Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Evidence for functional and physical association between Caenorhabditis elegans SEL-10, a Cdc4p-related protein, and SEL-12 presenilin

Mutations in either of two human presenilin genes (PS1 and PS2) cause Alzheimer’s disease. Here we describe genetic and physical interactions between Caenorhabditis elegans SEL-10, a member of the Cdc4p family of proteins, and SEL-12, a C. elegans presenilin. We show that loss of sel-10 activity can suppress the egg-laying defective phenotype associated with reducing sel-12 activity, and that SEL-10 can physically complex with SEL-12. Proteins of the Cdc4p family have been shown to target proteins for ubiquitin-mediated turnover. The functional and physical interaction between sel-10 and sel-12 therefore offers an approach to understanding how presenilin levels are normally regulated.

A database and tools for 3-D protein structure comparison and alignment using the Combinatorial Extension (CE) algorithm

The database reported here is derived using the Combinatorial Extension (CE) algorithm which compares pairs of protein polypeptide chains and provides a list of structurally similar proteins along with their structure alignments. Using CE, structure–structure alignments can provide insights into biological function. When a protein of known function is shown to be structurally similar to a protein of unknown function, a relationship might be inferred; a relationship not necessarily detectable from sequence comparison alone. Establishing structure–structure relationships in this way is of great importance as we enter an era of structural genomics where there is a likelihood of an increasing number of structures with unknown functions being determined. Thus the CE database is an example of a useful tool in the annotation of protein structures of unknown function. Comparisons can be performed on the complete PDB or on a structurally representative subset of proteins. The source protein(s) can be from the PDB (updated monthly) or uploaded by the user. CE provides sequence alignments resulting from structural alignments and Cartesian coordinates for the aligned structures, which may be analyzed using the supplied Compare3D Java applet, or downloaded for further local analysis. Searches can be run from the CE web site, http://cl.sdsc.edu/ce.html, or the database and software downloaded from the site for local use.

The RESID Database of protein structure modifications and the NRL-3D Sequence–Structure Database

The RESID Database is a comprehensive collection of annotations and structures for protein post-translational modifications including N-terminal, C-terminal and peptide chain cross-link modifications. The RESID Database includes systematic and frequently observed alternate names, Chemical Abstracts Service registry numbers, atomic formulas and weights, enzyme activities, taxonomic range, keywords, literature citations with database cross-references, structural diagrams and molecular models. The NRL-3D Sequence–Structure Database is derived from the three-dimensional structure of proteins deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The NRL-3D Database includes standardized and frequently observed alternate names, sources, keywords, literature citations, experimental conditions and searchable sequences from model coordinates. These databases are freely accessible through the National Cancer Institute–Frederick Advanced Biomedical Computing Center at these web sites: http://www.ncifcrf.gov/RESID, http://www.ncifcrf.gov/ NRL-3D; or at these National Biomedical Research Foundation Protein Information Resource web sites: http://pir.georgetown.edu/pirwww/dbinfo/resid.html, http://pir.georgetown.edu/pirwww/dbinfo/nrl3d.html

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

Apoplastic α-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three α-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic α-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli α-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-α-d-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside revealed that the acidic α-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

The genomic and physical organization of Ty1-copia-like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms.

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Purification and physical properties of the male and female double sex proteins of Drosophila.

The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.

Structure, inheritance, and transcriptional effects of Pce1, an insertional element within Phanerochaete chrysosporium lignin peroxidase gene lipI.

A 1747-bp insertion within a lignin peroxidase allele of Phanerochaete chrysosporium BKM-F-1767 is described. Pce1, the element, lies immediately adjacent to the fourth intron of lip12. Southern blots reveal the presence of Pce1-homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (one to five copies), and conserved transposase motifs are lacking. The mutation transcriptionally inactivates lip12 and is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element that may play a significant role in generating ligninolytic variation in certain P. chrysosporium strains.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Stability of Coulomb systems in a magnetic field.

I study N electrons and M protons in a magnetic field. It is shown that the total energy per particle is bounded below by a constant independent of M and N, provided the fine structure constant is small. Here, the total energy includes the energy of the magnetic field.

Bioactive compounds and physical and chemical characteristics of mini tomatoes: influence of postharvest treatments

Tomatoes are among the most cultivated and used vegetables in the world. They are very succeptible to post harvest losses due to high perishability, therefore the use of postharvest treatments may contribute to conservation of this fruit, however the treatments might affect significantly physico-chemical, sensory and nutritional characteristics of tomatoes. Given the perishability of tomato and the economic importance of small tomato fruits, the purpose of the present study was to determine the effect of gamma radiation, carnauba coating and 1-MCP treatments on tomato fruit quality during storage. The study may be divided into two parts. In the first, mini tomatoes cv. Sweet Grape were harvested at breaker stage, divided into 4 grous and treated with gamma radiation (0.6 kGy), carnauba coating (1 L 1000 kg-1) and 1-MCP (500 nL L-1) and then stored at 25±2°C for 30 days with a control group of tomatoes. In the seconnd part, tomatoes harvested at light-red stage were submitted to the same treatments and storage period. Every 6 days tomatoes were evaluated for color modifications, fruit firmness, souble and total pectin (only for light-red tomatoes), mass loss, titratable acidity (TA), soluble solids (SS), SS/TA ratio, carotenoids profile, formation of lycopene isomers, total phenolic compounds, ascorbic acid and antioxidant capacity. For tomatoes harvested at breaker stage and submitted to the treatments the results showed mass loss was delaying mainly by carnauba wax, and to a lesser extend by 1-MCP. Fruit firmness were better retained for 1-MCP treated fruits and carnauba treatment showed a transient effect in preserving fruit firmness. SS/TA of tomatoes treated with gamma radiation and carnauba presented no differences from control values, and were lower with the application of 1-MCP. Color was negatively affected by 1-MCP and earlier changed (6th day) when gamma radiation was applied. In relation to bioactive compounds of tomatoes harvest at breaker stage, results indicated gamma radiation and 1-MCP decreased the final content of lycopene and produced more (Z)-isomers of lycopene. Gamma radiation also induced a decreased in ?-carotene and an increased in phenolic compounds by the end of storage period. 1-MCP treatment promoted a slow down increase in ascorbic acid content during storage. Antioxidant capacity of the hydrophilic fraction was not dramatically affected by treatments and the lipophilic fraction was lower, especially for 1-MCP fruits. In addition, contents of ?-carotene, lycopene, (Z)-isomers of lycopene, ascorbic acid and antioxidant capacity increased during the period of storage while contents of lutein and phenolic compounds tended to decrease. Regarding tomatoes harvest at light-red stage, the most effective treatments for delaying fruit firmness and mass loss was carnauba and 1-MCP, while gamma radiation was the treatment with higher mass loss and the less fruit firmness, which could be associated with the higher solubilization of pectins promoted by radiation treatment. Color (L* and Hue) was mainly affected by 1-MCP treatment which delayed color development, however, by the end of storage, the values were not different from the other treatments. SS/TA ratio was lower for fruits treated with 1-MCP and TA was not so dramatically affected by treatments. Furthermore, mini tomatoes harvested at light-red stage, demonstrated irradiation induced changes in the final content of lycopene, increasing it, and formed less (13Z)-lycopene, while 1-MCP and carnauba coating slow down the increase in lycopene and slown down the decrease of ascorbic acid and phenolic compounds. Antioxidant capacity of lipophilic fraction was not affected by treatments and the hydrophilic fraction was lower for irradiated fruits only on day 0 as well as phenolic compounds. In the other days, no differences among treatments were observed for hydrophilic antioxidant capacity. Considering the results, the best combination of SS and TA and fruit preservation for mini tomatoes harvest at breaker stage was promoted by carnauba coating, which seems to be the treatment that causes fewer changes in bioactive compounds of breaker tomatoes. However, when mini tomatoes were harvested at light-red stage, SS/TA ratio and color were better and, to preserve the quality of these fruits, besides carnauba coating, 1-MCP also could be indicated

Orbital and Physical Properties of the σ Ori Aa, Ab, B Triple System

We provide a complete characterization of the astrophysical properties of the σ Ori Aa, Ab, B hierarchical triple system and an improved set of orbital parameters for the highly eccentric σ Ori Aa, Ab spectroscopic binary. We compiled a spectroscopic data set comprising 90 high-resolution spectra covering a total time span of 1963 days. We applied the Lehman-Filhés method for a detailed orbital analysis of the radial velocity curves and performed a combined quantitative spectroscopic analysis of the σ Ori Aa, Ab, B system by means of the stellar atmosphere code FASTWIND. We used our own plus other available information on photometry and distance to the system for measuring the radii, luminosities, and spectroscopic masses of the three components. We also inferred evolutionary masses and stellar ages using the Bayesian code BONNSAI. The orbital analysis of the new radial velocity curves led to a very accurate orbital solution of the σ Ori Aa, Ab pair. We provided indirect arguments indicating that σ Ori B is a fast-rotating early B dwarf. The FASTWIND+BONNSAI analysis showed that the Aa, Ab pair contains the hottest and most massive components of the triple system while σ Ori B is a bit cooler and less massive. The derived stellar ages of the inner pair are intriguingly younger than the one widely accepted for the σ Orionis cluster, at 3 ± 1 Ma. The outcome of this study will be of key importance for a precise determination of the distance to the σ Orionis cluster, the interpretation of the strong X-ray emission detected for σ Ori Aa, Ab, B, and the investigation of the formation and evolution of multiple massive stellar systems and substellar objects.