue samples, type-specific deoxyribozyme cleavable substrates were generated by genomic PCR using a chimeric primer containing three bases of RNA. The RNA component enabled each amplicon to be cleavable in the presence of its matching deoxyribozyme. In this format, the specificity of deoxyribozyme cleavage is defined by Watson-Crick interactions between one substrate-binding domain (arm I) and the polymorphic sequence which is amplified during PCR. Deoxy-ribozyme-mediated cleavage of amplicons generated by this method was used to examine the HPV status of genomic DNA derived from Caski cells, which are known to be positive for HPV16. This method is applicable to many types of nucleic acid sequence variation, including single nucleotide polymorphisms.

Capsid Proteins↗

PubMed2000

The influence of arm length asymmetry and base substitution on the activity of the 10-23 DNA enzyme.

A small oligodeoxyribonucleotide derived from in vitro selection has been shown to be capable of efficient sequence-specific cleavage of RNA at purine-pyrimidine junctions. As the reaction readily takes place under simulated physiologic conditions, this molecule described as the 10-23 general purpose RNA-cleaving DNA enzyme, has potential as a therapeutic agent. To further explore the character of this prototype, we examined the influence of base substitution and binding arm length asymmetry on its RNA cleaving activity. Surprisingly, substitution of the proximal nucleotide on the 3'-arm, to allow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired purine in the RNA substrate cleavage site was found to have only a subtle influence on the rate of catalysis, with a slight decrease observed when a G at this position was changed to an A, nucleotide substitution (G to C) in the core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and extent of helix asymmetry. Where the cleavage rate of one substrate was impaired by truncation of the deoxyribozymes 5'-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncation of the 3'-arm, however, had no effect on the reaction rate of the one substrate tested yet nearly halved the cleavage rate in another substrate.

Animals↗

PubMed2000

Catalytic nucleic acids: from lab to applications.

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.

Animals↗

PubMed1999

Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) and is known as the "10-23" general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3'-3'-internucleotide inversion at the 3'-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.

Animals↗

PubMed1999

Protein-DNA footprinting of the human epsilon-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents.

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein-DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy epsilon-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences - both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein-DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5'- and 3'-ends of the CCAAT box in K562 cells - this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the epsilonF1 DNA binding motif in K562 cells.

Bleomycin↗

PubMed1999

Target site selection for an RNA-cleaving catalytic DNA.

A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.

Animals↗

PubMed1998

The DNA sequence specificity of hedamycin damage determined by ligation-mediated PCR and linear amplification.

The DNA sequence specificity of hedamycin damage was compared using two techniques: Ligation-mediated PCR (LMPCR) and Linear Amplification (LA). The electrophoretic mobility of LA products were made comparable with LMPCR products by using a primer with a 27 bp non-binding 5'-extension. By direct comparison of the damage intensity (as represented by each of the methods), considerable bias was found in the LMPCR system with respect to LA--resulting in the under representation of lesions with T as the base 3' to the damage site. In view of this observation some caution should be exercised in the interpretation of data for DNA damage/repair specificity derived by LMPCR. In addition the extended primer LA method used in these experiments, could be applied to generate a dideoxy sequencing ladder for analysis of LMPCR products. This would negate the need to prepare Maxam-Gilbert chemical sequencing fragments for amplification through LMPCR.

Alkylating Agents↗

PubMed1998

Detection of protein-DNA interactions at beta-globin gene cluster in intact human cells utilizing hedamycin as DNA-damaging agent.

The DNA sequence specificity of hedamycin (HDM) damage was investigated in the single-copy human beta-globin gene cluster in an erythroid cell line, a nonerythroid cell line, and purified genomic DNA. The target DNA sequences for this study were the beta-globin gene locus control region (LCR) hypersensitive site 2 (HS-2) and the beta-globin gene promoter. The DNA fragments produced by HDM damage in these target sequences were selectively amplified by the ligation-mediated polymerase chain reaction (LMPCR) and analyzed at nucleotide resolution by DNA-sequencing gel electrophoresis. The DNA sequences damaged by HDM in the cellular environment were found to be similar to that observed in the purified genomic DNA. However, substantial differences did occur between the intensity of cellular and purified genomic DNA reaction products at discrete regions corresponding to transcription factor-binding motifs. This was most apparent in the LCR HS-2 at the tandem NF-E2/AP-1 motif, where the DNA damage activity of HDM was severely impaired. This motif has been shown to bind to the erythroid-specific nuclear factor-erythroid 2 (NF-E2) and the widely distributed activator protein-1 (AP-1). The HDM damage protection patterns or "genomic footprints" observed at this motif were probably caused by protein-DNA interactions with one or both of these transcription factors. This result indicates that the DNA damaging activity of HDM in cells is sensitive to bound nuclear factors. Because HDM can enter intact cells, where its DNA damaging activity is modulated by protein-DNA interactions, it may have application in genomic footprinting experiments.

Alkylating Agents↗

PubMed1997

Protein-DNA interactions in the human beta-globin locus control region hypersensitive site-2 as revealed by four nitrogen mustards.

Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other sites of protection and enhancement that corresponded to known transcription factor binding sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.

Aminacrine↗

PubMed1996

Influence of chromatin structure on bleomycin-DNA interactions at base pair resolution in the human beta-globin gene cluster.

The DNA sequence specificity of bleomycin was examined in human cells and in purified genomic DNA. In each case, DNA damage sites were determined at nucleotide resolution in the human single-copy beta-globin promoter and the locus control region (LCR) hypersensitive site 2 (HS-2). Exponential amplification of gene-specific genomic fragments was achieved by ligation-mediated PCR, and labeled reaction products were analyzed directly by sequencing gel electrophoresis. Bleomycin was found to cleave DNA preferentially at GC, GT, and GA dinucleotides. This study represents the first occasion that the sequence specificity of bleomycin has been determined in intact human cells at the single-copy gene level. The intensity of bleomycin damage sites in the LCR HS-2 was found to differ substantially between intact cells and purified DNA at putative transcription factor binding sites. Bleomycin activity was greatly reduced in cells at a tandem NF-E2/AP1 DNA sequence element. This footprint was strongest in K562 cells where the nuclear factor-erythroid 2 (NF-E2) is thought to bind. Protection and enhancement were also observed at other sequence elements in the HS-2 that associate with erythroid-specific and ubiquitous transcription factors. These results suggest that the activity of bleomycin is significantly reduced at the site of protein-DNA interactions in intact cells. This property of bleomycin is extremely useful in genomic "footprinting", where it has significant advantages over other commonly used agents.

Base Composition↗

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

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↗

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

Quantitation and three-dimensional reconstruction of Ch4 nucleus in the human basal forebrain.

The basal nucleus of Meynert, incorporating the Ch4 group of cholinergic neurons, was examined in six patients with no signs of neurological abnormalities. The ages of the patients ranged from 20 to 80 years. Despite a number of descriptions of these neurons, few age-related studies have been dedicated to the analysis of the entire anteroposterior extent of the nucleus. Staining with cresyl violet and acetylcholinesterase histochemistry, alone or in combination, was used to identify the cytoarchitectural organization of the Ch4. Computer-assisted morphometry was used for three-dimensional visualization and quantitation. The three-dimensional computer reconstructions revealed a continuous ribbon of neurons with a highly variable density. Four distinct subregions could be clearly identified in all cases by their cytoarchitecture and cellular morphology, although these subgroups were different to those previously described. There were no quantitative differences between the hemispheres in volume, density or cell number of the Ch4, although equivalent levels varied in area and density. The measures were similar in all cases with the exception of the case aged 80 years old. The data demonstrate individual variability in three dimensions and confirm previous studies that found only a mild decline of the Ch4 in old age.

Acetylcholinesterase↗

PubMed1984

The effects of phenylpropanolamine and other sympathomimetics on food consumption and motor activity in mice.

The effects of phenylpropanolamine on motor activity and on food intake were compared with those of S-amphetamine, ephedrine, 2-aminoindane and fenfluramine in groups of mice. Motor activity was additionally measured in mice pretreated with levodopa and benserazide, and food intake in mice pretreated with alpha-methyl-p-tyrosine. Amphetamine (2.5 mg kg-1) increased motor activity, phenylpropanolamine (10-40 mg kg-1) and 2-aminoindane (2.5-10 mg kg-1) decreased activity whilst ephedrine (2.5-40 mg kg-1) had a biphasic effect. Fenfluramine (10-40 mg kg-1) had negligible effect on activity. In mice pretreated with levodopa and benserazide both phenylpropanolamine and 2-aminoindane caused a massive increase in motor activity whilst fenfluramine's action was not affected in the same way. Whilst the anorectic action of fenfluramine was considerably potentiated in mice pretreated with alpha-methyl-p-tyrosine, that of amphetamine, ephedrine, 2-aminoindane and phenylpropanolamine was either unaffected or initially antagonized. It is concluded that the mechanisms of motor and anorectic actions of phenylpropanolamine are similar to those of amphetamine.

Animals↗