The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.     

Molecular cytogenetic analysis of radiation-induced wheat-rye terminal and intercalary chromosomal translocations and the detection of rye chromatin specifying resistance to Hessian fly

Radiation-induced wheat-rye chromosome translocation lines resistant to Hessian fly, Mayetiola destructor (say), were analyzed by in situ hybridization using total genomic and highly repetitive rye DNA probes pSc119 and pSc74. In situ hybridization analysis revealed the exact locations of the translocation breakpoints and allowed the estimation of the sizes of the transferred rye segments. T6BS·6BL-6RL and T4BS· 4BL-6RL are terminal translocations with either most of the complete long arm of rye chromosome 6R or only the distal 57% of the 6RL arm attached to the long arms of wheat chromosomes 6B and 4B, respectively. The breakpoint in T6BS·6BL-6RL is located at a fraction length (FL) of 0.11 in the long arm of T6BS 6BL-6RL and at FL 0.46 in the long arm of T4BS·4BL-6RL. Ti4AS·4AL-6RL-4AL is an intercalary translocation with the breakpoint located at FL 0.06 in the long arm of wheat chromosome 4A. The inserted 6RL segment, with the Hessian fly resistance gene, has a size of 0.7 m, and is the smallest and, so far, the first radiation-induced intercalary translocation identified in wheat.by R. Apples     

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

 Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae. Received: 21 February 1996 / Accepted: 12 July 1996     

Evaluation of cell surface-displayed protein stability against simulated gastric fluid

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed β-glucosidase resisted pepsin digestion compared with secreted, free β-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted β-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed β-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted β-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed β-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.     

Monitoring the Active Conformation of Green Fluorescent Protein (GFP) and β-Glucosidase Adsorbed on Soil Particles

In order to determine the effect of various soil components on the activity of proteins, we monitored the fluorescence and the enzymatic activity of, respectively, green fluorescent protein (GFP) and β-glucosidase adsorbed on fine soil particles. We also monitored the activity of these proteins in the presence of components that are representative of soil colloids: a montmorillonite clay, goethite and organic matter extracted from soil. Upon adsorption on clay and goethite, GFP lost its fluorescence properties while β-glucosidase suffered only a partial loss of its catalytic activity. Extractable organic matter had an inactivating role on GFP while it did not cause inactivation of β-glucosidase. When GFP and β-glucosidase adsorbed on particles from natural soil samples, their behaviour was consistent with the behaviour observed for these proteins in the presence of the separate components, suggesting that the macroscopic activity of proteins adsorbed on soil particles corresponds to an average of the activities of proteins adsorbed on a mixture of surfaces. The monitoring of the proteins on soil particles with different organic matter contents has also shown that organic matter can have different effects (protecting or inactivating) on different proteins.     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

Membrane processing for purification and concentration of β-glycosidases from barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Tangential flow ultrafiltration with a polyethersulfone (100 kDa) membrane was used for the purification and concentration of β-glycosidases (β-galactosidase and β-glucosidase) from aqueous extract of barley. The performances of mode 1 (concentration followed by diafiltration) and mode 2 (diafiltration followed by concentration) were compared. In mode 1 activity recoveries of 91.44 and 88% as well as purifications of 1.84 and 1.77-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 9 h. In mode 2 activity recoveries of 95.68 and 91.76% with purifications of 4.56 and 4.38-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 6 h. The removal of total carbohydrates and protein was 56.74 and 50.73%, respectively, in mode 1, whereas it was 81.46 and 79.04%, respectively, in mode 2. The diafiltration volume and volume concentration of 3 were maintained in both mode 1 and 2. Flux decline was severe in mode 1 and led to a long processing time of about 9 h. These results indicate that mode 2 was better than mode 1 for purification of β-glycosidases.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Regulation of b- and a-Glycolytic Activities in the Sediments of a Eutrophic Lake

Temporal changes in α-and β-glucosidase activities, dissolved organic matter content, and bacterial biomass were studied in the superficial sediment layer of a eutrophic lake during the period of anoxia. The mean α-and β-glucosidase activities were 30.7±11.0 and 15.1±6.2 nmol h⁻¹ g⁻¹ of dry sediment, respectively. The specifc β-glucosidase activity seemed to be stimulated by carbohydrates (r=0.80, P<0.05), whereas the specifc α-glucosidase activity was negatively correlated with the dissolved protein concentration (r=−0.72, P<0.10). To test the effect of organic matter on hydrolytic activities under controlled conditions, changes in specific activities were studied in relation to the concentrations of different types of organic matter: phytoplankton, polymers (proteins, cellobiose, and starch) and monomers (glucose and amino acids). The specifc α-and β-glucosidase activities were strongly induced by their natural substrates (starch and cellobiose, respectively) (P<0.05) and were not inhibited by glucose. Proteins inhibited these activities (P<0.05), whereas supplementation with amino acids had no effect on specifc glycolytic activities.     

Prediction of "hot spots" of aggregation in disease-linked polypeptides

Background  

The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as β2-microglobulin, lysozyme, transthyretin or the prion protein, whereas β-amyloid peptide, amylin or α-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets.     

Construction of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> cell-surface display system using a putative GPI-anchored protein

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.     

Developmental expression of β-glucosidase in olive leaves

Plant β-glucosidases catalyze the hydrolysis of glycosidic linkages and play a vital role in defense against pathogens and stress. The present work investigated the relationship between leaf development and β-glucosidase protein content in Olea europea L. (cv. Picual) leaves. The total chlorophyll content increased with leaf age in current-season leaves. Immunoblot analysis revealed that the content of 61 kD protein of β-glucosidase also increased with leaf age, and that the enzyme existed in three isoforms (pI 5.8–6.2). Statistical analysis indicated a strong correlation between chlorophyll and β-glucosidase protein contents.     

Identification and functional analysis of a gene encoding β-glucosidase from the brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.     

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol⁻¹. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.     

Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.