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Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies.

High mobility group protein B1 (HMGB1) binds to the internucleosomal linker DNA in chromatin and abuts the nucleosome. Bending and untwisting of the linker DNA results in transmission of strain to the nucleosome core, disrupting histone/DNA contacts. An interaction between H3 and HMGB1 has been reported. Here we confirm and characterize the interaction of HMGB1 with H3, which lies close to the DNA entry/exit points around the nucleosome dyad, and may be responsible for positioning of HMGB1 on the linker DNA. We show that the interaction is between the N-terminal unstructured tail of H3 and the C-terminal unstructured acidic tail of HMGB1, which are presumably displaced from DNA and the HMG boxes, respectively, in the HMGB1-nucleosome complex. We have characterized the interaction by nuclear magnetic resonance spectroscopy and show that it is extensive for both peptides, and appears not to result in the acquisition of significant secondary structure by either partner.

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys(2) and Lys(11) in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced approximately 15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys(2) or Lys(11) abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys(2) and Lys(11) are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.

The mechanism underlying negative regulation of HMGB1-DNA interaction by the acidic C-terminal tail is ill defined. To address this issue, we have devised a novel NMR chemical-shift perturbation mapping strategy to elucidate interactions between the tail, which consists solely of aspartic acid and glutamic acid residues, and the two well characterized HMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing from each other by five residues was generated. Chemical-shift perturbation mapping using these mutants shows that tails of different lengths bind with different affinities. Nevertheless, the truncated tails bind along the same path on the HMG boxes as the full-length tail, differences in length being manifested in differences in the "reach". The tail makes extensive contacts with the DNA-binding surfaces of both HMG boxes, thus explaining the basis of negative regulation of HMGB1-DNA interaction by the tail.

MeCP2, whose methylated DNA-binding domain (MBD) binds preferentially to DNA containing 5Me-CpG relative to linear unmethylated DNA, also binds preferentially, and with similar affinity, to unmethylated four-way DNA junctions through the MBD. The Arg133Cys (R133C) mutation in the MBD, a Rett syndrome mutation that abolishes binding to methylated DNA, leads to only a slight reduction in the affinity of the MBD for four-way junctions, suggesting distinct but partially overlapping modes of binding to junction and methylated DNA. Binding to unmethylated DNA junctions is likely to involve a subset of the interactions that occur with methylated DNA. High-affinity, methylation-independent binding to four-way junctions is consistent with additional roles for MeCP2 in chromatin, beyond recognition of 5Me-CpG.

Arrays of neighbouring histone H1 molecules are present in rat liver chromatin, and can be cross-linked to each other by bisimidoesters. The product, poly(H1), can be extracted from cross-linked chromatin in high yield with 5% perchloric acid and thus distinguished from cross-linked oligomers of the core histones. On analysis in dodecyl sulphate/polyacrylamide gels poly(H1) gives a striking pattern of alternating strong and weak bands which have been shown, by the use of cleavable cross-linked reagents and two-dimensional gel electrophoresis, to arise respectively from H1 homopolymers and H1 polymers linked to the nucleosome core histones. H1-H1 proximities as measured by cross-linking exist both at low ionic strength where the nucleosome filament is extended, and at higher ionic strengths at which it is folded into a 30-nm-diameter fibre, probably in the form of a solenoid, although some additional H1-H1 contacts seem to occur in the folded form. A similar pattern of H1-H1 cross-linking is observed for nucleosome oligomers too short to form a complete turn of a solenoid. The lack of any strong dependence of the H1 cross-linking pattern on ionic strength, and the results for short oligomers, suggest that in a solenoid the major H1-H1 interactions are lateral (i.e. they occur between neighbouring nucleosomes along a turn) although additional vertical contacts (between successive turns) are not excluded. For a short nucleosome oligomer containing n nucleosomes, the number of H1 molecules that may be cross-linked into an array at low ionic strength (approximately 15 mM) is n, consistent with the presence of one H1 per nucleosome. H1-H1 cross-linking occurs only within the chromatin framework; when cross-linking is carried out in 0.5 M NaCl so that H1 is dissociated from chromatin, no poly(H1) is formed. When H1 which has been dissociated in 0.5 M NaCl is allowed to reassociate by gradual lowering of the ionic strength, the poly(H1) is identical with that in native chromatin, suggesting faithful rebinding of H1 molecules. H1-H1 proximities do not seem to be mediated by the presence of bound high-mobility-group non-histone proteins because the poly(H1) pattern persists when cross-linking is carried out at 365 mM ionic strength, when these high-mobility-group proteins are dissociated from chromatin. The H1 cross-linking pattern described here could prove to be a useful assay for the native arrangement of H1 molecules in reconstituted chromatin and for changes in H1-H1 contacts that might result from cell-cycle modifications (e.g phosphorylation) of H1. It may not prove a good assay for higher-order structure because of the relative insensitivity to ionic strength of the poly(H1) pattern as studied here. A detailed analysis will be necessary to determine whether there are any subtle differences in the interactions between H1 molecules in the folded and extended nucleosome filament.

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p “domains” differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high α-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.

We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin. This supports a model in which GH5 contacts two duplexes on the nucleosome. However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones. We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different. GH5 protects a 166 bp chromatosome in both “bulk” and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine. Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished. Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster. Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes.

In order to assess the potential of hydropsychid capture net anomalies as a bioindicator of chronic toxicity in streams and rivers, the effects of 2,4-dichlorophenol (2,4-DCP) exposure on the net-spinning behavior of Hydropsyche slossonae were examined for anomalies after 0, 5, 10, 15, and 20 exposure days to gradient concentration of 2,4-dichlorophenol. The net-spinning behavior was significantly affected when larvae were exposed to 1.0, 10, 25, and 50 microg small middle dotL(-1), as expressed by the occurrence of two distinct abnormalities. The first one was a distortion of the midline meshes, where the normal diamond-shape structure is disrupted and the meshes are separated by extra strands (called "midline anomaly). The second aberration observed was called "chaotic net, where the nets are highly irregular without any real structure or well-defined areas. A good correlation was found between the chaotic net frequencies and the reduction of ATP concentrations in the larvae, indicating possible uncoupling effects of 2,4-DCP on the oxidative phosphorylation process. Toxicity curves demonstrate that the sensitivity threshold of chaotic net frequencies ranged from 3.5 to 7 microg small middle dotL(-1), which is highly sensitive compared with other sublethal effects of 2,4-DCP on other aquatic species.

The aim of this study was to characterize polypeptide components of the capture net spun by trichopteran larvae Hydropsyche slossonae (Banks) (Trichoptera: Hydropsychidae). Thirty-one polypeptide bands were identified by SDS – polyacrylamide gel electrophoresis (SDS–PAGE) from extracted net material, with molecular weights ranging from 8500 to 179 000. Comparison with published data on Bombyx mori (L.) (Lepidoptera: Bombycidae) silk, treated under similar denaturing conditions, shows that six low molecular weight polypeptides ranging between 8500 and 18 800 in the silk of H. slossonae are absent from that of B. mori; furthermore, two high molecular weight polypeptides (210 000 and 220 000) detected in the silk of B. mori are not present in that of H. slossonae. Differences between both groups are probably related to their mode of living and to the specific use of silk (in air versus under water). Our findings are consistent with the current trend in the literature that silk spun by aquatic and terrestrial insects, as well as those spun by different species, is apparently made of different biopolymers according to the protein constituents. Hence, the polypeptide characterization of silk, combined with sequence data and (or) antibodies cross-reactivity data, could represent a potential tool for taxonomic classification improvement of aquatic insects. These results could eventually be used to characterize hydropsychid capture net anomalies induced by environmental pollution.

Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.

The pneumococcus undergoes spontaneous phase variation between an opaque and a transparent colony form. In an animal model of systemic infection following intraperitoneal inoculation of mice, the opaque phenotype was significantly more virulent than the transparent for each of 3 strains examined. The opaque phenotype was associated with 1.2- to 5.6-fold greater amounts of capsular polysaccharide compared with the transparent using a sandwich ELISA. A similar technique comparing the amount of total teichoic acid showed that the transparent phenotype had 2.1- to 3.8-fold more immunodetectable teichoic acid. This difference was confirmed by comparing the incorporation of [3H]choline into teichoic acid. Cell fractionation revealed that variation in quantity of incorporated choline was due to differences in cell wall-associated teichoic acid. Results suggest that the pneumococcus phase varies between a virulent form with more capsular polysaccharide and less teichoic acid and an avirulent form with less capsular polysaccharide and more teichoic acid.

The asymmetric catalyzed metallo-ene reaction was studied. Enantioselectivities up to 47% were observed using the Pd-ene reaction applied on the substrate 4. © 1997 Elsevier Science Ltd.

We have labelled a DNA-binding site on the globular domain of histone H5 (GH5) by ultraviolet-activated cross-linking of a self-complementary 5-bromodeoxyuridine (5BrU)-substituted oligonucleotide with the sequence 5'-AGCGA5BrUATCGCT-3'. Cross-linking was to His62, mainly to the protein backbone. This observation provides further support for the mode of binding of GH5 to DNA proposed on the basis of the similarity between the X-ray crystal structure of GH5 and the DNA-bound structures of catabolite activator protein and hepatic nuclear factor 3 gamma [Ramakrishnan, V. (1994) Curr. Opin. Struct. Biol. 4. 44-50].

We have identified a clear preference of histone H1 for CpG-methylated DNA, irrespective of DNA sequence. The conditions under which this preference is observed allowed cooperative binding of H1; the H1-DNA complexes formed were shown earlier to be 'tramlines' of two DNA duplexes bridged by an array of H1 molecules, and multiples of these. The preference for methylated DNA is clear in sedimentation assays, which also show that the preference is greater with increased methylation level, and in gel retardation assays with an oligonucleotide containing a single methyl-CpG pair; it is shared by the globular domain which also binds cooperatively to DNA. A small intrinsic preference of H1 for methylated DNA is also apparent in Southwestern assays where the immobilized H1 presumably cannot bind cooperatively. Methylated DNA in H1-DNA complexes was partially protected (relative to unmethylated DNA) against digestion by MspI but not by enzymes whose cutting sites were not methylated, consistent with a direct interaction of H1 with methylated nucleotides; this was also true of GH1-DNA complexes. H1 variants (spH1 and H5) from transcriptionally repressed nuclei have a stronger preference than H1 for methylated DNA, suggesting that this may be relevant to the stabilization of chromatin higher order structure and transcriptional repression.

HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain. Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns. They show qualitatively similar properties with quantitative differences. We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold [Weir et al. (1993) EMBO J. 12, 1311-1319]. We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy. The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D [Jones et al. (1994) Structure 2, 609-627]. There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains [Teo et al. (1995) Eur. J. Biochem. 230, 943-950] and will be the subject of further investigation. NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II. These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes [Love et al. (1995) Nature 376, 791-795; Werner et al. (1995) Cell 81, 705-714].

High-mobility-group protein 1 (HMG1) is an abundant, non-sequence-specific, chromosomal protein with two homologous, HMG-box, DNA-binding domains, A and B, and an acidic tail. The HMG-box motif also occurs, as a single copy, in some sequence-specific transcription factors, e.g. the sex-determining factor, SRY. We have investigated whether or not there are differences in the DNA-binding properties of the isolated A and B HMG-box domains of HMG1 and SRY and whether, in the case of A and B, there might also be differences due to different sequence contexts within the native protein. The basic regions that flank the HMG1 B box, giving B', enhance its DNA-binding, supercoiling and DNA-bending activities, and promote the self-association of the DNA-bound B-box. All the HMG-box domains bind with structure specificity to four-way junctions, but the structure selectivity is significantly greater for A and the SRY box than for the HMG1 B or B' domains, as judged by competition with excess plasmid DNA. The domains self-associate to different extents on supercoiled DNA and this may explain differences in the ability to discriminate between four-way junctions and supercoiled DNA. The HMG1 A, B and B' domains constrain negative superhelical turns in DNA, but the SRY HMG box does not. Only the full B domain (B') bends DNA in a ligase-mediated circularisation assay; the minimal B box, the A domain and the SRY box do not. Thus, despite a common global fold, the HMG box appears to have been adapted to various functions in different protein contexts.

We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.

The asymmetric catalyzed metallo-ene reaction was studied. Enantioselectivities up to 47% were observed using the Pd-ene reaction applied on the substrate 4. © 1997 Elsevier Science Ltd.

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. (15)N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a "sandwich"-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.

Direct discharge of untreated sewage to surface waters is a common practice in many parts of the world. However, relatively little is known about the behaviour of synthetic organic pollutants under these conditions. This paper describes a sampling campaign designed to track changes in water quality in a surface water system in Vientiane (Lao PDR) receiving significant quantities of untreated waste water. The study was based on following in-channel transport using a fluorescent tracer injected as a pulse, with a focus on the anionic surfactant linear alkylbenzene sulphonate (LAS) and ammonia. Water samples were collected at a number of stations with sampling times estimated to coincide with solute time-of-travel. The reduction in LAS concentration with flow-time could be approximated by first-order kinetics with a half life of about 7 h. Free ammonia concentrations decreased more slowly than LAS and remained above the level believed to be toxic for sensitive aquatic species along the entire channel. Changes in the ratios of LAS alkyl chain homologues to total LAS concentrations suggest a preferential removal of longer chain lengths. The role of biodegradation in the removal of LAS was confirmed by the presence of LAS metabolites (sulphophenylcarboxylates, SPCs) which increased systematically (as a fraction of LAS remaining) with flow-time.

The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA.

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous “HMG-box” DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.

A laboratory study on the sublethal effects of malathion on the net-spinning behavior of the caddisfly larvae Hydropsyche slossonae was conducted in order to assess the potential of net anomalies as an indicator of chronic exposure to organophosphorus insecticides. Two anomalies were identified after chronic exposure to 0.01, 0.05, 0.1, 0.5 and 1.0 µg l(-1) malathion. The first was a distortion of the midline meshes where the normal diamond shape structure was disrupted and the meshes were separated by extra strands (called 'midline' anomaly). The second aberration observed was a significant decrease in net symmetry. Both anomalies were highly correlated to the toxic action of malathion, i.e. inhibition of the acetylcholinesterase enzyme (AChE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of capture nets did not show any modification of silk polypeptides after exposure to malathion, indicating that net distortions were not related to silk composition. Both anomalies seem to represent the symptoms of the specific toxic action of malathion; nevertheless, they can serve as an index of the physiological condition of the larvae, especially the midline anomaly. The symmetry of the nets decreased significantly after exposure to 0.5 and 1.0 µg l(-1). However, the toxicity curves (EC(50)) showed that the sensitivity threshold for the midline anomaly ranged from 0.11 to 0.28 µg l(-1), which reflect more realistic exposure to concentrations expected to occur in the field. Hence, the use of capture net anomalies of hydropsychid larvae could represent a valuable indicator of sublethal toxicity induced by malathion and other organophosphorus insecticides in running waters.

We have investigated the nature of the “structure-specific” binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-way junction DNA. AB didomain binding favours the open, planar form of the junction, as shown by reaction with potassium permanganate. Site-directed cleavage of the DNA by a 1,10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain interacts with the widened minor groove at the centre, contacting one of the four strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolated B domain can, however, bind to the centre of the junction. The preferential binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably at least partly due to the higher positive surface potential in the DNA-binding region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged phosphates surrounding the central hole of the junction) and partly to differences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes.

A laboratory study on the sublethal effects of cadmium (Cd) on the net-spinning process of the larvae of Hydropsyche slossonae was conducted in order to assess the potential of net anomalies as an indicator of chronic exposure to Cd. Two major anomalies with different frequency levels were identified after chronic exposure to 0.37, 1.2, 11.6, 21.4 and 43.3 microg l(-1) of Cd. The first was a distortion of the midline meshes where the diamond-shape structure is disrupted and the meshes are separated by extra strands (called 'midline' anomaly). The second aberration consisted of a distortion of the rectilinear structure of net opening by strands being fused or added over the meshes (called 'crossover' anomaly). The midline distortion may be linked to a physiological stress caused by Cd, which can affect the control of the net-spinning process. It was not possible to relate the crossover aberrations to a specific toxic action of Cd, but data indicated that both anomalies are independent from each other and that two modes of action could be implicated. Protein analyses of capture nets have revealed silk polypeptide modifications at the highest Cd concentration tested, indicating a possible effect of Cd interaction with silk proteins. However, neither a gradient-concentration nor a time-dependent response could be established with both aberration frequencies. Silk protein modifications would rather play a secondary role in the appearance of both net anomalies, and mostly at a high concentration level. Finally, the toxicity curves (EC(50)) show that the sensitivity threshold for both types of aberration ranged from 1 to 5 microg l(-1) which is highly sensitive compared with other sublethal effects of Cd on other macroinvertebrate species. Hence, the use of capture-net anomalies of hydropsychid larvae would represent a valuable indicator of sublethal toxicity induced by Cd and possibly by other metals in running waters.

High-mobility-group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA-binding HMG-box domains, A and B, linked by a short basic region to an acidic carboxy-terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG-box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (approximately 20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure-specific preferences of single and tandem HMG boxes for four-way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non-sequence specific) HMG-box proteins.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.

We have compared transcriptionally competent and repressed genes with respect to their linker histone content and their ability to fold into higher-order structures. Histones were cross-linked covalently to DNA in chicken erythrocyte and oviduct nuclei by UV irradiation, and the DNA that was immunoprecipitated with anti-H1 and (for erythrocytes) anti-H5 antibodies was analysed for particular DNA sequences. None of the sequences investigated was free of H1 (H5). However, in mature erythrocytes the tissue-specific adult beta-globin gene (beta A) appears to be partially depleted of H5, and both the beta-globin gene and the H5 gene (also tissue-specific), as well as the 'housekeeping' beta-actin gene, appear to be partially depleted of H1 relative to inactive genes; in oviduct slight H1-depletion is detected on the ovalbumin gene relative to genes that are inactive in this tissue and the actin gene. Transcriptionally competent erythrocyte chromatin fragments, in contrast to inactive fragments, are unable to self-associate into 'pseudo-higher-order structures'. This is likely to be a consequence of the partial depletion of H5 and/or H1 in active chromatin, resulting in the breakdown of (probably cooperative) interactions between H5 and/or H1 molecules that otherwise mediate the assembly of pseudo-higher-order structures in vitro and a stable 30 nm chromatin filament in vivo.

A three-stage chemical modification procedure [Lambert, S. F. & Thomas, J. O. (1986) Eur. J. Biochem. 160, 191–201; Thomas, J. O. & Wilson, C. M. (1986) EMBO J. 5, 3531–3537] for selectively radiolabelling lysine residues that interact with DNA has been used to investigate core histone – DNA interactions in sea urchin sperm chromatin, in particular to determine the binding site of the long N-terminal domain of sperm-specific H2B. Comparison of the patterns of radiolabelling of core histones from extended chromatin and nucleosome core particles (which lack linker DNA) reveals the regions of the histones involved in interactions with the linker. The results show that the N-terminal domain of H2B is bound to DNA outside the 146-bp nucleosome core, presumably to the linker DNA. H2A and H4 make no substantial contacts with the linker in extended chromatin; the N-terminal tail of H4 is bound within the core particle, but the N-terminal tail of H2A is not bound in core particles or in extended chromatin, and may therefore have a role in higher-order structure. H3, like H2B, makes contacts with DNA outside the 146-bp nucleosome core in its N-terminal region, as well as elsewhere, and probably interacts with the two 10-bp extensions that complete the two turns of DNA in the nucleosome and/or with the linker.

The nature of the complexes formed between histone H1 and linear double-stranded DNA is dependent on ionic strength and on the H1 : DNA ratio. At an input ratio of less than about 60% (w/w) H1 : DNA, there is a sharp transition from non-co-operative to co-operative binding at a critical salt concentration that depends on the DNA size and is in the range 20 to 50 mM-NaCl. Above this critical ionic strength the H1 binds to only some of the DNA molecules leaving the rest free, as shown by sedimentation analysis. The ionic strength range over which this change in behaviour occurs is also that over which chromatin folding is induced. Above the salt concentration required for co-operative binding of H1 to DNA, but not below it, H1 molecules are in close proximity as shown by the formation of H1 polymers upon chemical cross-linking. The change in binding mode is not driven by the folding of the globular domain of H1, since this is already folded at low salt in the presence of DNA, as indicated by its resistance to tryptic digestion. The H1-DNA complexes at low salt, where H1 is bound distributively to all DNA molecules, contain thickened regions about 6 nm across interspersed with free DNA, as shown by electron microscopy. The complexes formed at higher salt through co-operative interactions are rods of relatively uniform width (11 to 15 nm) whose length is about 1.6 times shorter than that of the input DNA, or are circular if the DNA is long enough. They contain approximately 70% (w/w) H1 : DNA and several DNA molecules. These thick complexes can also be formed at low salt (15 mM-NaCl) when the H1 : DNA input ratio is sufficiently high (approximately 70%).

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.

We have asked whether histone H1 is tightly bound in chromatin at relatively low ionic strength (below physiological) or whether it can exchange between binding sites. We have studied this question in chromatin fragments generated by digestion with micrococcal nuclease, by mixing two fragments of known H1 content and different length (either a fragment radioactively labelled in all its histones with an unlabelled fragment, or two labelled fragments, only one of which contains H1) and then separating them again by centrifugation in sucrose gradients in order tom reexamine their H1 contents. At very low ionic strength (5 m m -Tris · HCl (pH 7.1), 1 m m -Na2EDTA, 0.5 m m -phenylmethylsulphonyl fluoride) there was very little (less than 5 to 10%) exchange of H1. In contrast, the presence of 70 m m -NaCl in the buffer caused rapid and complete equilibration of H1 between sites in less (possibly much less) than about one hour. At 30 m m added NaCl, the result was intermediate between those at 0 m m and 70 m m added NaCl, partial exchange occurring with a half-time of one to two hours. The results were essentially the same whether or not both fragments contained H1. We infer from these results that at physiological ionic strength (~150 m m ) there will be rapid and complete equilibration of H1 between sites in chromatin. We do not know whether the exchange of particular H1 subtypes is restricted to a particular class of binding site.

The DNA bound on the surface of the histone octamer in the nucleosome core particle partially protects the epsilon-amino side-chains of a subset of the lysine residues from reductive methylation. Most of the strongly protected lysines, which probably define the path of the DNA on the octamer surface, are in the globular ('structured') regions of the core histones rather than in the N-terminal or C-terminal 'tails'. Analysis of the protected peptides shows that the three strongest lysine-containing DNA-binding sites in the core histones contain the sequence-Lys/Arg-Lys-Thr/Ser-. On the assumption that the lysine-containing regions protected from chemical modification are also those found in lysine-DNA cross-links in another study [Mirzabekov et al. (1978) Proc. Natl Acad. Sci. USA 75, 4184-4188], particular DNA-protected peptides may be tentatively assigned to particular DNA contact points. This leads to a more detailed description of the DNA-binding regions on the octamer surface in the nucleosome core particle. Strong contacts, reflected in strongly protected lysines, may well contribute to the distortion of the DNA from smooth bending [Richmond et al. (1984) Nature (Lond.) 311, 532-537].

Tetramers of the arginine-rich histones H3 and H4 associate with supercoiled SV40 DNA either singly, giving tetrameric nucleoprotein complexes or in pairs giving octameric complexes, both of which are visualized as beads in the electron microscope. The relative amounts of the two complexes may be revealed by complete cross-linking of the proteins, followed by analysis in SDS-polyacrylamide gels. By electron microscopy of unmodified and of cross-linked complexes, both the tetrameric and the octameric complexes are shown to have a diameter of 8-9 nm and to contain about 145 base pairs (a nucleosome core length) of DNA. The compaction of the DNA in both cases is thus similar to that in the nucleosome, which has a diameter of about 12.5 nm and contains 200 base pairs of DNA.

We have characterised the histone and DNA contents of neuronal and glial nuclei from ox cerebral cortex which have, respectively, repeat lengths of 162 base pairs and 201 base pairs. Although the neuronal population cannot be obtained completely free of glial nuclear contamination, the degree of contamination is easily determined by counting, and has been allowed for in all the methods used here. By diphenylamine assay and flow cytofluorometry we find that the DNA contents of both nuclear types are essentially equal, and equivalent to the diploid value, contrary to some reports. By quantification of the core histones in known numbers of nuclei with respect to an added external standard, we have shown that the ratio of core histone octamers in the two nuclear types, neuronal and glial, is the inverse of the ratio of repeat lengths. Thus the same proportion of DNA is associated with core histone octamers in the two nuclear types, most simply all of the DNA. By complete radiolabelling of the lysine side chains of the histones with methyl [1-3H]acetimidate we have determined the stoichiometry of H1 relative to the core histones. Neuronal nuclei have a low H1 content of 0.45 molecule H1/nucleosome on average; glial nuclei have the 'normal' 1 H1 molecule/nucleosome. In neuronal nuclei about half of the nucleosomes therefore probably lack H1. Whether there is any relation between the low H1 content and the short DNA repeat length of neuronal nuclei, on the one hand, and their high transcriptional capacity (at least when assayed in vitro), on the other, remains to be established.

Recent work in many laboratories [l-8] has confirmed the central role of the arginine-rich histones H3 and H4 in the nucleosome [9]. H3 and H4 occur in solution as the tetramer (H3),(H4)a [lO,l l] and the protein core of the nucleosome probably comprises one such tetramer and two molecules each of the lysine-rich histones H2A and H2B (for references see [ 121 or [ 131). It is generally assumed that those properties of chromatin that are regenerated by reconstitution of DNA and histones H3 and H4 alone, such as nuclease sensitivity [ 11, a low angle X-ray diffraction pattern [6], a beaded appearance in the electron microscope [3-s], and insertion of supercoils into closed circular DNA [3,4,8], are generated by the combination of one tetramer with a nucleosome core length of DNA (about 140 base pairs). However, without better characterisation of reconstituted complexes it is not possible to assess the extent to which a single tetramer is capable of conferring nucleosome-like properties on 140 base pairs of DNA. We report here the preparation and partial characterisation of nucleosome-like particles containing two tetramers of H3 and H4 and about 140 base pairs of DNA; these are distinct from a complex containing one tetramer and 140 base pairs. The particles have been prepared in two ways by mild procedures involving dissociation of the lysine-rich histones from chromatin or nucleosome core particles with salt, thus ensuring minimal manipulation of, and damage to, H3 and H4.

1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed.