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Biomedical subjects

A D Malcolm

Publications and source records attributed to A D Malcolm.

At least 73 records · Page 4Linked to original sources
PubMed1984

Supercoiling and the mechanism of restriction endonucleases.

We have used topoisomerase I in the presence of netropsin and ethidium bromide to generate DNA molecules of varying superhelical density. Digestion by endonuclease EcoRI is sensitive to supercoiling, being maximal for the relaxed form. Endonucleases AvaI and BamHI, by contrast, are relatively unaffected. The results are interpreted in terms of the base composition of the DNA in the vicinity of these sites. dA + dT-rich regions are more susceptible to deformation than are dG + dC-rich ones. Analysis of the rates of disappearance of linear molecules confirms a two-step mechanism for EcoRI cleavage but suggests that BamHI and AvaI cleave both strands simultaneously.

Base Sequence↗
PubMed1984

Mitral leaflet prolapse.

Mitral leaflet prolapse (MLP) is a common condition but sudden or subtle incapacity will occasionally present due to (a) arrhythmia, (b) systemic emboli, (c) sudden onset of severe mitral regurgitation due to chordal rupture, (d) non-anginal chest pain. It seems reasonable to allow asymptomatic subjects with MLP to maintain unrestricted flying status provided that they have no family history of sudden death, no evidence of significant arrhythmia on Holter electrocardiographic monitoring, a normal exercise electrocardiogram, and no increase in left atrial or left ventricular dimensions on the echocardiogram. The Holter recording, exercise test and echocardiogram may need to be repeated at intervals. Any departure from these requirements might require restriction to multi-crew operation only.

Adult↗
PubMed1982

Differential inhibition of a restriction enzyme by quinoxaline antibiotics.

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.

Anti-Bacterial Agents↗
PubMed1982

Preparative scale, high resolution purification of low molecular weight DNA fragments.

A commercially available continuous electroelution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80-95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1-4 x 10(5) (200-700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.

Acrylic Resins↗
PubMed1981

Differential reactivities at restriction enzyme sites.

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and phi X174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in phi X174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than dose the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.

Base Sequence↗
PubMed1981

Properties of methyl acetimidate and its use as a protein-modifying reagent.

The rate of hydrolysis of the imido ester methyl acetimidate and its rate of amidination of denatured aldolase were investigated under different conditions of temperature, pH and ionic strength. Both rate constants increase greatly with temperature, whereas ionic strength has no effect on either. The effect of pH is more complex. Between pH 6.8 and 8.8 the rate of hydrolysis decreases and the rate of amidination increases. These results are discussed in terms of the reaction mechanisms involved.

Chemical Phenomena↗
PubMed1981

Cation dependence of restriction endonuclease EcoRI activity.

Restriction endonuclease EcoRI cleaves the DNA sequence 5'd(-G-A-A-T-T-C-) under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI activity when 5'd(-A-A-T-T-) is cut. A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and Co2+ and Zn2+ were also found to act as cofactors for EcoRI. A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. The Km of EcoRI for ColE1 DNA, in the presence of Co2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double-stranded scission/min with Co2+ compared to eight/min with Mg2+. The implications of all these findings on the enzyme's mechanism are discussed.

Cations, Divalent↗
PubMed1981

The essential carboxyl group in restriction endonuclease EcoRI.

We have carried out studies on type II restriction endonuclease EcoRI, which cleaves the DNA sequence 5'd(-G-A-A-T-T-C-)3', as indicated. The active form of the enzyme consists of two subunits, each 31063 molecular weight. A water-soluble reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-sulphonate, which reacts with carboxyl groups and also with tyrosine and cysteine residues, has been found to inactivate this enzyme. Results are presented which show the following. (1) This specific inactivation is not due to modification of tyrosine or cysteine residues. (2) There is one carboxyl group per subunit which, when modified with carbodiimide, inactivates the enzyme. (3) phi X174 DNA (which does not contain EcoRI sites) partially protects the enzyme from the carbodiimide; protection is unaffected by the additional presence of Mg2+, but significantly greater with Co2+ and phi X174 DNA.

Bacteriophage phi X 174↗
PubMed1981

The isolation, mapping and transcription in vitro of a beta 0-thalassaemia globin gene.

The red blood cell precursors of a patient with homozygous beta 0-thalassaemia have previously been shown to contain nuclear, but not cytoplasmic, beta-globin-specific transcripts. We describe the isolation of a beta-globin gene from this patient as a recombinant bacteriophage chromosome. Restriction-enzyme cleavage-site mapping experiments demonstrate no detectable deletions, insertions or major rearrangements in this thalassaemia gene. Two different techniques show that the gene isolated is transcribed as efficiently in vitro as the normal beta-globin gene.

Base Sequence↗
PubMed1980

Kinetic analysis of effectors of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.

The activity of phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxy-lyase (phosporylating) EC 4.1.1.31) purified from Bryophyllum fedtschenkoi has been measured in the presence of various concentrations of phosphoenolpyruvate, L-malate and glucose 6-phosphate. At high pH, the enzyme is competitively inhibited by L-malate and activated by glucose 6-phosphate. A reaction scheme describing the interaction of enzyme, substrate and effectors is proposed. Values for the appropriate equilibrium constants have been calculated for the enzyme acting at pH 7.8, which is one of its two pH optima. The kinetics are more complicated at low pH, partly because of non-linear reaction rates and partly because inhibition by L-malate is not competitive. Activation by glucose 6-phosphate is similar at high and low pH values. The behaviour of a wide range of other possible effectors is described briefly.

Carboxy-Lyases↗
PubMed1980

Non-specific binding of restriction endonuclease EcoR1 to DNA.

Restriction endonuclease, EcoRl cleaves the DNA sequence (see formula in text) at the points indicated. Under certain conditions, EcoRl activity is observed when (see formula in text) is cut. Mg2+ is required for both activities. We find that in addition to binding to the above sites, EcoRl will also bind, although less strongly, to DNA containing neither site. Methyl acetimidate, which reacts specifically with lysine residues, inactivates the enzyme. This specific effect can be prevented by SV40 DNA and lambda DNA which contain EcoRl and EcoRl sites, by 0X174 DNA, which has only EcoRl sites and also by Polyd(AT) and polyd(GC) containing neither site. Protection occurs in the absence or presence of magnesium. The significance of this non-specific binding, both for the use and mechanism of EcoRl will be discussed.

Base Sequence↗