Formaldehyde in the plant kingdom.

Formaldehyde, at its dimedone adduct, formaldemethone, has been detected by thin-layer and high-performance liquid chromatography in extracts of all species tested of marine algae, macrofungi, lichens, bryophytes, pteridophytes, gymnosperms and angiosperms. The yields of formaldehyde recorded in this study varied from 30 microg/g to 4060 microg/g, fresh weight.

The use of HPLC for the detection and quantification of formaldehyde in the pteridophyta.

Formaldehyde, as its dimedone adduct, formaldemethone, has been detected and quantified in all species of Pteridophyta examined. The procedure involved the use of an Hypersil C-18 column, methanol-water (60 : 40 v/v) as the mobile phase and an UV detector set at 258 nm. Quantification was based on peak height. Yields varied from 30 microg/g fresh weight for Polystichum setiferum to 5370 microg/g fresh weight for Selaginella viticulosa.

Isolation, partial characterisation and complement inhibiting activity of a new glycoprotein from Cuscuta europea.

A new complement inhibiting factor (CIF) was isolated from the seeds of Cuscuta europea parasitic plant. When activated via both classical and alternative pathway, the complement activity was completely depleted by CIF at a concentration of 0,25 mg per ml serum. Studies concerning the precipitation showed that CIF developed one or two precipitin bands against human sera. It was established that the precipitation is as a result of the specific association of CIF to the C3 component of complement. A partial characteristic of the CIF was carried out. It is a glycoprotein with molecular weight between 27000 and 28000 Da. Its molecule consists of one polypeptide chain.

An automated method for the determination of deoxyribonuclease activity as exemplified by fractionation of the components of the medicament Varidase.

The activity of most deoxyribonuclease enzymes can be monitored by measuring the change in absorbance at 260 nm which accompanies the breakdown of the double-stranded structure of native DNA. An automated method for determining deoxyribonuclease activity, based on such an absorbance change, which can overcome problems of inhibition arising from the presence of inorganic cations, is described. Variations in inorganic cation concentration is a particular problem when measuring the activity of chromatographic fractions eluted via a salt gradient. A comparison is made between the automated and a manual method for the assay of deoxyribonuclease active constituents, of the medicament 'Varidase', eluted from a Cellex-D (Bio-Rad Laboratories Ltd) anionic exchange resin using a 0.05-1.0 M sodium chloride gradient.

The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.

Isolation, characterisation and growth-related changes of an HMG-like protein from microplasmodia of Physarum polycephalum.

An alanine, lysine and glutamic acid-rich nuclear protein (P2) of Mr approximately 19,500 co-extracts with the histones from nuclei of Physarum polycephalum when using the CaCl2 method for histone extraction [1] and was found to have the composition previously ascribed to a putative histone H1(0) isolated from microplasmodia using 5% PCA (Yasuda, H., Mueller, R.D., Logan, K.A. and Bradbury, E.M. (1986) J. Biol. Chem. 261, 2349-2354). P2 has very similar electrophoretic properties to chicken erythrocyte histone H5, calf thymus histone H1(0) and the Physarum HMG-like protein AS-2, but does not appear to be immunologically or structurally similar to H5 or H1(0). An increase in the abundance of P2 was observed during exponential growth in microplasmodia, reaching an approximately 1:1 ratio with histone H1 by 48 h of culture. Standard amino acid analysis and NMR show that P2 is more HMG-like than H1-like and CD measurements demonstrated that P2 contains only 5% secondary structure in its maximally structured state and is, therefore, essentially unstructured under in vivo conditions. Also possible clustering of acidic residues is detected using CD and may be of functional significance. Analysis of post-translational modification of P2 shows that it is phosphorylated at up to three sites as isolated from immature spherules. The relationship of P2 to the HMG family of proteins and AS-2 is discussed.

Differential synthesis of histone H1 during early spherulation in Physarum polycephalum.

H1 and P2 (an H1 degree/HMG-like protein) accumulate during exponential growth of Physarum microplasmodia (unpublished results), indicating that these proteins may play a role in differentiation (spherulation). To test this hypothesis, pulse labelling using [14C]lysine was used to determine whether any differential histone synthesis occurs during salts-induced spherulation. A peak in the uptake of [14C]lysine into microplasmodia was detected between 12 and 24 h following salts-induction. During the same interval, incorporation of label into the CaCl2-extracted histones occurred, with H1 being synthesised at approx. 3 times the level of the core histones and P2. Densitometry of SDS-PAGE gels showed that high levels of H1 were maintained up to 40 h in salts medium, beyond the observed peak in synthesis. The synthesis and accumulation of high levels of H1 during early spherulation indicates a role for this histone in the initiation and maintenance of a transcriptionally inactive differentiated state.

Polyamines permit the preparation of stable Physarum core particles which have a structure similar to those from higher eukaryotes.

The inherent instability of Physarum nucleosome core particles prepared by micrococcal nuclease digestion in Na+/Ca2+ buffers can be overcome by the addition of 0.15 mM spermine and 0.5 mM spermidine. Neutron scattering, circular dichroism, nuclease digestion and thermal denaturation studies carried out on these stable monosomes show them to be very similar to those obtained from higher eukaryotes.

Physical studies by NMR and circular dichroism determining three structurally different domains in Physarum polycephalum histone H1.

Combined studies which include, NMR spectroscopy, circular dichroism, amino acid analysis and polyacrylamide gel electrophoresis together show that the protein designated as histone H1 from Physarum polycephalum has many of the features of histone H1 derived from other sources. The molecular masses of the globular peptide and the whole molecule were found to be 9000 +/- 1000 Da and 33000 +/- 3000 Da respectively. NMR melting experiments showed that the half-melt temperature was 53 +/- 1 degree C and the enthalpy of melting was 100 kJ . mol-1. Unusual facets of the molecule are the relatively large numbers of histidine residues (6 or 7) and the mono, di and trimethylation of some of the lysines, the major type of modification being trimethylation of 9 +/- 2 residues. The conditions necessary for structuring Physarum H1 are not the same as the histone H1 from calf thymus. It is suggested that titration of the histidine residues is the most decisive step for the development of tertiary folding of the globular unit.

A further assessment of the sub-nucleosomal product, "peak A", from Physarum polycephalum.

Nuclease digestion studies of Physarum polycephalum nuclei (1-3) and nucleoli (4) over the past few years have been centred on a number of modified nucleosomal products which have been related to active-gene regions of the genome. We have re-investigated one such particle, peak A, using the techniques of differential melting and polyacrylamide gel electrophoresis and show that this material is unlikely to be a specific histone:DNA complex as suggested by earlier authors.

Selective depletion and reconstitution of nucleosome core particles.

A method for the complete and specific removal of histones H2A and H2B from nucleosome core particles is presented. Reconstitution of the separated products of depletion form a particle which has the same structure as native core particles as judged by a number of physical and biochemical criteria. The technique described also minimises the possibility of the formation of reconstituted core particles with different histone stoichiometries. These experiments are important as they demonstrate a procedure which can be extended to prepare core particles with selectively deuterated components while maintaining complete integrity of structure. When prepared, and studied by neutron scattering, selectively-deuterated core particles can give detailed information with respect to the relative positions and structure of the histone fractions within the core particle.

Neutron-scattering studies of accurately reconstituted nucleosome core particles and the effect of ionic strength on core particle structure.

Chicken erythrocyte nucleosome core particles can be dissociated quantitatively into histones (H3, H4)2 bound to 146 base pairs of DNA, and 2(H2A, H2B). Reconstitution of core particles from the two components produces an 85% yield of particles which neutron scattering studies show to be accurate stoichiometrically and indistinguishable from native core particles: the radii of gyration of the shape, the protein components and the DNA components of the particles are 4.02 nm, 3.3 nm and 4.95 nm respectively. The largest distance and most probable distance which can be drawn in the particles are 11.5 nm and 4.3 nm respectively. The molecular weight of the particles is identical to that of control 'native' core particles. All of these values, within limits of error, are the same as known values for 'native' core particles. These experiments confirm the essential role of histones H3 and H4 in the initial organisation of core-particle structure, make possible the manufacture of perfectly pure and homogeneous core-particle preparations and allow the 100% incorporation of labelled or modified histones. Neutron scattering studies of core particles at high contrast (in D2O and H2O) have been carried out over a range of ionic strengths and pH. No change in structure is detected down to pH 5.5 in 20 mM NaCl or down to ionic strength 2.0 mM at pH 7.

Organisation of subunits in chromatin.

There is considerable current interest in the organisation of nucleosomes in chromatin. A strong X-ray and neutron semi-meridional diffraction peak at approximately 10 nm had previously been attributed to the interparticle specing of a linear array of nucleosomes. This diffraction peak could also result from a close packed helical array of nucleosomes. A direct test of these proposals is whether the 10 nm peak is truly meridional as would be expected for a linear array of nucleosomes or is slightly off the meridian as expected for a helical array. Neutron diffraction studies of H1-depleted chromatin support the latter alternative. The 10 nm peak has maxima which form a cross-pattern with semi-meridional angle of 8 to 9 degrees. This is consistent with a coil of nucleosomes of pitch 10 nm and outer diameter of approximately 30 nm. These dimensions correspond to about six nucleosomes per turn of the coli.

Physical studies of chromatin. The recombination of histones with DNA.

Experiments have been carried out to define clearly which histone combinations can induce a higher order structure when combined with DNA. The criterion for a higher order structure being the series of low-angle X-ray diffraction maxima nominally at 5.5 nm, 3.7 nm, 2.7 nm and 2.2 nm. Such a pattern, with resolution similar to that of H1-depleted chromatin, is readily attainable by recombining histones H2A + H2B + H3 + H4 with DNA using a salt-gradient dialysis method. However, the use of urea in the recombination procedure is shown to be detrimental to the production of a higher order structure. Low-angle ring patterns are not obtained by recomgining DNA with single pure histones or any combination of histone pairs exept H3 + H4. The diffraction maxima from the latter are, however, weaker than those from chromatin and there are pronounced semi-equatorial arcs. The presence of a third histone, either H2A or H2B in the H3 + H4 recombination mixture tends to distort the recognised low-angle pattern. It is concluded that the histone pair H3 + H4 is essential for the formation of a regular higher order structure in chromatin, although for a complete structural development the presence of H2A + H2B is also required.