Symmetry, flexibility and permeability in the structure of yeast retrotransposon virus-like particles.

The virus-like particles (VLPs) of the yeast retrotransposon Ty are genetically, structurally and functionally analogous to retroviral nucleocapsids or cores. Like retroviral cores Ty-VLPs package and possibly promote the enzyme activities for reverse transcription and integration, as well as encapsulating the RNA that is the intermediate in retrotransposition. Here we show that Ty-VLPs assemble into symmetrical structures across a broad distribution of particle sizes. This spread of sizes violates the principle of quasi-equivalent packing. In addition, RNase accessibility experiments suggest that these particles form an open structure that does not protect the encapsulated RNA. These features distinguish Ty-VLPs from typical spherical viral capsids in both structure and function.

A potent new dipeptide inhibitor of cell sickling and haemoglobin S gelation.

A dipeptide L-lysine-L-phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sickle-cell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys-Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys-Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys-Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3-bisphosphoglycerate. The effect of Lys-Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys-Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane-mediated antisickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume. The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.

Chemical synthesis and cloning of a gene for human beta-urogastrone.

A DNA duplex coding for the 53 amino acids of human beta-urogastrone has been synthesised. Computer assisted design of the gene included restriction endonuclease sites for plasmid insertion, a termination codon and two triplets coding for lysine at the 5'-end of the structural gene. The synthesis involved preparation of 23 oligodeoxyribonucleotides by phosphotriester procedures coupled to rapid HPLC techniques. The gene was constructed in two halves by enzymatic ligation of the oligonucleotides and cloned into a specially constructed chimeric plasmid vector. Escherichia coli K12 MRC8 was transformed by the plasmid and clones containing the full gene sequence were isolated and characterised.

Light scattering measurements supporting helical structures for chromatin in solution.

Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.

The structure of the chromatin core particle in solution.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.

Structural investigations of chromatin core protein by nuclear magnetic resonance.

A complex derived from chromatin containing one molecule of each of histones H2A, H2B, H3, and H4, termed core protein, was studied by 13C and 1H nuclear magnetic resonance. 13C line widths, when analyzed and compared with those of native and thermally unfolded representative globular proteins, showed that regions of the core protein possess considerable mobility. Studies of Calpha and Cbeta line widths, and Calpha spin-spin relaxation times, show that this mobility arises from sections of random-coil polypeptide. It is argued that these regions are N-terminal "tails", attached to C-terminal globular polypeptides. The 270-MHz 1H nuclear magnetic resonance spectrum shows numerous ring current shifted resonances, indicating that the C-terminal globular domain has a precise tertiary structure. The globular domain most likely forms the histone "core" of the chromatin monomer particle, whilst the basic tails probably wind around the grooves of the double helix, enabling the basic side chains to interact with the DNA phosphate groups. Some biological implications of this model are considered.

Low-angle neutron scattering from chromatin subunit particles.

Monomer chromatin particles containing 140 base pairs of DNA and eight histone molecules have been studied by neutron scattering. From measurements in various H2O/D2O mixtures, radii of gyration and the average scattering density of the particle were determined. The radius of gyration under conditions when scattering from the DNA dominates is 50A, and when scattering from the protein dominates, 30A. Consequently the core of the particle is largely occupied by the histones while the outer shell consists of DNA together with some of the histone.

An investigation of the conformational and self-aggregational processes of histones using 1H and 13C nuclear magnetic resonance.

Histone self-aggregation processes have been studied by 13C and 1H nuclear magnetic resonance (NMR) as a function of ionic strength and protein concentration. Thus has led to a model involving apolar aggregation between structured regions of these molecules. This analysis supports the validity of the acquistion of conformational data on histones by the simulation of 13C NMR spectra at high concentration. Solution conformations for histones F2B and F3 are presented.

The structure and properties of histone F2a comprising the heterologous group F2a1 and F2a2 studied by 13C nuclear magnetic resonance.

(13)C n.m.r. spectra have been obtained for aqueous solutions of histones F2a(1) and F2a(2), for the group F2a, for the appropriate amino acid mixturesand for the corresponding hydrolysates. These, when compared with computer simulated spectra give good agreement for secondary structure with that calculated from the known primary structure of the proteins. Evidence based on the spectra obtained at various salt concentrations leads to the conclusion that F2a is not a simple mixture but an interacting heterologous group of histones F2a(1) and F2a(2).