The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements.

BACKGROUND: A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained. RESULTS: Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences. CONCLUSION: The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.

Eight prion strains have PrP(Sc) molecules with different conformations.

Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

Development of chromosome-specific BAC resources for genomics of bread wheat.

The large bread wheat genome (1C approximately 17 Gbp) contains a preponderance of repetitive DNA and the species is polyploid. These characteristics together serve to hamper the molecular analysis of the wheat genome. Its complexity can, however, be reduced by using flow cytometry to isolate individual chromosomes, and these can be exploited to construct chromosome-specific BAC libraries. Such libraries simplify the task of physical map construction, positional cloning and the targeted development of genetic markers. Rapid improvements in the efficiency and cost of DNA sequencing provide an opportunity to contemplate sequencing the wheat genome by preparing sequence-ready physical maps for each chromosome or chromosome arm in turn. The quality of the chromosome-specific libraries depends on their chromosome coverage and the mean insert size. First-generation libraries suffered from a relatively low mean insert size, but improvements to the protocol have generated a second wave of libraries with a significantly increased mean insert size and better chromosome coverage. Each chromosome (arm)-specific library is composed of a manageable number of clones, and so represents a practical tool in the area of wheat genomics.

A synthetic peptide initiates Gerstmann-Sträussler-Scheinker (GSS) disease in transgenic mice.

The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.

Scrapie infectivity and prion protein are distributed in the same pH range in agarose isoelectric focusing.

We separated lysed synaptosomal-microsomal membrane fraction from scrapie-infected hamster brain in preparative agarose isoelectric focusing. We also studied the distribution of PrP27-30 and scrapie infectivity in 13 regions of the gel in the range of pH 3.5 to 9.3. Most of the infectivity remained in the trough, where it had been placed at the beginning of the electrophoresis, along with PrP27-30. Scrapie infectious particles that encountered the gel demonstrated charge heterogeneity and were distributed in the range of pH 5.4 to 9.3. Analysis of charge heterogeneity of PrP27-30 after sodium dodecyl sulfate solubilization showed an isoelectric pattern in the same pH range as that for scrapie infectious particles. The similarity in charge heterogeneity between infectivity and PrP27-30, together with copurification, support the idea that PrP27-30 is an essential component of the scrapie infectious agent.

Scrapie-associated precursor proteins: antigenic relationship between species and immunocytochemical localization in normal, scrapie, and Creutzfeldt-Jakob disease brains.

We describe the antigenic properties and detection of a normal isoform of scrapie-associated precursor protein (PrP33-35C) in normal, and both normal and scrapie isoforms in scrapie- or Creutzfeldt-Jakob disease (CJD)-infected mouse, hamster, and human brains, using a variety of specific antibodies. Polyclonal antibodies raised against mouse and hamster PrP27-30 and against a synthetic peptide of the N-terminal sequence of this protein were used as immunologic probes. PrP27-30 purified as a primary immunogen corresponded to the lower molecular mass peptide, with Mr between 9.3 and 13.5 kd as estimated by size-exclusion high-pressure liquid chromatography. ELISA and immunoblot techniques demonstrated that antibodies recognized homologous antigens as well as precursor proteins from brains (PrP33-35C) and the scrapie isoform of scrapie-associated proteins (PrP33-35Sc/CJD and PrP27-30) from scrapie- and CJD-infected brains. The normal, scrapie, and CJD isoforms of scrapie-associated proteins share common epitopes with varying degrees of interspecies homology. Specific antigen detected in neurons indicated that these proteins are synthesized primarily in these cells. In infected brains, extracellular amyloid deposits formed by the scrapie isoform of PrP protein also strongly reacted with anti-PrP antibodies.

Immunohistochemical localization of prion protein in spongiform encephalopathies and normal brain tissue.

We used polyclonal antibodies raised against hamster and mouse PrP27-30 as immunologic probes to study the localization of intracellular and extracellular deposits of prion protein in normal and scrapie-infected mouse and hamster brains and in Creutzfeldt-Jakob disease (CJD)-infected mouse brains. In addition, we examined normal human brain and brain tissues from patients with CJD, kuru, Alzheimer's disease, and idiopathic chronic encephalitis. There was positive staining in the cytoplasm of neurons of normal and scrapie- and CJD-infected mice, and in the neurons of normal and scrapie-infected hamsters. The staining pattern suggests the localization of PrP in an intracellular membrane compartment, most likely the rough endoplasmic reticulum or Golgi apparatus. Antibodies raised against a 15-amino-acid synthetic peptide of the N-terminal of hamster PrP27-30 displayed a similar pattern of staining in mouse brain sections. We observed no intracellular staining in human brain sections obtained at autopsy. Antibodies prepared against mouse and hamster PrP27-30 reacted with amyloid plaques in scrapie-infected mouse and kuru- and CJD-infected human brain sections but not with amyloid plaques in the brain of a patient with Alzheimer's disease.

Subcellular distribution and physicochemical properties of scrapie-associated precursor protein and relationship with scrapie agent.

We studied the biologic properties of hamster-adapted scrapie (strain 263K) and its relationship to the precursor protein of scrapie (PrP33-35Sc). The highest titer of infectious material and the greatest concentration of PrP33-35Sc were in the fractions containing microsomal and synaptosomal membranes. We found traces of infectivity in the absence of PrP33-35Sc associated with matrix protein. Partitioning of membranes with neutral chloroform-methanol resulted in concentration of PrP33-35Sc and infectivity within the interphase layer. Recombination of membrane glycoproteins (interphase) with lipids extracted from homologous brains decreased infectivity greater than or equal to 4 logs. Temperature-dependent phase separation of infected synaptosomal and microsomal membranes with Triton X-114 yielded a phospholipid-rich phase containing a high concentration of PrP33-35Sc and greatest infectivity titers. This material spontaneously formed liposomes, indicating that PrP33-35Sc and PrP33-35C precursor proteins are highly hydrophobic intrinsic membrane components integrated with phospholipids. Homologous membrane phospholipids appear to prevent aggregation of the scrapie isoform of PrP and maintain high levels of infectivity.

Quantitative traits of prion strains are enciphered in the conformation of the prion protein.

Variations in prions, which cause different disease phenotypes, are often referred to as strains. Strains replicate with a high degree of fidelity, which demands a mechanism that can account for this phenomenon. Prion strains differ by qualitative characteristics such as clinical symptoms, brain pathology, topology of accumulated PrP(Sc), and Western blot patterns of glycosylated or deglycosylated PrP(Sc). Since none of these qualitative features can directly explain quantitative strain traits such as incubation time or dose response, we analyzed conformational parameters of PrP(Sc) and the rate of accumulation in different prion strains. Using the conformation-dependent immunoassay (CDI), we were able to discriminate among PrP(Sc) molecules from eight different prion strains propagated in Syrian hamsters. CDI quantifies PrP isoforms by simultaneously following antibody binding to both the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupied a unique position, indicating that each strain accumulated different concentrations of particular PrP(Sc) conformers. This conclusion was supported by a unique pattern of equilibrium unfolding of PrP(Sc) found within each strain. By comparing the PrP(Sc) levels before and after limited proteinase K digestion, we found that each strain produces a substantial fraction of protease-sensitive PrP(Sc). We asked whether this fraction of PrP(Sc) might reflect those PrP(Sc) molecules that are most readily cleared by cellular proteases. When the protease-sensitive PrP(Sc) fraction was plotted as a function of the incubation time, a linear relationship was found with an excellent correlation coefficient (r = 0.94). Combined with the data on time courses of prion infection in Tg(MHu2M) and Tg(SHaPrP) mice, the results argue that different incubation times of various prion strains may arise predominantly from distinct rates of PrP(Sc) clearance rather than from different rates of PrP(Sc) formation.

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres.

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.

Effect of saliva contamination on the bond of dentin to resin-modified glass-ionomer cement.

This in vitro study compared the shear bond strength of a resin-modified glass-ionomer restorative material (Fuji II LC) bonded to saliva-contaminated dentin versus non-contaminated dentin. Seventy-five extracted human molar teeth were randomly divided into five groups of 15 samples each. The dentin was treated with 10% polyacrylic acid for 20 seconds, rinsed, and dried. The acid-treated dentin surfaces in Groups 1-4 were contaminated with saliva. In Group 1, the saliva was air thinned. In Groups 2-4, saliva was dried completely with compressed air. The saliva-contaminated dentin in Group 3 was rinsed and dried. The saliva-contaminated dentin in Group 4 was rinsed, dried, treated with 10% polyacrylic acid, and dried. Specimens in Group 5 received no contamination. The resin-modified glass-ionomer cement restorative material was mixed and applied to the dentin surfaces. Following placement of the restorative material and 7 days of storage, the specimens were thermo-cycled 300 times. Using the Instron Universal Testing Machine, a shear force was applied to the restorative material. Shear bond strength values were compared among the groups using a one-way ANOVA and Student-Neuman-Keuls Multiple Range Test (alpha = 0.05). The non-contaminated specimens (Group 5) were significantly stronger than the contaminated specimens (Groups 1-4). There were no significant differences in bond strength among the groups containing contaminated specimens. Salivary contamination occurring after dentin etching significantly reduced the bond strength of the resin-modified glass-ionomer restorative material to dentin. Neither rinsing nor rinsing and re-etching resulted in bond strengths as great as to non-contaminated dentin.

Revealing smuggled nuclear material covered by a legitimate radioisotope shipment using CdTe-based gamma-ray spectrometry.

Illicit trade of nuclear materials (NM) represents a serious challenge to radiation monitoring upon scenarios, when legitimate radioisotope shipments are used to obscure the weak radiation of NM. Planar and hemispherical Cd(Zn)Te detectors with a portable mini-multichannel analyzer were proven to be suitable, in measuring times of 10min order, for revealing the presence of low-enriched or natural U-bearing reactor fuel pellets in amounts of kg order, placed beside transport containers of lead or depleted uranium, which contain high activity 60Co (10GBq range) or 192Ir (TBq range) radioisotope sources. Such a hand-held or portable device may help authorities combating illicit trafficking of nuclear materials.