Histone-histone interactions within chromatin. Crosslinking studies using ultraviolet light.

Irradiation of either whole cells or chromatin at 280 nm results in the covalent linkage of histones 2A and 2B, presumably at their mutual binding sites. The reaction is specific and proceeds with high yield (about 80%). Irradiation of reconstituted nucleohistone containing only H2A, H2B and DNA also yields the H2A-H2B dimer. The cross-linking event is sensitive to the conformation of the H2A-H2B pair since the histones must be bound to DNA for maximum cross-linking specificity at low ionic strength. However, the histones must first interact with each other before being deposited on the DNA, since separate addition of the histones to the DNA yields no dimer upon irradiation. If irradiation is conducted at 254 nm rather than 280 nm, DNA-histone cross-linking appears to dominate.     

Histone-histone proximity in chromatin as seen by imidoester cross-linking

After treatment of purified chromatin with dimethyl adipimidate (a reversible crosslinking reagent) a number of histone oligomers could be isolated in the soluble form from the chromatin. It was shown that all five histones take part in the dimethyl adipimidate-induced oligomer formation. Only a few kinds of histone oligomers (with unknown composition) could be isolated in the soluble form after treatment of chromatic with formaldehyde.     

Histone-histone interaction mediates chromatin unfolding at physiological ionic strength

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.     

Histone-histone interactions as revealed by formaldehyde treatment of chromatin

Histone oligomers produced by formaldehyde treatment of chromatin were studied. It was shown that these histone oligomers could be converted into monomers by boiling in 0.1 NH₂SO₄. Dimers H2B-H4 and H2B-H2A were identified by two-dimensional polyacrylamide gel electrophoresis.Abbreviations. Histones Nomenclature H1 formerly histone F1 - H2B formerly histone F2b - H2A formerly histone F2a2 - H3 formerly histone F3 - H4 formerly histone F2a1This nomenclature has been proposed at the recent Symposium on the Structure and Function of Chromatin. Ciba Foundation. London. April 1974.     

Subunit associations among chromatin particles.

The self-association of oligonucleosomal chromatin particles in solution has been studied by light scattering and sedimentation. In the absence of magnesium ions no association is observed. In the presence of 70mM sodium or 2mM magnesium ions mono, di, tri and tetranucleosomes self-associate only if they contain bound histone 1. This association leads to the formation of compact aggregates and is continuous and non-cooperative. The relevance to higher order arrangements of nucleosomes is discussed.     

Endosymbiotic associations within protists

The establishment of an endosymbiotic relationship typically seems to be driven through complementation of the host''s limited metabolic capabilities by the biochemical versatility of the endosymbiont. The most significant examples of endosymbiosis are represented by the endosymbiotic acquisition of plastids and mitochondria, introducing photosynthesis and respiration to eukaryotes. However, there are numerous other endosymbioses that evolved more recently and repeatedly across the tree of life. Recent advances in genome sequencing technology have led to a better understanding of the physiological basis of many endosymbiotic associations. This review focuses on endosymbionts in protists (unicellular eukaryotes). Selected examples illustrate the incorporation of various new biochemical functions, such as photosynthesis, nitrogen fixation and recycling, and methanogenesis, into protist hosts by prokaryotic endosymbionts. Furthermore, photosynthetic eukaryotic endosymbionts display a great diversity of modes of integration into different protist hosts.In conclusion, endosymbiosis seems to represent a general evolutionary strategy of protists to acquire novel biochemical functions and is thus an important source of genetic innovation.     

Complex formation between flavodoxin and cytochrome c. Cross-linking studies

Complex formation between Azotobacter vinelandii flavodoxin and horse cytochrome c has been demonstrated through cross-linking studies with dimethyl suberimidate, dimethyl adipimidate, 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide, and dimethyl-3,3'-dithiobispropionimidate. Essentially quantitative cross-linking of cytochrome c and flavodoxin was observed at low ionic strengths with the carbodiimide cross-linking reagent. An association constant of 4 X 10(4) M-1 was obtained between cytochrome c and flavodoxin at 88 mM ionic strength from analysis of the cross-linking studies. This value is similar to the association constant determined kinetically during the electron transfer reaction between cytochrome c and flavodoxin (Simondsen, R.P., Weber, P.C., Salemme, F.R., and Tollin, G. (1982) Biochemistry 21, 6366-6375), and suggests that the cross-linked complex may be similar to the precursor complex identified kinetically. A structural model for the flavodoxin-cytochrome c complex proposed by these workers is shown to be compatible with the present cross-linking results.     

Amino acid contacts between histones are the same for plants and mammals. Binding-site studies using ultraviolet light and tetranitromethane.

Leek chromatin has been cross-linked by UV light and tetranitromethane. The same major H2A--H2B and H2B--H4 cross-linked dimers are formed as in mammalian chromatin. CNBr peptide mapping shows that the cross-links occur in the same regions of the histone sequence for both plants and mammals. Interspecies complexes formed between leek and calf H2A and H2B can be cross-linked by UV light with the same specificity as intraspecies H2A--H2B complexes. We conclude that certain geometric features of histone-histone binding sites are conserved precisely during evolution despite large changes in the overall histone sequence. Moreover, our data show that identification of cross-linked amino acids using binding-site probes such as UV light and tetranitromethane can yield significant information about thermodynamically important contacts within histone-histone binding sites.     

Cross-linking studies with the uvrA and uvrB proteins of E. coli

The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.     

Cross-linking evidence for motional constraints within chemoreceptor trimers of dimers

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.     

Electron-microscopic studies on primate neurons: microtubule-pigment associations.

The relationship between microtubules and the lipoprotein pigment found in senile Cynomolgus brain and senile human brain biopsy material was investigated. Numerous microtubules were present in all parts of the cytoplasm and within the pigment areas, running parallel or obliquely to the pigment bodies and being associated with its lateral aspects. Microtubules also occurred in the periphery of neurons or appearing to enter the perineuronal oligodendrocytes. These observations indicate a possible role of microtubules in the transport of pigment bodies. The oligodendrocyte from human brain biopsy material has definitely taken the role of a phagocyte in ingesting pigment bodies. Its numerous microtubules may offer a fasting moving system for disposing off pigment residures to the capillary endothelium.     

Gastropod associations within Malham Tarn, Yorkshire

A survey of gastropod species associations was undertaken in one locus, i.e. Malham Tarn, Yorkshire. This ranged through both the major weed beds and the rocky shores. A semiquantitative analysis, in terms of both presence-absence and relative abundance indices, suggested a clear distinction between the snail fauna present in the weed beds and that on the rocky shores, and also indicated a separation of faunas within these two major habitat types. The latter was closely correlated with degree of exposure. From this, and other evidence, it was suggested that wind, via wave action, may be a critical determinant of both the qualitative and quantitative aspects of biocoenotic structure in large bodies of fresh water.     

Cross-linking in adhesive quinoproteins: studies with model decapeptides

Mytilus edulis foot protein-1 (mefp1) is a major component of the byssus, an adhesive holdfast in mussels. The recent report of 5, 5'-di(dihydroxyphenyl-L-alanine) (diDOPA) cross-links in byssus [McDowell et al. (1999) J. Biol. Chem. 274, 20293] has raised questions about the relationship of these to mefp1. About 80% of the primary structure of mefp1 consists of a tandemly repeated consensus sequence Ala(1)-Lys(2)-Pro(3)-Ser(4)-Tyr(5)-Pro(6)-Pro(7)-Thr(8)-Tyr(9)-Lys(10 ) with varying degrees of posttranslational hydroxylation to hydroxyprolines in positions 3, 6, and 7 and to DOPA in positions 5 and 9. Six natural or synthetic variants of this decapeptide were subjected to oxidation by tyrosinase or periodate. DOPA is the only residue to suffer losses in all oxidized peptides. Moreover, using MALDI TOF mass spectrometry, oxidized decapeptides all showed evidence of multimer formation and a mass loss of 6 Da per coupled pair of peptides. Multimer formation was inhibited by addition of DOPA-like o-diphenols, but addition of simple amines such as free Lys had no effect. The results are consistent with aryloxy coupling to diDOPA followed by reoxidation to diDOPA quinone. There are subtle but noteworthy variations, however, in multimer formation among the peptide congeners. Decapeptides with Pro(3) modified to trans-4-hydroxyproline do not form multimers beyond dimers; they also exhibit significant Lys losses following oxidation of DOPA. Moreover, in Ala-Lys-Hyp-Ser-Tyr-DiHyp-Hyp-Thr-DOPA-Lys, Tyr appears to be protected from oxidation by tyrosinase.     

DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.     

The reaction of tetranitromethane with human chorionic gonadotropin.

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding.     

Cross-linking studies of the protein topography of rat liver microsomes

A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0°C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an M_I of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with M_r of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercapto-ethanol or dithiothreitol were not successful.