John C.W. Shepherd

Exchange of solubilized water and aqueous electrolyte solutions between micelles in apolar media

Abstract By means of fluorescent measurements, using Tb 3+ as a probe, it has been shown that there is a rapid exchange of electrolyte solutions and water between aerosol OT micelles in iso-octane occurring during collisions. This process is considered to be analogous to a collision between liquid spheres and a theoretical discussion of this model treats the resultant deformation as a vibration induced during the time of collision. The surfactant molecules move outward from the point of impact during the collision, thus opening a channel through which the solubilizate has time to diffuse from one micelle to the other.

Zwitterionic dipoles as a dielectric probe for investigating head group mobility in phospholipid membranes

For phospholipid membranes with zwitterionic head groups, the dipole can be considered as a specific label for tracing the changes in the dynamic behaviour of this region of the bilayer in its various phases. Measurements of the dielectric properties of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the frequency range 1--50 MHz show a dispersion which is attributed to the motion of the phosphocholine dipoles in the plane of the bilayers. When the temperature is varied, both the permittivity and loss factor increase sharply at the pretransition (35 degrees C) and the main transition (42 degress C). The relaxation time and amplitude were also determined for this dispersion and these further reflect the structural changes occurring with temperature. The relaxation times varied between 4 ns at 30 degrees C and 2.3 ns at 50 degrees C. Due to steric hindrances a restriction in the angle of head group rotation occurs at lower temperatures but is greatly reduced above the main transition.

Periodic correlations in DNA sequences and evidence suggesting their evolutionary origin in a comma-less genetic code

SummaryStrong rhythms with a period of three bases have been seen while correlating the relative positions of purines and pyrimidines and of the four individual bases in the complete DNA sequence of the viruses øX174, G4 and fd. Generally weaker variations of the same type have been found in the DNA virus SV40, the plasmid pBR322, the RNA virus MS2, and elsewhere in procaryotes and eucaryotes (e.g. in a ribosomal protein gene cluster ofE. coli and the sea urchin histone genes). From the interrelation of four-base with purine-pyrimidine rhythms it seems that the purine-pyrimidine relationships have a basic significance. An explanation is proposed in terms of the former use of a comma-less genetic code (i.e. readable only in one frame) of the general form RNY (R = purine, Y = pyrimidine and N = purine or pyrimidine). In spite of subsequent mutation, there appears to be still enough of the primitive messages remaining to produce these periodic variations with their characteristic properties in phase and amplitude. Particularly good evidence for this hypothesis is provided by the fact that the phases for the stronger rhythms are the same in all the genomes tested and can be successfully predicted by a simple consideration of the original RNY pattern. With regard to amplitude it can be similarly foreseen which variations will be more clearly marked than others. The observed behaviour of the amplitude as the separation between correlated bases increases is also explained by the insertions, deletions and point mutations which have occurred. Additionally it is possible to account for some notable features of the non-random use of codons for the same amino acid by this theory.

DNA recognition and cleavage by the EcoP15 restriction endonuclease.

Abstract Eco P15 is a restriction-modification enzyme coded by the P15 plasmid of Escherichia coli . We have determined the sites recognized by this enzyme on pBR322 and simian virus 40 DNA. The enzyme recognizes the sequence: In restriction, the enzyme cleaves the DNA 25 to 26 base-pairs 3′ to this sequence to leave single-stranded 5′ protrusions two bases long.

The influence of cholesterol on head group mobility in phospholipid membranes

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0--70 degrees C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42 degrees C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42 degrees C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42 degrees C, however, a decrease in f2 at cholesterol contents up to 20--30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.

The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence.

The EcoA restriction enzyme from Escherichia coli 15T‐ has been isolated. It proves to be an unusual enzyme, clearly related functionally to the classical type I restriction enzymes. The basic enzyme is a two subunit modification methylase. Another protein species can be purified which by itself has no enzymatic activities but which converts the modification methylase to an ATP and S‐adenosylmethionine‐dependent restriction endonuclease. The DNA recognition sequence of EcoA has an overall structure that is very similar to previously determined type I sequences. It is: 5′‐GAGNNNNNNNGTCA‐3′ 3′‐CTCNNNNNNNCAGT‐5′ where N can be any nucleotide. Modification methylates the adenosyl residue in the specific trinucleotide and the adenosyl residue in the lower strand of the specific tetranucleotide.

Two type I restriction enzymes from Salmonella species: Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.

The DNA sequence recognised by the HinfIII restriction endonuclease

HinfIII is a type III restriction enzyme (Kauc & Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be: 5′-C-G-A-A-T-3′·····3′-G-C-T-T-A-5′ In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.

New Variations on an Old Theme: Type I Restriction Enzymes and their Recognition Sequences

Restriction enzymes have revolutionised molecular biology over the past decade through their ability to dissect ge.noniic DNA into well defined, experimentally accessible fragments. Close to two hundred different enzymes have been described but very few of them have been more than cursorily characterised; in most cases all that is known of the enzymes is their most important characteristic from a practical point of view, that is, the DNA sequence that they recognise. Most of the known enzymes are relatively simple in that they are composed of a single subunit and recognise and cleave their recognition sequence with the aid of no cofactor more complicated than magnesium. A handful, however, have more complex enzyme structures and reaction mechanisms and it is some of these enzymes that form the subject of this paper.

The Head Group Mobility in Phosphatidylcholine Membranes in the Presence of Cholesterol as Seen by a Dielectric Investigation

For phospholipid membranes with zwitterionic head groups, the dipole can be considered as a specific label for tracing the changes in the dynamic behaviour of this region of the bilayer in its various phases. Measurements of the dielectric properties of fully hydrated 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the frequency range 1–50 MHz show a dispersion which is attributed to the motion of the phosphocholine dipoles in the plane of the bilayers. When the temperature is varied, both the permittivity and loss factor increase sharply at the pretransition (35°C) and the main transition (42°C). The relaxation time and amplitude were also determined for this dispersion,and these further reflect the structural changes occurring with temperature. Due to steric hindrances a restriction in the angle of head group rotation occurs at lower temperatures but is greatly reduced above the main transition. When cholesterol is added the abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in f2 are found at 20 or 33 mole % cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, an initial decrease in f2 at cholesterol content up to 20–30 mole % is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.