Intergenic transcribed spacer PCR ribotyping for differentiation of Saccharomyces species and interspecific hybrids

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.     

Constraints on long range interactions mediating contour detection

Contour detection may be mediated by lateral interactions between neighboring cortical neurons whose receptive fields have collinear axes of preferred orientation. This hypothesis was tested in psychophysical experiments and computer simulations using a contour detection task in which observers searched for groups of Gabor patches that followed spatially extended contour paths embedded in noise consisting of several hundred Gabor patches with random positions and orientations. The orientation-selective units in the simulated neural network were linked by facilitatory interconnections whose strength depended on the geometry (distance, curvature, change in curvature) of smooth curves connecting the orientation axes of units in a pairwise fashion. Psychophysical detection performance was much higher for contour signal groups that followed closed rather than open-ended paths. However, just two sudden changes in orientation of neighboring Gabor patch elements in closed-path contours reduced detection performance to the same levels obtained with open-ended contours. These psychophysical data agreed with the results of the neural network simulations. Furthermore, the simulations also accounted for previous findings that removal of a single Gabor patch element from a closed-path contour group significantly degraded detection performance. We conclude that closure alone is not sufficient to enhance the visibility of a contour. However, if a closed contour meets certain geometric constraints, then lateral interactions based on these constraints can generate facilitation that reverberates around the closed path, thereby enhancing the contour's visibility.     

Evolution of porosity in long freezing range alloys

In an earlier publication, [1] the occurrence of shrinkage porosity was described by a simple “geometric” model, which was based primarily on experimental data available for low-carbon steel alloys. In this publication, the predictions of the “geometric” model are compared to experimental data for long freezing range alloys. The comparison and a theoretical analysis show that the earlier results do not apply to the solidification of these alloys. A dimensionless group has been developed to characterize the freezing range, the original “geometric” model has been modified, and a new analysis is presented to characterize shrinkage porosity formation in long freezing range alloys.     

Cross-slip phenomena in L12 long range ordered alloys

Cross-slip of screw superlattice dislocations in long range ordered alloys is investigated by using the weak-beam darkfield imaging method. A Ni₃(Al,Ti) alloy and Ni₃Fe were selected. The former alloy belongs to the category whose representatives are ordered up to the melting temperature; the latter alloy has an order/disorder transition at 503°C. Ni₃(Al,Ti) exhibits an anomalous increase of the yield stress with increasing temperature. Cross slip of screw superlattice dislocations onto cube planes is a characteristic feature which was found to occur with high frequency at the temperature of the onset of the anomaly. Screws dissociated on cube planes are predominantly observed. In the temperature regime below where the flow stress slightly decreases with increasing temperature octahedral cross-slip was found. In Ni₃Fe there is almost no temperature dependence of the flow stress. Octahedral cross-slip is frequently observed but cube cross-slip is rarely found. The edge superlattice dislocations are fourfold dissociated.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Determination of long range order in Ni-Base ternary alloys by X-ray anomalous diffraction using synchrotron radiation

Long range order (LRO) parameters in three ternary Ll₂ alloys [Ni₇₅Al₁₅Ti₁₀, Ni₇₀Al₂₀Cr₁₀ and (Ni₃Fe)_96.4Cr_3.6] have been measured using a new method. This method takes advantage of the variation in atomic scattering factors for X-rays near absorption threshold and opens a new area of research with the development of synchrotron facilities. The results show that Ti substitutes mostly, but not exclusively, for Al in NiAlTi. In NiAlCr, Cr occupies both Ni and Al sites. In NiFeCr, the order is not perfect; the Cr atoms are distributed on both types of sites, with a slight preference for Fe sites.     

Ultra-broadband near infrared phosphor with wide spectral range and long peak wavelength achieved by double-site occupation

The miniaturization and intelligence of near infrared(NIR) devices urgently require an excellent broadband NIR phosphor.However,emission spectra of most NIR phosphors cover less than 1200 nm with a full width at half maximum(FWHM) less than 200 nm and a peak wavelength less than 900 nm.Here,we successfully developed ultra-broadband NIR phosphors ScNbO₄:Cr³⁺which can overcome the above shortcomings based on a double-site occupancy strategy.The phosphors achieve ultra-broadba...     

A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping

Oligonucleotide primers complementary to conserved regions of the 16S and 23S ribosomal RNA genes were used to amplify the 16S-23S intergenic spacer region of bacterial pathogens. The amplification patterns produced were compared for their potential use in molecular epidemiologic analysis. This method, polymerase chain reaction (PCR) ribotyping, was applied to isolates of Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter species. Length polymorphisms in the amplified DNA distinguished unrelated strains of all bacteria. The banding patterns of 3 S. aureus isolates from the blood of 1 patient on 3 consecutive days were identical. Plasmid analysis, biotyping, and antibiograms were also obtained on the Enterobacter isolates. All three of these methods showed considerable variability after in vitro passage of bacteria, but PCR ribotypes remained stable. Results demonstrate the utility of the conserved primers for PCR ribotyping, a widely applicable method for the molecular epidemiology of genetically diverse bacteria.     

A numerical study of long range internal stresses associated with subgrain boundaries

A simple two-dimensional numerical model is developed to examine the stress fields associated with subgrain boundaries. It is shown that when low angle tilt boundaries are biased by an applied stress, long range internal stresses are developed. These stress fields have a periodicity of the order of the subgrain size, and are consistent with measurements of creep anelasticity and studies of in situ dislocation motion. The behavior of single subgrains and finite arrays of subgrains is examined.     

Comparison of Vibrio cholerae O1 isolates by polymerase chain reaction fingerprinting and ribotyping

AIM: To determine reproducibility and accuracy of the Nikon Retinomax autorefractor when used with children who were made cycloplegic. METHODS: Autorefraction and retinoscopy or subjectively refined retinoscopy (where, under the patient's direction, the refraction was varied until the best visual acuity was achieved) were performed on the right eye of 47 children, age 11-93 months. Autorefraction was performed using the Nikon Retinomax, which provides up to eight measured values of refractive error and one representative measurement of refractive error. RESULTS: Autorefractor measurements were successfully obtained from 7/9 children age 3 years or younger, and from all older children. Vector methods were used to calculate differences. Retinomax reproducibility averaged 0.43 D. Unbiased Retinomax and retinoscopy measurements differed by an average of 0.82 D. Unbiased Retinomax and subjectively refined retinoscopy differed by an average of 1.03 D. CONCLUSIONS: Reproducibility of Retinomax measured values in children is comparable with reproducibility of retinoscopy, subjective refraction, and autorefraction measurements in adults. Agreement between Retinomax and retinoscopy and agreement between Retinomax and subjective refinement in children is comparable with agreement between autorefraction and subjective refraction in adults. The study indicates that the Retinomax is a useful instrument for measuring refractive errors in young children.     

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.     

Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae

Prokaryotes have three amino acid-specific class II tRNAs that possess a characteristic long variable arm, tRNASer, tRNALeuand tRNATyr, while eukaryotes have only two, tRNASerand tRNALeu. Because of such a phylogenetic divergence in the composition of tRNA, the class II tRNA system is a good candidate for studying how the tRNA recognition manner has evolved in association with the evolution of tRNA. We report here a cross-species aminoacylation study of the class II tRNAs, showing the unilateral aminoacylation specificity between Escherichia coli and a yeast, Saccharomyces cerevisiae. Both SerRS and LeuRS from E.coli were unable to aminoacylate yeast class II tRNAs; in contrast, the yeast counterparts were able to aminoacylate E.coli class II tRNAs. Yeast seryl-tRNA synthetase was able to aminoacylate not only E.coli tRNASerbut also tRNALeuand tRNATyr, and yeast LeuRS was able to aminoacylate not only E.coli tRNALeubut also tRNATyr. These results indicate that the recognition manner of class II tRNA, especially the discrimination strategy of each aminoacyl-tRNA synthetase against noncognate class II tRNAs, is significantly divergent between E.coli and yeast. This difference is thought to be due mainly to the different composition of class II tRNAs in E.coli and yeast.     

Inositol trisphosphate and cyclic ADP ribose as long range messengers generating local subcellular calcium signals

The process of messenger-mediated release of Ca2+ from intracellular stores, which is of great importance in virtually all cell types including neurons, can best be studied in cells lacking voltage-gated Ca2+ channels in the plasma membrane. In pancreatic acinar cells agonist-evoked repetitive cytosolic Ca2+ spikes are due to release of Ca2+ via inositoltrisphosphate (IP3) and ryanodine receptors and reuptake into the stores via thapsigargin-sensitive Ca2+ pumps. At low acetylcholine (ACh) or cholecystokinin concentrations the cytosolic Ca2+ spikes are mostly confined to the secretory granule area of the polarized pancreatic acinar cells. Similar results can be obtained by intracellular infusion of IP3 (or one of its non-metabolizable analogues) or cyclic ADP ribose. This suggests that high affinity IP3 and ryanodine receptors are concentrated in the secretory granule area. We have generated an 'artificial synapse' on isolated acinar cells by having a cell-attached patch pipette filled with ACh on the basal membrane. Initially, ACh is prevented from making contact with the receptors by the negative potential applied to the pipette. When the pipette polarity is switched to positive ACh can bind to its receptors. Using digital Ca2+ imaging it could be seen that the first cytosolic rise often occurred in the secretory granule area, a considerable distance away from the site of the agonist-receptor interaction. This shows the long-range action of the messenger(s) IP3 and or cyclic ADP ribose generated by the ACh-receptor interaction. The local Ca2+ spikes in the secretory granule area are sufficient for exocytotic secretory responses as seen in capacitance measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.