Prions are secreted in milk from clinically normal scrapie-exposed sheep.

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

Generation and characterisation of monoclonal antibodies to Rainbow trout (Oncorhynchus mykiss) prion protein.

We report the production and characterisation of three monoclonal antibodies to the prion protein (PrP) of Rainbow trout (Oncorhynchus mykiss), a piscine protein with characteristic structural features common to mammalian prion protein. All of the antibodies were used to detect PrP in ELISA, Western blot and by immunohistochemistry. The antibodies showed specificity for certain genera of the Salmonidae, binding to PrP of Rainbow trout and Atlantic salmon (Salmo salar) but not to that from Arctic char (Salvelinus alpinus). Using the immunoreagents in Western blots, we demonstrated that O. mykiss PrP protein is a 64 kDa protein present in brain, spinal chord and optic nerve. PrP was not detected in a range of peripheral tissues: eye, heart, stomach, intestine, liver, kidney, spleen, muscle and skin. Furthermore, PrP could be detected in all brain regions studied: optic lobe, cerebrum/olfactory lobe, cerebellum, hypothalamus/pituitary and medulla oblongata and was widespread within these tissues as determined by immunohistochemistry. These immunoreagents provide specific tools to study the biology of Rainbow trout and Atlantic salmon PrP and any possible transmissible spongiform encephalopathy-like disease of these economically important fish species.

Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide.

Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus. A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C-terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate. An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates. The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114). The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site. Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative. However, the antibody did react with one isolate with the motif (112)KRQ(114). There was no apparent reactivity with any of the avirulent isolates tested. We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates.

FHY1: a phytochrome A-specific signal transducer.

Phytochromes are plant photoreceptors that regulate plant growth and development with respect to the light environment. Following the initial light-perception event, the phytochromes initiate a signal-transduction process that eventually results in alterations in cellular behavior, including gene expression. Here we describe the molecular cloning and functional characterization of Arabidopsis FHY1. FHY1 encodes a product (FHY1) that specifically transduces signals downstream of the far-red (FR) light-responsive phytochrome A (PHYA) photoreceptor. We show that FHY1 is a novel light-regulated protein that accumulates in dark (D)-grown but not in FR-grown hypocotyl cells. In addition, FHY1 transcript levels are regulated by light, and by the product of FHY3, another gene implicated in FR signaling. These observations indicate that FHY1 function is both FR-signal transducing and FR-signal regulated, suggesting a negative feedback regulation of FHY1 function. Seedlings homozygous for loss-of-function fhy1 alleles are partially blind to FR, whereas seedlings overexpressing FHY1 exhibit increased responses to FR, but not to white (WL) or red (R) light. The increased FR-responses conferred by overexpression of FHY1 are abolished in a PHYA-deficient mutant background, showing that FHY1 requires a signal from PHYA for function, and cannot modulate growth independently of PHYA.

Resetting of the circadian clock by phytochromes and cryptochromes in Arabidopsis.

The authors sought to investigate the role of phytochromes A and B (phyA and phyB) and cryptochromes 1 and 2 (cryl and cry2) in the synchronization of the leaf position rhythm in Arabidopsis thaliana. The seedlings were transferred from white light-dark cycles to free-running conditions with or without exposure to a light treatment during the final hours of the last dark period. The phase advance caused by a far-red light treatment was absent in the phyA mutant, deficient in the fhy1 and fhy3 mutants involved in phyA signaling, and normal in the cryl and cryl cry2 mutants. The phase shift caused by blue light was normal in the cry2 mutant; reduced in the phyA, cryl, phyA cry1, and cry1 cry2 mutants; and abolished in the phyA cryl cry2 triple mutant. The phase shift caused by red light was partially retained by the phyA phyB double mutant. The authors conclude that cryl and cry2 participate as photoreceptors in the blue light input to the clock but are not required for the phyA-mediated effects on the phase of the circadian rhythm of leaf position. The signaling proteins FHY1 and FHY3 are shared by phyA-mediated photomorphogenesis and phyA input to the clock.

fhy3-1 retains inductive responses of phytochrome A.

The fhy3 mutation of Arabidopsis impairs phytochrome A (phyA)-mediated inhibition of hypocotyl growth without affecting the levels of phyA measured spectrophotometrically or immunochemically. We investigated whether the fhy3-1 mutation has similar effects on very low fluence responses (VLFR) and high irradiance responses (HIR) of phyA. When exposed to hourly pulses of far-red light, etiolated seedlings of the wild type or of the fhy3-1 mutant showed similar inhibition of hypocotyl growth, unfolding of the cotyledons, anthocyanin synthesis, and greening upon transfer to white light. In the wild type, continuous far-red light was significantly more effective than hourly far-red pulses (at equal total fluence). In the fhy3-1 mutant, hourly pulses were as effective as continuous far-red light, i.e. the failure of reciprocity typical of HIR was not observed. Germination was similarly promoted by continuous or pulsed far-red in wild-type and fhy3-1 seeds. Thus, for hypocotyl growth, cotyledon unfolding, greening, and seed germination, the fhy3-1 mutant retains VLFR but is severely impaired in HIR. These data are consistent with the idea that VLFR and HIR involve divergent signaling pathways of phyA.

Sheep monoclonal antibody fragments generated using a phage display system.

Monoclonal sheep antibodies have great potential for biomedical, veterinary and agricultural purpose. Although conventional sheep monoclonal antibodies can be generated by a modified hybridoma technology, the procedures are not routine. Here, we describe a method to generate recombinant sheep antibody fragments from immunised animals using a modified phage display system. Total RNA from pooled spleens of sheep immunised with the model antigens human serum albumin and conalbumin were used to amplify immunoglobulin V gene repertoires and an efficient two-step cloning method was employed to rapidly construct a phage display single-chain Fv (scFv) library. A total of 14 different scFvs were isolated and characterised. Sequence analysis indicated typical ovine immunoglobulin characteristics. Thirteen Vlambda and 11 VH genes were identified that could be grouped into the sheep Vlambda families I, II, VI and a single VH family. Soluble monomeric scFvs, produced in the periplasm of Escherichia coli, were subjected to affinity measurement via surface plasmon resonance analysis and affinities typical of the secondary immune response were observed. The method described here should be of value for the study of sheep immunology as well as for biorecognition in general.

High affinity ScFvs from a single rabbit immunized with multiple haptens.

We report the generation of single-chain Fv (scFv) fragments with high affinities against four different hapten molecules from a single immunised rabbit. The rabbit was immunised with a mixture of protein conjugates of four different haptens, namely the herbicide mecoprop and derivatives of the herbicides atrazine, simazine, and isoproturon. An scFv phage display library was constructed, and several scFvs with high affinity against each hapten were isolated. For each hapten, a single binder was selected by k(off) ranking and used for affinity determination. The affinities were in sub-nanomolar range and the lowest K(d) value obtained was 6.75 x 10(-10) M. An unusual feature of one of the anti-isoproturon scFvs was its ability to retain binding activity at pH1.7. The utility and potential of using a single animal and immunisation with multiple antigens for the production of multiple, specific, high affinity scFvs by phage display is discussed.

Cytoplasmic expression of a soluble synthetic mammalian metallothionein-alpha domain in Escherichia coli. Enhanced tolerance and accumulation of cadmium.

Bacteria are commonly used for bioremediation of heavy metal pollution and strategies to improve their performance in this respect are desirable. In this study, an Escherichia coli strain was engineered to express a common metallothionein-alpha domain. The metallothionein-alpha domain was over-expressed in the cytoplasm of E. coli as a fusion to the carboxyl terminal of maltose binding protein. The fusion protein was highly soluble in the cytoplasm of E. coli. When grown in the presence of cadmium, cells expressing the metallothionein-alpha fusion protein showed increased viability compared with control cells. Cells expressing the metallothionein-alpha also demonstrated increased accumulation of cadmium.

Selection of phage antibodies to surface epitopes of Phytophthora infestans.

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana.

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.

Selection of phage-display peptides that bind to cucumber mosaic virus coat protein.

Several discrete peptides that bind specifically to the coat protein of cucumber mosaic virus (CMV) were isolated from a diverse phage library displaying random nonapeptides on the major coat protein VIII. Enrichment was shown by polyclonal phage enzyme linked immunosorbent assay (ELISA) after three rounds of selection. Sequencing of the genes encoding 10 of these peptides revealed an absence of any conserved motifs, although nine of them contained a high proportion of proline residues. Some of the selected peptides were displayed at the N-terminus of thioredoxin and expressed in the cytoplasm of Escherichia coli. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CMV and CMV present in crude leaf extracts from infected plants. By dot blot analysis, a thioredoxin-peptide fusion could readily detect as little as 5 ng of CMV. The peptides did not bind to other plant viruses. These peptides have been shown to be specific and highly sensitive tools in the detection of CMV and, as well as their diagnostic potential, they could form the basis for a novel disease resistance strategy.

Photomophogenesis: Phytochrome takes a partner!

How light signals are transduced by phytochromes is still poorly understood. Recent studies have provided evidence that a PAS domain protein, PIF3, physically interacts with phytochromes, plays a role in phytochrome signal transduction and might be a component of a novel signalling pathway in plants.

Phytochrome D acts in the shade-avoidance syndrome in Arabidopsis by controlling elongation growth and flowering time.

Shade avoidance in higher plants is regulated by the action of multiple phytochrome (phy) species that detect changes in the red/far-red ratio (R/FR) of incident light and initiate a redirection of growth and an acceleration of flowering. The phyB mutant of Arabidopsis is constitutively elongated and early flowering and displays attenuated responses to both reduced R/FR and end-of-day far-red light, conditions that induce strong shade-avoidance reactions in wild-type plants. This indicates that phyB plays an important role in the control of shade avoidance. In Arabidopsis phyB and phyD are the products of a recently duplicated gene and share approximately 80% identity. We investigated the role played by phyD in shade avoidance by analyzing the responses of phyD-deficient mutants. Compared with the monogenic phyB mutant, the phyB-phyD double mutant flowers early and has a smaller leaf area, phenotypes that are characteristic of shade avoidance. Furthermore, compared with the monogenic phyB mutant, the phyB-phyD double mutant shows a more attenuated response to a reduced R/FR for these responses. Compared with the phyA-phyB double mutant, the phyA-phyB-phyD triple mutant has elongated petioles and displays an enhanced elongation of internodes in response to end-of-day far-red light. These characteristics indicate that phyD acts in the shade-avoidance syndrome by controlling flowering time and leaf area and that phyC and/or phyE also play a role.

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis.

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector.

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.

Phytochromes and photomorphogenesis in Arabidopsis.

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red-far-red light-absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome-encoding genes, PHYA-PHYE. Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.

Phytochrome E influences internode elongation and flowering time in Arabidopsis.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion.

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains of Staphylococcus aureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein delta pIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.

Fluorescence spectroscopy and photochemistry of phytochromes A and B in wild-type, mutant and transgenic strains of Arabidopsis thaliana.

Phytochrome (P) was characterized in etiolated seedlings of wild-type, mutant and transgenic strains of Arabidopsis with the use of low-temperature (85 K) fluorescence spectroscopy and photochemistry. The position (lambda max) of the Pr emission spectrum, its intensity (F0) proportional to [P tot] and the extent of the Pr-->lumi-R phototransformation at 85 K (gamma 1) were shown to vary depending on the plant strains and tissues used, while the extent of the Pr-->Pfr transformation at 273 K (gamma 2) remained relatively constant. Depletion of phyA (fre1-1 in Nagatani et al., Plant Physiol. 102 (1993) 269-277, and fhy2-2 in Whitelam et al., Plant Cell 5 (1993) 757-768) resulted in a steep decrease of F0 to approximately equal to 10%. The phyB mutant (hy3-B064 in Reed et al., Plant Cell 5 (1993) 147-157) revealed a slight reduction (by approximately equal to 20%) of F0 while lambda max and gamma 1 remained practically unaffected. In phyAphyB mutuant no P emmission was observed. Overexpression of oat phyA (13k7 and 21k15 in Boylan and Quail, Proc. Natl. Acad. Sci. USA 88 (1991) 10806-10810) brought about an increase of F0 by two or three times, a shift of lambda max to 685 nm and an increase of gamma 1 to 0.3-0.4. On the contrary, an increase of F0 (up to 40%) in Arabidopsis and rice phyB overexpressors (ABO and RBO in Wagner et al., Plant Cell 3 (1991) 1275-1288) was followed by a decrease of gamma 1 values to 0.13-0.14. These data together with the results on phyB (lh) mutant of cucumber prove the existence of the two phyA populations with high (phyA') and low (phyA") photochemical activity at low temperatures. PhyB emits maximally in the same region as phyA in Arabidopsis (approximately equal to 683 nm) and at shorter wavelength (< 680 nm) in rice. It is characterized by low photochemical activity at 85 K (gamma 1 < or = 0.05) and can be attributed in this respect to the same pigment type as phyA".