A Highly Sensitive Plant Hybrid Protein Assay System Based on the \u3cem\u3eSpm\u3c/em\u3e Promoter and TnpA Protein for Detection and Analysis of Transcription Activation Domains

TnpA is a multifunctional DNA binding protein encoded by the maize Suppressor-mutator (Spm) transposable element. TnpA is required for transposition and is a repressor of the unmethylated Spm promoter. While analyzing protein domains using a yeast GAL4-based hybrid system in transiently transformed tobacco cells, we found that TnpA represses the \u3e10-fold transcriptional activation observed when the GAL4 DNA-binding domain is used alone. By contrast, compared to the backgroundless TnpA DNA-binding domain alone, 33- to 45-fold activation of the Spm promoter was observed when the VP16 activation domain was fused to it. TnpA-binding sites, but no TATA box, were required for transcription activation. Among the TnpA deletion derivatives tested, those retaining the coding sequences for the DNA-binding and protein dimerization domains gave the highest level of transcription activation when fused with the VP16 activation domain. The TnpA gene and TnpA-binding sites in the short Spm promoter therefore provide a novel, highly sensitive single-hybrid system for identifying and studying plant transcription activation domains in plant cells

Concerted Formation of Macromolecular \u3cem\u3eSuppressor-mutator\u3c/em\u3e Transposition Complexes

Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range

Grassland Root Communities: Species Distributions and How They Are Linked to Aboveground Abundance.

There is little comprehensive information on the distribution of root systems among coexisting species, despite the expected importance of those distributions in determining the composition and diversity of plant communities. This gap in knowledge is particularly acute for grasslands, which possess large numbers of species with morphologically indistinguishable roots. In this study we adapted a molecular method, fluorescent fragment length polymorphism, to identify root fragments and determine species root distributions in two grasslands in Yellowstone National Park. Aboveground biomass was measured and soil cores (2 cm diam) were collected to 40 cm and 90 cm in an upland, dry grassland and a mesic, slope-bottom grassland, respectively, at peak foliar expansion. Cores were subdivided and species that occurred in each 10 cm interval were identified. The results indicated that the average number of species in 10 cm intervals (31 cm3) throughout the sampled soil profile was 3.9 and 2.8 at a dry and a mesic grassland, respectively. By contrast, average species number per 0.5 m2 determined by the presence of shoot material was 6.7 and 14.1 at dry and mesic sites, respectively. There was no relationship between soil depth and number of species per 10 cm interval in either grassland, despite the exponential decline of root biomass with soil depth at both sites. There also was no relationship between root frequency (i.e., the percentage of samples in which a species occurred) and soil depth for the vast majority of species at both sites. The preponderance of species were distributed throughout the soil profile at both sites. Assembly analyses indicated that species root occurrences were randomly assorted in all soil intervals at both sites, with the exception that F. idahoensis segregated from A. tridentata and P. spicata in 10-20 cm soil at the dry grassland. Root frequency throughout the entire sampled soil profile was positively associated with shoot biomass among species. Together these results indicated the importance of large, well proliferated root systems in establishing aboveground dominance. The findings suggest that spatial belowground segregation of species probably plays a minor role in fostering resource partitioning and species coexistence in these YNP grasslands

Nuclear Transition Matrix Elements for Double-β Decay Within PHFB Model

Employing the projected-Hartree-Fock-Bogoliubov (PHFB) approach, nuclear transition matrix elements (NTMEs) have been calculated to study the three complementary modes of β−β− decay, namely two neutrino β−β− (2νβ−β−) decay, neutrinoless β−β− (0νβ−β−) decay within mass mechanism and Majoron accompanied 0νβ−β− (0νβ−β−χ) decay. Reliability of HFB wave functions generated with four different parametrizations of the pairing plus multipolar type of effective two-body interaction has been ascertained by comparing a number of nuclear observables with the available experimental data. Specifically, the calculated NTMEs M(2ν) of 2νβ−β− decay have been compared with the observed data. Effects due to different parametrizations of effective two-body interactions, form factors and short-range correlations have been studied. It has also been observed that deformation plays a crucial role in the nuclear structure aspects of 0νβ−β− decay. Uncertainties in NTMEs calculated with wave functions generated with four different parametrizations of the pairing plus multipolar type of effective two-body interaction, dipole form factor and three different parametrizations of Jastrow type of short-range correlations within mechanisms involving light Majorana neutrinos, heavy Majorana neutrinos, sterile neutrinos and Majorons have been statistically estimated

Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Study of electrode effect within the atmospheric surface layer.

The thesis is divided into four chapters. A brief description of the past work on electrode effect is presented in the chapter-1.A description of topographical feature of the field station at Gulmarg and the instrumentation used is presented in the chapter-2. The Study of diurnal variation of some fair weather electrode effect parameters at Gulmarg constitute the subject matter of the chapter-3.In the chapter-4, the study of vertical profiles of atmospheric electric field, positive and negative small ions densities, electric conductivity and space charge density at Gulmarg field station estimated under turbulent and non-turbulent conditions are given. The thesis presents tables, figures, abstract and references in the end

Blast-induced flyrock: risk evaluation and management

Blasting is an integral unit operation of the Mine-Mill Fragmentation System and is a predominant method of rock breakage in mining and civil excavations. Blast sizes in terms of number of holes per blast range from few small diameter holes with small quantity of explosive in civil excavations to hundreds of large diameter holes with tonnes of explosives in large opencast mines. While only 20%–25% of the available explosive energy is used for fragmentation and throw, the rest of energy is manifested in unwanted effect. Optimum fragmentation and throw are inherent to blasting that are desired, while as undesired effects like ground vibration, air overpressure, fumes, and flyrock are undesired that need to be controlled. Flyrock, the subject of this chapter, is an unwanted throw of individual rock fragments that travel beyond the projected distances from the blast face and have a capability to damage structures and even cause fatalities. There have been several attempts by researchers to define the safety of blasting with respect to flyrock. One of the methods adopted for this is Risk Analysis. Risk can be defined in terms of probabilities of flyrock occurrence and the cost of damage that can occur due to a flyrock incident. The probabilities of flyrock further present a matrix of probabilities and can be defined to a better degree of reliability. However, the cost of damage due to flyrock is a very subjective matter, particularly, in case of fatalities. Such issues can be solved by adopting different methods like rules for safety or unsafe conditions. This chapter will discuss the intricacies of flyrock risk assessment while reviewing the existing state of the art and lay foundations for future research possibilities to address flyrock in detail. A new concept of Risk Management for flyrock prevention has been introduced here

Epigenetic Regulation of the Maize \u3cem\u3eSpm\u3c/em\u3e Transposable Element: Novel Activation of a Methylated Promoter by TnpA

Spm is epigenetically inactivated by C-methylation near its transcription start site. We have investigated the interaction between TnpA, an autoregulatory protein that can reactivate a silent Spm, and the promoter of the element. The promoter undergoes rapid de novo methylation and inactivation in stably transformed plants, but only if it includes a GC-rich sequence downstream of the promoter. TnpA activates the inactive, methylated promoter and leads to reduced methylation. By contrast, TnpA represses the active, unmethylated Spm promoter. Only the internal DNA-binding and dimerization domains of the protein are required for repression, while activation requires an additional C-terminal sequence. TnpA is therefore a unique regulatory protein with a conventional transcriptional repressor activity and a novel ability to activate a methylated, inactive promoter