Two's company, three's a crowd: co-occurring pollinators and parasite species in Breynia oblongifolia (Phyllanthaceae).

BACKGROUND: Obligate pollination mutualisms (OPMs) are specialized interactions in which female pollinators transport pollen between the male and female flowers of a single plant species and then lay eggs into those same flowers. The pollinator offspring hatch and feed upon some or all of the developing ovules pollinated by their mothers. Strong trait matching between plants and their pollinators in OPMs is expected to result in reciprocal partner specificity i.e., a single pollinator species using a single plant species and vice versa, and strict co-speciation. These issues have been studied extensively in figs and fig wasps, but little in the more recently discovered co-diversification of Epicephala moths and their Phyllanthaceae hosts. OPMs involving Epicephala moths are believed occur in approximately 500 species of Phyllanthaceae, making it the second largest OPM group after the Ficus radiation (> 750 species). In this study, we used a mixture of DNA barcoding, genital morphology and behavioral observations to determine the number of Epicephala moth species inhabiting the fruits of Breynia oblongifolia, their geographic distribution, pollinating behavior and phylogenetic relationships. RESULTS: We found that B. oblongifolia hosts two species of pollinator that co-occurred at all study sites, violating the assumption of reciprocal specificity. Male and female genital morphologies both differed considerably between the two moth species. In particular, females differed in the shape of their ovipositors, eggs and oviposition sites. Phylogenetic analyses indicated that the two Epicephala spp. on B. oblongifolia likely co-exist due to a host switch. In addition, we discovered that Breynia fruits are also often inhabited by a third moth, an undescribed species of Herpystis, which is a non-pollinating seed parasite. CONCLUSIONS: Our study reveals new complexity in interactions between Phyllantheae and Epicephala pollinators and highlights that host switching, co-speciation and non-pollinating seed parasites can shape species interactions in OPMs. Our finding that co-occurring Epicephala species have contrasting oviposition modes parallels other studies and suggests that such traits are important in Epicephala species coexistence.

Clathrin cubes: an extreme variant of the normal cage.

Clathrin triskelions form polyhedral cages with hexagonal and pentagonal faces when dialyzed against suitable assembly buffers. However, when the buffer is made 12% saturated in ammonium sulfate and the dialysis is performed at 4 degrees C, clathrin polymerizes into cubes. The cube is constructed from eight triskelions with one at each corner. The edge length of the cube is approximately 45 nm, equivalent to the length of the leg of a triskelion. Thus, each edge of the cube is composed of two antiparallel legs overlapping over their whole length. The interactions between the legs in the cube are a subset of those postulated to occur in cages. Indeed, the cube can be derived from a pentagonal dodecahedron by removing 12 of the 20 triskelions with only slight adjustment of the legs of the remaining triskelions. The cube forms regular arrays and appears to be a favorable species for crystallization of clathrin.

A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated.

Monoclonal antibodies against the 110-kD component of the yeast spindle pole body (SPB) were used to clone the corresponding gene SPC110. SPC110 is identical to NUF1 (Mirzayan, C., C. S. Copeland, and M. Synder. 1992. J. Cell Biol. 116:1319-1332). SPC110/NUF1 has an MluI cell cycle box consensus sequence in its putative promoter region, and we found that the transcript was cell cycle regulated in a similar way to other MluI-regulated transcripts. Spc110p/Nuflp has a long central region with a predicted coiled-coil structure. We expressed this region in Escherichia coli and showed by rotary shadowing that rods of the predicted length were present. The 110-kD component is localized in the SPB to the gap between the central plaque and the sealed ends of the nuclear microtubules near the inner plaque (Rout, M., and J. V. Kilmartin. 1990. J. Cell Biol. 111:1913-1927). We found that rodlike structures bridge this gap. When truncations of SPC110 with deletions in the coiled-coil region of the protein replaced the wild-type gene, the gap between the central plaque and the ends of the microtubules decreased in proportion to the size of the deletion. This suggests that Spc110p connects these two parts of the SPB together and that the coiled-coil domain acts as a spacer element.

Assembly and analysis of conical models for the HIV-1 core.

The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.

DNA recognition by the oestrogen receptor: from solution to the crystal.

BACKGROUND: The steroid/nuclear hormone receptors are a large family of conserved ligand-activated transcription factors that regulate gene expression through binding to response elements upstream of their target genes. Most members of this family bind to DNA as homodimers or heterodimers and recognize the sequence, spacing and orientation of the two half-sites of their response elements. The recognition and discrimination of the sequence and arrangements of these half-sites are mediated primarily by a highly conserved DNA-binding domain. RESULTS: Here we describe the DNA-binding properties of the isolated DNA-binding domain of the oestrogen receptor, the ERDBD, and its refined NMR structure. This domain is monomeric in solution, but two molecules bind cooperatively to specific DNA sequences; this cooperativity determines the arrangement of half-sites that is recognized by the ERDBD. The 10 carboxy-terminal residues and a region of 15 residues within the domain are disordered in the solution structure, yet are important for DNA binding. CONCLUSION: The cooperative nature of ERDBD binding to DNA is important. The previously-determined X-ray structure of the ERDBD dimer bound to DNA shows that the 15 internal residues disordered in solution make contact both with DNA and with the corresponding region of the other monomer. These results suggest that these residues become ordered during the process of binding to DNA, forming the dimer interface and thus contributing to the cooperative interaction between monomers.

Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.

Crystal structure of uncleaved ovalbumin at 1.95 A resolution.

Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.

Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure.

To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.

Nuclear localization signal recognition causes release of importin-alpha from aggregates in the cytosol.

Importin-alpha is a cytosolic receptor that recognizes classical Nuclear Localization Signals (NLSs) and mediates import into the nucleus. We have used a number of methods to investigate the aggregation state of Xenopus importin-alpha both as a recombinant, purified protein and in cytosolic extracts. We have found that recombinant importin-alpha aggregates at a protein concentration similar to that estimated to be present in the Xenopus cytoplasm, and that the importin-alpha aggregation is relieved by NLS peptide binding, with the importin-alpha then binding the NLS as a monomer. We have also found that in HeLa cytosolic extracts, importin-alpha is present in an aggregated form. Similarly to the purified importin-alpha aggregation, NLS peptides relieve the aggregation of importin-alpha in the cytosol. These observations indicate that aggregation of importin-alpha in the cytosol may be an intrinsic property of the import receptor and may be functionally related to NLS binding.Our results suggest a novel mechanism for NLS recognition, whereby NLSs mediate disassembly of importin-alpha aggregates in the cytosol.

Crystal structure of the nucleosome core particle at 16 A resolution.

The crystal structure of the nucleosome core particle has been studied by neutron diffraction to a resolution of 16 A. By using H2O/D2O solvent contrast variation, the structures of the DNA and histone core were analysed separately. The DNA, as seen at this resolution, forms a super-helix of pitch 25.8 A, radius 42.1 A and 1.8 turns in length. The histone core itself is approximately helical and follows the DNA along the inside of the super-helix, giving the nucleosome core particle an overall 2-fold axis of symmetry. Four regions can be distinguished in the protein density, which we interpret as dimers of histones within the octameric core. The dimers have been assigned on the basis of other evidence as being of two kinds, (H2A-H2B) and (H3-H4). Because solvent contrast variation can distinguish between hydrophobic and hydrophilic regions in the protein density, our results suggest that the interface between the monomers of each dimer is probably quite hydrophobic in character, while the interaction between dimers is weaker and/or more hydrophilic. The protein is in contact with most of the DNA and there are some regions where it may penetrate between the turns of the super-helix. In particular, the tetramer (H4-H3)-(H3-H4) is in close contact with the central part of the DNA, but significant contacts are seen also between the histones H3 and the extremities of the super-helix, thus explaining the stability of a nucleosome-like particle depleted of H2A and H2B. Significant departures from the molecular 2-fold axis of symmetry occur in the relative arrangements of the two (H2A-H2B) dimers.

Role of base-backbone and base-base interactions in alternating DNA conformations.

Sequence-specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar-phosphate backbone. The TT step is inflexible and B-like, as it has two methyl groups which interlock with each other and with the sugar-phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A-conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine-pyrimidine, pyrimidine-purine, purine-pyrimidine steps, respectively, but to less extent, depending on the number of non-A:T basepairs in the steps.

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Single crystals of long DNA molecules.

We have grown single crystals of long DNA molecules, 54 and 65 base pairs in length which encompass the binding site of the Xenopus protein transcription factor IIIA (TFIIIA). These molecules are considerably longer than those previously crystallised. X-ray diffraction shows that the crystals are ordered to a resolution of 9 A. The DNA molecules are arranged side to side in layers which are orientated at 120 degrees with respect to adjacent layers. The phosphate backbones of molecules in adjacent layers are interdigitated in both the major and minor grooves. The intensity distribution indicates that the average structure is B-like, although CD spectra in solution are more consistent with the structure, intermediate between A- and B-, found in crystals of a nonamer fragment of the binding site. The B-character in our crystals may therefore be impressed by the tight packing between layers.