Prions and the prion disorders

One of us remembers sitting in a high school biology class in 1977 being taught about scrapie, a naturally occurring disorder of sheep. The teacher had no particular interest in agriculture, but was pointing out some peculiar characteristics of this disease as a biological curiosity on a wet Friday afternoon. The prion disorders captured the imagination of a range of biologists (including that teacher) well before the epidemic of bovine spongiform encephalopathy (BSE) and the appearance of a new variant of the human prion disease, Creutzfeldt Jakob disease (CJD), in the UK, because of their extraordinary biology and the unique properties of the infectious agent. We review the results of studies leading to a convergence of evidence that the causative infectious agent, the `prion', is devoid of nucleic acid and is composed of an abnormal isoform of a host-encoded protein, the prion protein (PrP). Received: 2 March 1998 / Accepted: 2 March 1998     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae .     

The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete M_r 2 × 10⁶ dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.     

Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures

Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products using thermostable polymerase produces fragments that are shorter than expected when the enzyme prematurely detaches as it approaches the 5′-end of the DNA template. These premature terminations result in a substantially reduced reading length of the DNA sequence. Since some DNA templates spontaneously fold and form stable secondary structures at temperatures that are typically used for primer annealing, one factor that may cause premature terminations to occur is the formation of secondary structures in the template during the annealing step of the cycle sequencing reaction. We describe a simple and effective method for reducing premature terminations in DNA sequences. We demonstrate that maintaining the annealing temperature of the cycle sequencing reaction above a critical temperature reduces premature terminations in DNA sequences that regularly contain premature terminations when the temperature of the annealing step is 60°C. In the method described, annealing and extension of the primer along the template take place at the same temperature (72°C). This procedure for reducing premature terminations can be applied when sequencing with primers that are relatively long (at least 27 mer) and have high optimal annealing temperatures.     

Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.     

Ecotoxicological threshold levels of a mixture of herbicides (atrazine, diuron and metolachlor) in freshwater microcosms

Twelve indoor, plankton-dominated, freshwater microcosms (600 l) were used to study the effect of a mixture of herbicides on structural and functional aspects of these ecosystems. The EC50, 72 h values of the most susceptible standard test alga Selenastrum capricornutum (EC50, atrazine=54 μg l−1, EC50, diuron=15 μg l−1, EC50, metolachlor=56 μg l−1) were used as a starting point for the dosage applied in the microcosms (dosages: 0, 0.01, 0.03, 0.1, 0.3, 1× EC50). The microcosms were exposed to chronic levels for 28 days and subsequently monitored for 4 more weeks. The following effects were observed: (1) direct effects became apparent from an initial drop in photosynthesis efficiency, pH and oxygen concentration and a decrease in the abundance of several phytoplankton taxa at the 0.3 × EC50 treatment level and higher. (2) Fourteen days post application an increase in the abundance of several phytoplankton taxa (Chlamydomonas sp. and Stephanodiscus/Cyclotella) was observed; oxygen concentrations recovered while alkalinity, conductivity and total inorganic nitrogen were elevated. (3) Effects on fauna were minor. Daphnia galeata showed a decreasing trend and the cyclopoid copepods an increasing trend at the end of the experiment. Multivariate statistical analyses demonstrated no effects of any treatment level on the zooplankton community. Effects were reported for the phytoplankton community at dose levels of 0.3 × EC50 and higher. On species level the most sensitive taxon was Chlorophyceae coccales. For this taxon a NOEC at the dose level of 0.01 × EC50 was calculated. This effect however was relatively small in magnitude and merely based on an increase in numbers in the control and lowest treated microcosms rather than a decrease in numbers in all other treatments. The standards based on algal toxicity data, as adopted by the Uniform Principles, consist of a safety factors of 0.1 to be multiplied with the EC50. The NOEC of coccales was lower than 0.1 × EC50. All other observed variables in this aquatic ecosystem were sufficiently protected against the mixture of herbicides by the safety factor as proposed in the Uniform Principles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Plant defence signals and Batesian mimicry

In a game theory context, we investigated conditions for an evolutionarily stable equilibrium of defended, signalling plants, and plants mimicking these signals – that is, conditions for a stable mimicry complex. We modelled this in three steps. First, we analysed conditions for selection for defended, signalling plants, in a population of undefended plants. Second, we analysed conditions for when mimicking plants can invade a population of defended, signalling plants, leading to a stable equilibrium between the two strategies. Third, we analysed how sampling of signalling plants by herbivores affects the equilibrium between the strategies. The predictions show that mimicry of plant defence signals may be common, and even imperfect mimics could invade a population of defended, signalling plants. Whether the latter prediction holds or not depends on how herbivores generalize over signals, and on the length of their avoidance sequence'. The length of the avoidance sequence is the number of signalling plants that a herbivore avoids to attack, after attacking a defended plant. If herbivores always sample signalling plants, then mimicry cannot evolve, whereas if herbivores have a long avoidance sequence, this may allow selection even for imperfect mimics.