Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.     

Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ miniplasmid

A new restriction endonuclease, StyI, free of contaminating nuclease activities, has been isolated from Escherichia coli carrying the hsd+ miniplasmid of Salmonella typhi origin. In the presence of 10 mM Mg2+, it recognizes and cleaves a hexanucleotide sequence of 5'-C decreases C(AT)(AT)GG. The advantages of the StyI endonuclease include its stability, high yield (more than 2 X 10(3) units/g of wet cells), easy handling of producer cells, and the ability to recognize new sequences, CCAAGG and CCTTGG.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Only a subset of the PAB1‐mRNP proteome is present in mRNA translation complexes

We have previously identified 55 nonribosomal proteins in PAB1‐mRNP complexes in Saccharomyces cerevisiae using mass spectrometric analysis. Because one of the inherent limitations of mass spectrometry is that it does not inform as to the size or type of complexes in which the proteins are present, we consequently used analytical ultracentrifugation with fluorescent detection system (AU‐FDS) to determine which proteins are present in the 77S monosomal translation complex that contains minimally the closed‐loop structure components (eIF4E, eIF4G, and PAB1), mRNA, and the 40S and 60S ribosomes. We assayed by AU‐FDS analysis 33 additional PAB1‐mRNP factors but found that only five of these proteins were present in the 77S translation complex: eRF1, SLF1, SSD1, PUB1, and SBP1. eRF1 is involved in translation termination, SBP1 is a translational repressor, and SLF1, SSD1, and PUB1 are known mRNA binding proteins. Many of the known P body/stress granule proteins that associate with the PAB1‐mRNP were not present in the 77S translation complex, implying that P body/stress granules result from significant protein additions after translational cessation. These data inform that AU‐FDS can clarify protein complex identification that remains undetermined after typical immunoprecipitation and mass spectrometric analyses.     

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. Xuelian Zheng and Wei Deng contributed equally to this work and are considered co-first authors.     

PUF3 Acceleration of Deadenylation in Vivo Can Operate Independently of CCR4 Activity, Possibly Involving Effects on the PAB1-mRNP Structure

The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3′ untranslated region sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts through a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A)-binding protein (PAB1) in which removal of the RRM1 (RNA recognition motif 1) domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3-controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex, but not PAN2, interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.     

Dynamic Rearrangement Within the Antheraea pernyi Silk Fibroin Gene Is Associated with Four Types of Repetitive Units

We characterized a full-length gene encoding wild silkmoth Antheraea pernyi fibroin (Ap-fibroin) to clarify the conformation of repetitive sequences. The gene consisted of a first exon encoding 14 amino acid residues, a short intron (120 bp), and a long second exon encoding 2,625 amino acid residues. Three amino acids, alanine, glycine, and serine, amounted to 81% of the Ap-fibroin sequence. The Ap-fibroin, except for 155 residues of the amino terminus, was composed of 80 tandemly arranged polyalanine-containing units (motifs). A motif was a doublet of a polyalanine block (PAB) and a nonpolyalanine block (NPAB). Seventy-eight of the 80 motifs were classified into four types based on differences in the NPAB sequences. Although respective motifs were significantly conserved, many rearrangements were observed within the second exon, i.e., the triplication of a 558-bp-long sequence and other duplication events of shorter sequences. Chi-like sequences, GCTGGAG, might contribute to the rearrangement within the gene as described in human minisatellite loci, because they were found at specific sites of NPAB-encoding sequences in three of four types of motifs. The present results support the idea that the Ap-fibroin gene is unstable like minisatellite sequences and that the evolution of this gene is strongly associated with its instability. Received: 18 February 2000 / Accepted: 30 June 2000     

Drivers of Solidago species invasion in Central Europe—Case study in the landscape of the Carpathian Mountains and their foreground

AimThe invasion process is a complex, context‐dependent phenomenon; nevertheless, it can be described using the PAB framework. This framework encompasses the joint effect of propagule pressure (P), abiotic characteristics of the environment (A), and biotic characteristics of both the invader and recipient vegetation (B). We analyzed the effectiveness of proxies of PAB factors to explain the spatial pattern of Solidago canadensis and S. gigantea invasion using invasive species distribution models.LocationCarpathian Mountains and their foreground, Central Europe.MethodsThe data on species presence or absence were from an atlas of neophyte distribution based on a 2 × 2 km grid, covering approximately 31,200 km² (7,752 grid cells). Proxies of PAB factors, along with data on historical distribution of invaders, were used as explanatory variables in Boosted Regression Trees models to explain the distribution of invasive Solidago. The areas with potentially lower sampling effort were excluded from analysis based on a target species approach.ResultsProxies of the PAB factors helped to explain the distribution of both S. canadensis and S. gigantea. Distributions of both species were limited climatically because a mountain climate is not conducive to their growth; however, the S. canadensis distribution pattern was correlated with proxies of human pressure, whereas S. gigantea distribution was connected with environmental characteristics. The varied responses of species with regard to distance from their historical distribution sites indicated differences in their invasion drivers.Main conclusionsProxies of PAB are helpful in the choice of explanatory variables as well as the ecological interpretation of species distribution models. The results underline that human activity can cause variation in the invasion of ecologically similar species.