Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806

The 26 SmaI digest fragments of pTi-B6-806 plasmid have a total molecular weight (121 × 10⁶) which accounts for the size of the plasmid as determined by contour length measurements. We have determined the physical arrangement of all SmaI digest fragments with reference to HpaI digest fragments. Hybridization of individual labeled SmaI digest fragments to HpaI digest fragments (cellulose nitrate transfers) allowed the latter to be ordered and located the SmaI boundary fragments. Recleavage of isolated HpaI fragments with SmaI revealed the SmaI fragments located within each HpaI fragment. The order of these internal SmaI fragments within a given HpaI fragment was determined by partial digestion of the latter with SmaI and hybridization of the resulting fragments with SmaI boundary fragments. From the sizes of partial digest fragments containing each boundary, the order of occurrence of SmaI fragments from each end was deduced. The complete map of the SmaI digest fragments is presented. The map of the HpaI digest fragments is presented with the following ambiguity: The order of fragments 12, 15, and 16, which map within SmaI fragment 1, was not determined. The SmaI digest fragments that contain DNA sequences transferred to plant cells during tumor induction, fragments 3b and 10c, were found to be contiguous on the physical map.     

Inquiring into the causes of depressed folivory in a fragmented temperate forest

Folivory is lower in forest fragments of the Maulino forest than in continuous stands. We experimentally assessed whether depressed folivory is related to a reduction in foliar palatability caused by the more xeric microclimate of forest fragments. We compared leaf anatomy at fragments and continuous forest for four tree species (Aristotelia chilensis, Cryptocarya alba, Nothofagus glauca and Gevuina avellana), and evaluated consumption of leaves of the two different habitats by insect species (Sericoides viridis and Ormiscodes rufosignata).Anatomy of leaves of fragments differs from that from continuous forest in at least one of the traits, for all the plant species. However, not all species exhibit more sclerophyllous traits in forest fragments. A. chilensis exhibits the greatest number of changes, being more sclerophyllous in fragments. In palatability trials, there were no differences in the consumption of leaves of fragments versus leaves of continuous forest. Therefore, depressed folivory levels in forest remnants of the Maulino forest do not seem to be attributable to reduced foliar palatability, suggesting that changes in the insect assemblage, and not palatability, account for reduced herbivory in fragmented forests.     

Intrinsic factors influence the attachment of fragments of the green alga Caulerpa filiformis

Asexual reproduction via fragmentation is an integral component of the life history of many marine organisms. In south-eastern Australia the green macroalga Caulerpa filiformis (Suhr) Hering is abundant on many rocky shorelines and fragments are very abundant in the water column. We examined some of the factors that may influence the growth and successful reattachment of fragments of C. filiformis that potentially contribute to its success. Field surveys revealed that C. filiformis fragments were of variable morphologies (from small fronds to entire thalli) and sizes (0.1-60 cm in length). All fragments of C. filiformis were viable propagules that survived and grew well in the laboratory. However, some fragments grew more than others, in particular larger sizes (7.5 cm class) of certain morphologies (fragments that consisted only of a single frond). Rhizoids were produced by all fragments, but again, larger fragments produced more rhizoids than smaller fragments. The force required to detach fragments was proportional to the number (but not size) of rhizoids, demonstrating the importance of rhizoids for attachment. The rate of attachment of fragments was also fast, usually within 12 h, but the longer these fragments were attached to a substratum the greater the force required to dislodge them. Interestingly, fragments only produced rhizoids if they were resting on a substratum which may explain the small proportion of fragments with rhizoids in the field (< 5%). Ultimately, the long term success of C. filiformis fragments was low as only 1.6% of fragments attached within 24 h and none of these persisted for longer than 2 days. Nonetheless, the abundance and viability of fragments of C. filiformis available to attach suggest that asexual fragmentation is a successful reproductive strategy in this seaweed.     

Regeneration,colonisation and growth rates of allofragments in four common stream plants

Colonisation by stream plants occurs to a large extent from simple stem fragments. Allofragments are stem fragments formed by mechanical breakage. We studied regeneration, colonisation, and growth rates in four common stream plants: Elodea canadensis Michx., Myriophyllum spicatum L., Potamogeton perfoliatus L. and Ranunculus baudotii x pseudofluitans. The objectives of this study were to determine (1) if shoots with an apical tip have higher regeneration (growth of new shoots and rhizomes from allofragments) and colonisation (root attachment in sediment) abilities and higher relative growth rates (RGR) than shoots without an apical tip, and (2) if fragment size correlates with regeneration and colonisation abilities and with RGR of fragments. For all species, over 60% of fragments regenerated new shoots and colonised. Apical shoots and larger fragments generally had higher regeneration and colonisation abilities and higher RGR. Relative growth rate for E. canadensis and M. spicatum was between 0.06 and 0.09 d⁻¹ whereas it was about half this rate for Ranunculus and P. perfoliatus (0.02–0.04 d⁻¹).     

Adiponectin Receptor-1 C-Terminal Fragment (CTF) in Plasma: Putative Biomarker for Diabetes

Introduction

Polypeptide fragments from cell surface receptors when found in plasma may be indicators of receptor regulation in disease conditions. It is known that subjects with diabetes have significantly lower plasma concentrations of adiponectin, a hormone released by adipose tissue, compared with nondiabetic controls. This hormone interacts with cell surface receptors in muscle (AdipoR1) and liver (AdipoR2).

Methods

We analyzed the relative distribution of specific fragments of AdipoR1 in healthy and diabetic individuals using an immunoaffinity mass spectrometry approach. We used antibodies raised against AdipoR1 immobilized on pre-activated protein chip surfaces to determine the molecular weights of bound polypeptide fragments using immunomass spectrometry (immuno-MS).

Results

Initially, immuno-MS analyses using a polyclonal antibody revealed two peaks (m/z 3,902 and 7,812) in plasma from normal, healthy individuals (n?=?5) that were not present in the plasma of diabetics (n?=?5). To confirm the detection of these fragments, a monoclonal antibody was developed against the last 25 amino acids of the AdipoR1 C-terminal fragment (CTF). Using the immuno-MS method, the monoclonal antibody detected the AdipoR1 CTF (m/z 3475) in all healthy controls (n?=?10), but did not detect these fragments in the diabetic patients (n?=?10).

Discussion

These preliminary observations suggest that the plasma levels of this receptor fragment may serve as an indicator of diabetic condition.     

Functionally important segments in proteins dissected using Gene Ontology and geometric clustering of peptide fragments

We have developed a geometric clustering algorithm using backbone ,ψ angles to group conformationally similar peptide fragments of any length. By labeling each fragment in the cluster with the level-specific Gene Ontology 'molecular function' term of its protein, we are able to compute statistics for molecular function-propensity and p-value of individual fragments in the cluster. Clustering-cum-statistical analysis for peptide fragments 8 residues in length and with only trans peptide bonds shows that molecular function propensities ≥20 and p-values ≤0.05 can dissect fragments within a protein linked to the molecular function.     

A novel method for screening species-specific gDNA probes for species identification

We report a method called SSH array which combines the suppression subtraction hybridization (SSH) and DNA array techniques to find species-specific DNA probes from genomic DNA (gDNA) for species identification. The method first obtains the differential gDNA fragments between two species by SSH and then hybridizes the differential gDNA fragments with arrays made of multiple whole genomes from several species to screen the unique gDNA fragments for one species. The screened unique gDNA fragments can be used as species-specific probes to differentiate the species they represent from all other species. We used five species of the genus Dendrobrium, D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., as experimental materials to study the feasibility of the method. The results showed that the method could efficiently obtain different species-specific probes for each of the five species.     

Area-sensitivity of three reed bed bird species breeding in Mediterranean marshland fragments

Density and species-specific thresholds for three strictly reed bed (Phragmites australis)—related bird species (Ixobrychus minutus, Acrocephalus scirpaceus, Acrocephalus arundinaceus), known as area-sensitive and breeding in an archipelago of 16 Mediterranean marshland fragments (Central Italy), were studied with mapping method techniques. No individuals of these species occurred in fragments smaller than 1 ha. Marshland fragments larger than 10 ha hosted all the three study species. We observed a significant increase in mean density between 0–1 and 1–10 ha for Acrocephalus scirpaceus and between 1–10 and >10 ha for Acrocephalus arundinaceus. Habitat heterogeneity of the marshland fragments was directly correlated to their size area. Therefore, area-sensitivity for the reed bed species can be interpreted also as a heterogeneity-sensitivity effect at fragment scale. In Mediterranean landscapes, a suitable marshland for the entire selected set of reed bed area-sensitive species should have a size greater than 10 ha with a patchy mosaic of reed beds, open waters, and other edge features.     

Fungal Fragments as Indoor Air Biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Evidence of endozoochory in upland geese Chloephaga picta and white‐bellied seedsnipes Attagis malouinus in sub‐Antarctic Chile

Birds are known to act as potential vectors for the exogenous dispersal of bryophyte diaspores. Given the totipotency of vegetative tissue of many bryophytes, birds could also contribute to endozoochorous bryophyte dispersal. Research has shown that fecal samples of the upland goose (Chloephaga picta) and white‐bellied seedsnipe (Attagis malouinus) contain bryophyte fragments. Although few fragments from bird feces have been known to regenerate, the evidence for the viability of diaspores following passage through the bird intestinal tract remains ambiguous. We evaluated the role of endozoochory in these same herbivorous and sympatric bird species in sub‐Antarctic Chile. We hypothesized that fragments of bryophyte gametophytes retrieved from their feces are viable and capable of regenerating new plant tissue. Eleven feces disk samples containing undetermined moss fragments from C. picta (N = 6) and A. malouinus (N = 5) and six moss fragment samples from wild‐collected mosses (Conostomum tetragonum, Syntrichia robusta, and Polytrichum strictum) were grown ex situ in peat soil and in vitro using a agar Gamborg medium. After 91 days, 20% of fragments from A. malouinus feces, 50% of fragments from C. picta feces, and 67% of propagules from wild mosses produced new growth. The fact that moss diaspores remained viable and can regenerate under experimental conditions following the passage through the intestinal tracts of these robust fliers and altitudinal and latitudinal migrants suggests that sub‐Antarctic birds might play a role in bryophyte dispersal. This relationship may have important implications in the way bryophytes disperse and colonize habitats facing climate change.     

The role of recreational activities in creating fragments of invasive Caulerpataxifolia

Once non-indigenous species are introduced to a new area, secondary spread is important in determining their ecological and economic impacts. Recreational activities may facilitate the secondary spread of invasive species by creating and transporting viable propagules, however to date there are few manipulative experiments that demonstrate the mechanistic basis of their impact. We examined the effect of two of the main aquatic recreational activities, boating and swimming, on the abundance of propagules (fragments) of the invasive green alga, Caulerpataxifolia in southeastern Australia. Surveys of two infested estuaries each with locations of different levels of recreational activity demonstrated that locations with high recreational activity had a significantly greater abundance and biomass of fragments than locations with low recreational activity. Manipulative experiments using beyond BACI type designs showed that both boating and swimming created fragments. A single pass of a motor-boat over C.taxifolia beds significantly increased the biomass of fragments, but only in shallow water. Similarly, swimming in C.taxifolia beds for 5 min significantly increased the biomass of fragments, but only when background levels of fragments were low. This study has provided the first manipulative experiments confirming that recreational activities such as boating and swimming can create propagules of aquatic invasive species. These findings provide scientific justification for targeting management efforts to minimize secondary spread of aquatic invasive species by restricting recreational activities in invaded areas.     

Changes in the dynamics of functional groups in communities of dung beetles in Atlantic forest fragments adjacent to transgenic maize crops

We investigated the composition and structure of dung beetle communities (Coleoptera, Scarabaeidae, Scarabaeinae) inhabiting areas of forest fragments next to either conventional or Bt-transgenic maize crops. The purpose of the study was to examine possible impacts of transgenic plants on non-target organisms associated with mammals through their food chain. In February 2011, we collected a total of 1502 beetles belonging to 33 species in Campos Novos, Santa Catarina state (SC), Brazil. Beetles were captured using 200 pitfall traps distributed among 20 forest fragments, 10 fragments in each site type (adjacent to conventional vs. Bt crops). In the fragments adjacent to conventional maize, 805 dung beetles from 27 species were collected. In the fragments adjacent to Bt-transgenic maize, 697 dung beetles from 27 species were caught. Dung beetle community composition was affected by fragment size and environmental complexity, and by distance between fragments. However, it did not explain the differences related to the two crop types, i.e., the functional group of dwellers was significantly over-represented in the fragments surrounded by transgenic maize. Hence, the dweller species Eurysternus francinae and Eurysternus parallelus were more frequent and abundant in fragments located near the transgenic maize, while the tunneler species Onthophagus tristis, Uroxys terminalis, Ontherus sulcator and the roller species Canthon lividus seminitens were more frequent and abundant in fragments surrounded by conventional maize. This observed impact of transgenic crops on functional group dynamics within dung beetle communities could potentially lead to impaired capacity for feces removal, seed dispersal, edaphic aeration, and incorporation of organic matter in the soil in these areas, as such ecosystem services are not performed by the dominant functional group (i.e., dwellers).     

Old Fragments of Forest Inside an Urban Area Are Able to Keep Orchid Bee (Hymenoptera: Apidae: Euglossini) Assemblages? The Case of a Brazilian Historical City

Retention of habitat fragments within the urban matrix can provide critical resources for the maintenance of regional biodiversity while still providing socio-economic value. Euglossini bees are important components in a community as they are important pollinators for economically valuable plants as well as hundreds of orchid species. However, some species are very sensitive to environmental impacts like urbanization. This study presents the role of antique urban fragments in a historical city in Brazil and compares it with a conservation area on the aspects of orchid bee assemblage, such as richness, composition, and abundance. Four fragments inside the city of Ouro Preto and three inside Parque Estadual do Itacolomi (PEIT) were sampled for Euglossini bees. Sorensen similarity index was used to compare community composition. The Mantel test was applied to verify the hypothesis that an urban center is a barrier for the mobility of the individuals. Fourteen Euglossini species from the region were registered. Close to 75% of the sampled bees were collected from the PEIT sampling areas. The fragments presented differences in Euglossini richness and abundance. A majority of the sampled fragments were dominated by the Eulaema cingulata Fabricius, Eulaema nigrita Lepeletier, and Euglossa securigera Dressler species. We found differences on community composition between the fragments localized in PEIT and those located in the urban center. The data suggest that there is a possible flux of individuals between the sampled fragments. The various small forest fragments in Ouro Preto, primarily in backyards, may also serve as stepping stones between sampled fragments.     

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]₂) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]₂). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term.     

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight ( > 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.