Shear breakage of DNA

Determinations were made of the mean length of fragments produced after shearing long (greater than 100 kb) native Hela DNA in a VirTis homogenizer. (VirTis Co., Inc., Gardiner, N.Y.). The mean length (L) is a function of the speed of rotation of the homogenizer blades (omega), time of shearing (t), water concentration ([H2O]), solvent viscosity (eta), temperature (T), and energy of activation (E*), but not a function of the initial length so long as the starting molecules sustain an average of three or more breaks. The relationship of the parameters is expressed by the equation L = (b/omegat1/2eta1/2[H2O])eE*/2kBT, where kB is the Boltzmann constant and b is a constant of proportionality. The breakage rate constant k was determined to have the relationship k = (omega2L2eta[H2O]2/2b2)e-E*/kBT. These equations are valid throughout large ranges of the parameters, and a simple method is described which chooses a final mean length between at least 0.15 and 36 kb by choosing the appropriate shearing conditions and initial fragment length. The heterogeneity of shearing conditions within the shearing vessel permits use of the equations at all breakage rates tested. Based on the work of others using more homogeneous shearing conditions and initial fragment lengths, more complicated forms of the equations are necessary at low breakage rates but not at high ones. A proposed model of the breakage mechanism suggests that molecules with stress-induced localized denaturations break at a rate different from that for native DNA

Maximal rank Hermitian completions of partially specified hermitian matrices

AbstractIn this note it is shown that, for a given partially specified hermitian matrix P, the maximal rank for arbitrary (possibly nonhermitian, complex) completions can be attained by hermitian completions. A simple formula for the maximal rank for nonhermitian completions was computed previously by Cohen et al. We also discuss the same situation for symmetric matrices over an arbitrary field, and show that the field size may be critical in establishing the same formulas. Finally, we discuss the same questions under Toeplitz structure, and show that for the matrixH=1?1?1?1?1the maximal completion rank is 3 for complex hermitian Toeplitz completions, 3 for real symmetric completions, 3 for real Toeplitz completions, but only 2 for real symmetric Toeplitz completions

Marginality in Inquiry-Based Science Learning Contexts: The Role of Exclusion Cascades

Vygotskyan-inspired theories of learning have been applied in science education research, yet to more explicit attention to links between local social interactions and cultural-historical processes is needed advance critical theories of science learning. This microgenetic case study examined identity and motivation processes in a 7th grade inquiry science context with the goal of better historicizing these processes by describing the phenomenon of exclusion cascades in relation to two backgrounded cultural-historical processes, alienation and the social division of labor. Exclusion cascades highlighted the mutual constitution of competence and belonging. Implications are discussed with respect to challenging adaptationist ethos in science education

The evolution of core proteins involved in microRNA biogenesis

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs (ncRNAs) which play important roles in eukaryotic gene regulation. miRNA biogenesis and activation is a complex process involving multiple protein catalysts and involves the large macromolecular RNAi Silencing Complex or RISC. While phylogenetic analyses of miRNA genes have been previously published, the evolution of miRNA biogenesis itself has been little studied. In order to better understand the origin of miRNA processing in animals and plants, we determined the phyletic occurrences and evolutionary relationships of four major miRNA pathway protein components; Dicer, Argonaute, RISC RNA-binding proteins, and Exportin-5.</p> <p>Results</p> <p>Phylogenetic analyses show that all four miRNA pathway proteins were derived from large multiple protein families. As an example, vertebrate and invertebrate Argonaute (Ago) proteins diverged from a larger family of PIWI/Argonaute proteins found throughout eukaryotes. Further gene duplications among vertebrates after the evolution of chordates from urochordates but prior to the emergence of fishes lead to the evolution of four Ago paralogues. Invertebrate RISC RNA-binding proteins R2D2 and Loquacious are related to other RNA-binding protein families such as Staufens as well as vertebrate-specific TAR (HIV trans-activator RNA) RNA-binding protein (TRBP) and protein kinase R-activating protein (PACT). Export of small RNAs from the nucleus, including miRNA, is facilitated by three closely related karyopherin-related nuclear transporters, Exportin-5, Exportin-1 and Exportin-T. While all three exportins have direct orthologues in deutrostomes, missing exportins in arthropods (Exportin-T) and nematodes (Exportin-5) are likely compensated by dual specificities of one of the other exportin paralogues.</p> <p>Conclusion</p> <p>Co-opting particular isoforms from large, diverse protein families seems to be a common theme in the evolution of miRNA biogenesis. Human miRNA biogenesis proteins have direct, orthologues in cold-blooded fishes and, in some cases, urochordates and deutrostomes. However, lineage specific expansions of Dicer in plants and invertebrates as well as Argonaute and RNA-binding proteins in vertebrates suggests that novel ncRNA regulatory mechanisms can evolve in relatively short evolutionary timeframes. The occurrence of multiple homologues to RNA-binding and Argonaute/PIWI proteins also suggests the possible existence of further pathways for additional types of ncRNAs.</p

Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process

Dysautonomia, A Heuristic Approach to a Revised Model for Etiology of Disease

Dysautonomia refers to a disease where the autonomic nervous system is dysfunctional. This may be a central control mechanism, as in genetically determined familial dysautonomia (Riley-Day Syndrome), or peripherally in the distribution of the sympathetic and parasympathetic systems. There are multiple reports of a number of different diseases associated with dysautonomia. The etiology of this association has never been explained. There are also multiple publications on dysautonomia associated with specific non-caloric nutritional deficiencies. Beriberi is the prototype of autonomic dysfunction. It is the best known nutritional deficiency disease caused by an imbalance between ingested calories and the vitamins required for their oxidation, particularly thiamin. Long thought to be abolished in modern medical thinking, there are occasional isolated reports of the full-blown disease in developed Western cultures

Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils

Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans