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.

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.

Results of latissimus dorsi tendon transfer for irreparable cuff tears.

Rupture of the supraspinatus and infraspinatus tendon (and teres minor) can cause loss of active external rotation (ER), entailing severe functional disability in daily activities. Latissimus dorsi tendon transfer (LDTT), proposed by Gerber in 1988, appears to be the best adapted solution in these cases of irreparable posterior and superior cuff tears. Between 2001 and 2004, 30 patients were operated on by the technique described by Gerber, with the transfer fixed anteriorly to the subscapularis tendon and laterally to the greater tuberosity by transosseous suture. One patient, subsequently requiring revision with a reversed prosthesis, was considered as a failure. Twenty-six patients were reviewed with a mean follow-up delay of 34+/-12 months. There were 14 men and 13 women. Mean age was 55.5 years (36 to 71 years). Preoperatively, active ER was symmetric in seven cases, loss of active ER was moderate with positive lag sign in five cases, significant with positive dropping sign in six cases, and severe in nine cases. Fatty muscular degeneration was present and significant in all cases for the infraspinatus muscle and in 14 cases for the teres minor muscle (associated with significant ER loss). Subjectively, 85% of the patients were very satisfied or satisfied and the Subjective Shoulder Value (SSV) was 68+/-17%. The pain score improved from 4.8+/-3 preoperatively to 12.2+/-2 postoperatively, strength from 3.7+/-2 kg to 4.2+/-1.8 kg, mean Constant score from 50+/-12 to 74+/-9, and Constant score adjusted for age and gender from 62+/-15% to 91+/-11%. Mean active ER gain was 7 degrees (-30 degrees to +50 degrees). The loss of active ER was aggravated in one case, unchanged in three, improved in nine and corrected in six. Hornblower sign was corrected in six cases and persisted in nine. Postoperatively, 8% of the patients were unable to eat and drink, compared to 64.7% preoperatively. The results of this series are comparable to those found in the literature for first-intention cases. LDTT restored active ER, but the results were incomplete and variable. Improvement was better in case of severe preoperative active ER deficit and insufficiency of the teres minor muscle. Recovery of strength was not observed in the present series. A narrow subacromial space and grade-3 Hamada classification had negative impact. In spite of an expected tenodesis effect, LDTT did not recenter the humeral head. LDTT compensates the deficient teres minor muscle rather than the infraspinatus muscle. The optimal indication for LDTT is irreparable superior and posterior rotator cuff rupture with loss of active ER associated with a deficient teres minor muscle. It is debatable whether LDTT is indicated in the absence of active motion deficiency: improvement was observed in these cases, but only in terms of subjective criteria.

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.

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.).

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radkal and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in

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.

Branched polyamines cure prion-infected neuroblastoma cells.

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.

Mimicking dominant negative inhibition of prion replication through structure-based drug design.

Recent progress determining the structure of the host-encoded prion protein (PrP(C)) and the role of auxiliary molecules in prion replication permits a more rational approach in the development of therapeutic interventions. Our objective is to identify a new class of lead compounds that mimic the dominant negative PrP(C) mutants, which inhibit an abnormal isoform (PrP(Sc)) formation. A computational search was conducted on the Available Chemicals Directory for molecules that mimic both the spatial orientation and basic polymorphism of PrP residues 168, 172, 215, and 219, which confer dominant negative inhibition. The search revealed 1,000 potential candidates that were visually analyzed with respect to the structure of this four-residue epitope on PrP(C). Sixty-three compounds were tested for inhibition of PrP(Sc) formation in scrapie-infected mouse neuroblastoma cells (ScN2a). Two compounds, Cp-60 (2-amino-6-[(2-aminophenyl)thio]-4-(2-furyl)pyridine-3, 5-dicarbonitrile) and Cp-62 (N'1-(¿5-[(4, 5-dichloro-1H-imidazol-1-yl)methyl]-2-furyl¿carbonyl)-4 methoxybenzene-1-sulfonohydrazide), inhibited PrP(Sc) formation in a dose-dependent manner and demonstrated low levels of toxicity. A substructure search of the Available Chemicals Directory based on Cp-60 identified five related molecules, three of which exhibited activities comparable to Cp-60. Mimicking dominant negative inhibition in the design of drugs that inhibit prion replication may provide a more general approach to developing therapeutics for deleterious protein-protein interactions.

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.

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.

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice.

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.

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.

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.

Prion protein selectively binds copper(II) ions.

The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231). Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. Binding of Cu2+ also promotes the conformational shift from a predominantly alpha-helical to a beta-sheet structure. Equilibrium dialysis experiments indicate a binding stoichiometry of approximately 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.

Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice.

There is considerable concern that bovine prions from cattle with bovine spongiform encephalopathy (BSE) may have been passed to humans (Hu), resulting in a new form of Creutzfeldt-Jakob disease (CJD). We report here the transmission of bovine (Bo) prions to transgenic (Tg) mice expressing BoPrP; one Tg line exhibited incubation times of approximately 200 days. Like most cattle with BSE, vacuolation and astrocytic gliosis were confined in the brainstems of these Tg mice. Unexpectedly, mice expressing a chimeric Bo/Mo PrP transgene were resistant to BSE prions whereas mice expressing Hu or Hu/Mo PrP transgenes were susceptible to Hu prions. A comparison of differences in Mo, Bo, and Hu residues within the C terminus of PrP defines an epitope that modulates conversion of PrPC into PrPSc and, as such, controls prion transmission across species. Development of susceptible Tg(BoPrP) mice provides a means of measuring bovine prions that may prove critical in minimizing future human exposure.

Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form.

A fundamental step in the pathogenesis of spongiform encephalopathies (prion diseases) is the conversion of the cellular isoform of prion protein (PrPC) into the infectious form (scrapie isoform, PrPSc), apparently by a conformational mechanism. Comparison of the native secondary and tertiary structures of both proteins is essential to elucidate the molecular basis of this transformation. To obtain sufficient quantities of native-like PrPC, we have developed a semipreparative method to purify PrPC from hamster brains. PrPC was solubilized from purified synaptosomal and microsomal membranes by the nonionic detergent n-octyl- beta-glucopyranoside; the soluble fraction was loaded at pH 7.5 onto a semipreparative cation-exchange TSK-SP-5PW (HPLC) column. The fractions eluted by linear NaCl gradient and enriched for PrPC were sequentially purified using an immobilized ion-affinity HPLC column charged by Co2+, followed by wheat germ agglutinin (WGA)-affinity HPLC or size-exclusion HPLC (SE-HPLC) using a TSK G3000SW column. More than 95% purity was achieved after SE-HPLC as estimated by quantitative densitometry of the silver-stained SDS-PAGE gel; the recovery of total brain PrPC was >/=8%. The purified PrPC was a monomer with an intact N-terminus, and with a Stoke's radius of 26 A, corresponding to that expected from the molecular weight for a native protein. The presence of the native-like conformation was further verified by peptide mapping after limited trypsin proteolysis, and by the apparent unfolding in guanidine hydrochloride, as detected by SE-HPLC.

Intracerebral distribution of infectious amyloid protein in spongiform encephalopathy.

We studied the regional distribution of infectious amyloid protein by western immunoblots of brain tissue extracts from 37 patients with different forms of spongiform encephalopathy, i.e., 16 sporadic cases, 18 familial cases with a variety of mutations, and 3 iatrogenic cases. In sporadic and familial Creutzfeldt-Jakob disease, amyloid protein concentrations were usually highest in the frontotemporal regions of the cerebral cortex, whereas iatrogenic Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome had as high or higher concentrations in the deep cerebral nuclei and cerebellum. As a group, familial cases had lower amyloid protein concentrations than either sporadic or iatrogenic cases, and fatal familial insomnia patients had the lowest concentrations found in any form of disease. This hierarchy of amyloid protein concentrations corresponds to the experimental transmission rates observed for each form of disease and is consistent with the concept that the protein molecule is an integral component of the infectious agent. Regional amyloid protein pattern analysis of brain and spinal cord may help to distinguish sporadic from environmentally acquired infections, as for example, cases of human disease suspected to have arisen from exposure to sheep or cows infected with scrapie or bovine spongiform encephalopathy.