Search

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and aging. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study subtelomeric gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a genome-wide, comprehensive screen for subtelomeric-silencing factors. The approach was replicable and allowed detection of expression changes caused by previously described silencing factors. We also identified new molecular players affecting this process, most of which are related to functions underlying chromatin conformation. This was the case of LGE1, a novel silencing factor herein reported, associated with histone ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

C. glabrata is an opportunistic fungal pathogen and the second most common cause of opportunistic fungal infections in humans, that has evolved virulence factors to become a successful pathogen: strong resistance to oxidative stress, capable to adhere and form biofilms in human epithelial cells as well as to abiotic surfaces and high resistance to xenobiotics. Hst1 (a NAD

The chronological lifespan of budding yeast is a model of aging and age-related diseases. This paradigm has recently allowed genome-wide screening of genetic factors underlying post-mitotic viability in a simple unicellular system, which underscores its potential to provide a comprehensive view of the aging process. However, results from different large-scale studies show little overlap and typically lack quantitative resolution to derive interactions among different aging factors. We previously introduced a sensitive, parallelizable approach to measure the chronological-lifespan effects of gene deletions based on the competitive aging of fluorescence-labeled strains. Here, we present a thorough description of the method, including an improved multiple-regression model to estimate the association between death rates and fluorescent signals, which accounts for possible differences in growth rate and experimental batch effects. We illustrate the experimental procedure—from data acquisition to calculation of relative survivorship—for ten deletion strains with known lifespan phenotypes, which is achieved with high technical replicability. We apply our method to screen for gene-drug interactions in an array of yeast deletion strains, which reveals a functional link between protein glycosylation and lifespan extension by metformin. Competitive-aging screening coupled to multiple-regression modeling provides a powerful, straight-forward way to identify aging factors in yeast and their interactions with pharmacological interventions.

A fundamental question in biology is to understand how appropriate transcriptional regulation and dense packaging of the genetic material within the eukaryotic nucleus are achieved. The exquisite gene expression control and other metabolic processes of DNA require a highly complex, multilayered, three-dimensional architecture of the chromatin and its specific compartmentalization within the nucleus. Some of these architectural and sub-nuclear positioning mechanisms have been extensively co-opted by eukaryotic pathogens to keep fine expression control and expansion of virulence-related gene families in Plasmodium falciparum, Trypanosoma brucei and Candida glabrata. For example non-linear interactions between distant cis-acting regions and the formation of chromatin loops are required for appropriate regulation of the expression of virulence-related multi-gene families encoding cell surface proteins. These gene families are located near the chromosome ends and tethered to the nuclear periphery. Consequently, only one or very few genes of the family are expressed at a time. These genes are involved in antigenic variation in parasites and the generation of subpopulations of cells with diverse antigenic proteins at the surface in some pathogenic fungi, making them highly efficient pathogens.

Summary Dietary restriction is arguably the most promising nonpharmacological intervention to extend human life and health span. Yet, only few genetic regulators mediating the cellular response to dietary restriction are known, and the question remains which other regulatory factors are involved. Here, we measured at the genomewide level the chronological lifespan of Saccharomyces cerevisiae gene deletion strains under two nitrogen source regimens, glutamine (nonrestricted) and γ‐aminobutyric acid (restricted). We identified 473 mutants with diminished or enhanced extension of lifespan. Functional analysis of such dietary restriction genes revealed novel processes underlying longevity by the nitrogen source quality, which also allowed us to generate a prioritized catalogue of transcription factors orchestrating the dietary restriction response. Importantly, deletions of transcription factors Msn2, Msn4, Snf6, Tec1, and Ste12 resulted in diminished lifespan extension and defects in cell cycle arrest upon nutrient starvation, suggesting that regulation of the cell cycle is a major mechanism of chronological longevity. We further show that STE12 overexpression is enough to extend lifespan, linking the pheromone/invasive growth pathway with cell survivorship. Our global picture of the genetic players of longevity by dietary restriction highlights intricate regulatory cross‐talks in aging cells.

The aim of the present work is to obtain an integral representation of the field associated with the refraction of a plane wave by an arbitrary surface. To this end, in the first part we consider two optical media with refraction indexes n1 and n2 separated by an arbitrary interface, and we show that the optical path length, ϕ, associated with the evolution of the plane wave is a complete integral of the eikonal equation in the optical medium with refraction index n2. Then by using the k function procedure introduced by Stavroudis, we define a new complete integral, S, of the eikonal equation. We remark that both complete integrals in general do not provide the same information; however, they give the geometrical wavefronts, light rays, and the caustic associated with the refraction of the plane wave. In the second part, using the Fresnel–Kirchhoff diffraction formula and the complete integral, S, we obtain an integral representation for the field associated only with the refraction phenomena, the geometric field approximation, in terms of secondary plane waves and the k-function introduced by Stavroudis in solving the problem from the geometrical optics point of view. We use the general results to compute: the wavefronts, light rays, caustic, and the intensity associated with the refraction of a plane wave by an axicon and plano-spherical lenses.

Optical scalars are functions designed to analyze the behavior of geodesic congruences in general relativity. Refracted rays are three-dimensional congruences of light rays and they can be studied with this formalism. In this work we obtain the optical scalars for such congruences: the expansion $\mathrm{\ensuremath{\Theta}}$, the twist $\ensuremath{\omega}$, and the shear $\ensuremath{\kappa}$. Furthermore, we apply this machinery to study the aberrations of wavefronts to establish a link between them and the aberration function $W$.

SUMMARYDietary restriction is arguably the most promising non-pharmacological intervention to extend human life and health span. Yet, only few genetic regulators mediating the cellular response to dietary restriction are known, and the question remains which other transcription factors and regulatory pathways are involved. To gain a comprehensive view of how lifespan extension under dietary restriction is elicited, we screened the chronological lifespan of most gene deletions of Saccharomyces cerevisiae under two dietary regimens, restricted and non-restricted. We identified 472 mutants with enhanced or diminished extension of lifespan by dietary restriction. Functional analysis of such dietary-restriction genes revealed novel processes underlying longevity specifically by dietary restriction. Importantly, this set of genes allowed us to generate a prioritized catalogue of transcription factors orchestrating the dietary-restriction response, which underscored the relevance of cell-cycle arrest control as a key mechanism of chronological longevity in yeast. We show that the transcription factor Ste12 is needed for full lifespan extension and cell-cycle arrest in response to nutrient limitation; linking the pheromone/invasive growth pathway with cell survivorship. Strikingly, STE12 overexpression was sufficient to extend chronological lifespan under non-restricted conditions. Our global picture of the genetic players of longevity by dietary restriction highlights intricate regulatory cross-talks in aging cells.

In this work we obtain the equations for curvatures of refracted wavefronts for a plano arbitrary lens. The functions Hsub0/sub, Hsub1/sub, and Hsub2/subthat determine the caustic also determine the curvature of these wavefronts. The analysis performed in these calculations allows us to study the behavior of the Ronchigrams for the case of plane incident wavefronts. We apply this procedure for a plano-spherical lens, and we discover that it is possible to describe the behavior of the Ronchigrams based on the τ function, which labels the refracted wavefronts of the optical system.

In this work we use geometrical optics and the caustic touching theorem, introduced by Berry, to describe the internal structure of the null Ronchi grating for a plano–parabolic lens illuminated by a point light source placed on the optical axis. The aim of this work is to explain the role of the caustic region in the process of morphology change between image and object in computing the null Ronchi grating. To this end, we obtain the analytic expression of the null Ronchi grating, and after that we deeply study the change in morphology between a single straight fringe image at the Ronchigram and the multiple curve rulings that can generate it (one open and one closed). We analyze exactly how multiple rulings generate the same straight image fringe, or how an entire ruling collapses into a single point image. For this analysis, we take different observation planes at different positions with respect to the caustic region. Finally, we characterize this topological change as one of two possible kinds depending on the relative position between the observation plane and the caustic region.

Recently, it has been shown that if S(r, θ, ) is a two-parameter solution of the eikonal and Laplace equations, then is an exact solution of the scalar wave equation in an isotropic optical medium. In particular, if we take , the resulting solution is a superposition of non-diffracting beams in free space with different cone angle θ for the k vector. In this paper, we analyze the geometrical properties of a particular example, when O(θ, ) = 1 and g(θ,) = m( − π/2), which corresponds to the superposition of non-diffracting Bessel beams. That is, we compute its wavefronts, light rays and caustic. The Bessel beam is a special case of a non-diffracting beam, first proposed by Durnin in 1986, the superposition of two Bessel beams have been studied before in order to minimize the size of the central core. In this work, we present a beam resulting from the continuous superposition of Bessel beams with the same order m and different cone angle. To show the behavior of this beam, we simulate the intensity pattern at some different planes perpendicular to the Z and Y axes. Finally, we generate this beam experimentally, and we show that the caustic determines qualitatively the maximum of the corresponding intensity pattern.

In this work we compute the wavefronts and the caustics associated with the solutions to the scalar wave equation introduced by Durnin in elliptical cylindrical coordinates generated by the function A(ϕ)=ceν(ϕ,q)+iseν(ϕ,q), with ν being an integral or nonintegral number. We show that the wavefronts and the caustic are invariant under translations along the direction of evolution of the beam. We remark that the wavefronts of the separable Mathieu beams generated by A(ϕ)=ceν(ϕ,q) and A(ϕ)=seν(ϕ,q) are cones and their caustic is the z axis; thus, they are not structurally stable. However, in general, the Mathieu beam generated by A(ϕ)=ceν(ϕ,q)+iseν(ϕ,q) is stable because locally its caustic has singularities of the fold and cusp types. To show this property, we present the wavefronts and the caustics for the Mathieu beams with characteristic value aν=0 and q=0,0.2,0.3,0.5. For q=0, we obtain the Bessel beam of order zero; in this case, the wavefronts are cones and the caustic coincides with the z axis. For q≠0, the wavefronts are deformations of conical ones, and the caustic surface, for some values of q, has singularities of the cusp ridge type. Furthermore, we remark that the set of Mathieu beams with characteristic value aν=0 and 0≤q

The aim of this work is to present a geometrical characterization of parabolic non-diffracting beams. To this end, we compute the corresponding angular spectrum of the separable non-diffracting parabolic beams in order to determine the one-parameter family of solutions of the eikonal equation associated with this type of beam. Using this information, we compute the corresponding wavefronts and caustic, and find that qualitatively the caustic corresponds to the maximum of the intensity pattern and the wavefronts are deformations of conical surfaces.

The aim of this work is threefold: first we obtain analytical expressions for the wavefront train and the caustic associated with the refraction of a plane wavefront by an axicon lens, second we describe the structure of the ronchigram when the ronchiruling is placed at the flat surface of the axicon and the screen is placed at different relative positions to the caustic region, and third we describe in detail the structure of the null ronchigrating for this system; that is, we obtain the grating such that when it is placed at the flat surface of the axicon its associated pattern, at a given plane perpendicular to the optical axis, is a set of parallel fringes. We find that the caustic has only one branch, which is a segment of a line along the optical axis; the ronchigram exhibits self-intersecting fringes when the screen is placed at the caustic region, and the null ronchigrating exhibits closed loop rulings if we want to obtain its associated pattern at the caustic region.

Adherence to host cells is an important step in the pathogenicity of the opportunistic fungal pathogen Candida glabrata. This adherence is mediated by some members of the large family of cell wall proteins encoded by the EPA (Epithelial Adhesin) genes present in the C. glabrata genome. The majority of the EPA genes are localized close to different telomeres in C. glabrata, resulting in a negative regulation of transcription of these genes through chromatin-based subtelomeric silencing. In vitro, adherence to epithelial cells is mainly mediated by Epa1, the only member of the EPA family that is expressed in vitro. EPA1 forms a cluster with EPA2 and EPA3 at the subtelomeric region of telomere E-R. EPA2 and EPA3 are subject to silencing that propagates from this telomere in a process that depends on the Sir2, -3, -4, and Rif1 proteins, but surprisingly not on the yKu70 and yKu80 proteins. Here we describe that the yKu70/yKu80-independent silencing of telomere E-R is due to the presence of a cis-acting protosilencer (Sil2126) located between EPA3 and the telomere. This element can silence a reporter gene when placed 31.9 kb away from this telomere, but not when it is removed from the telomere context, or when it is placed near other telomeres, or inverted with respect to the reporter. Importantly, we show that the cis-acting Sil2126 element is required for the yKu70/80-independent silencing of this telomere, underscoring the importance of cis-elements for repressive chromatin formation and spreading on some telomeres in C. glabrata.

Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional activity of a specific locus in a variety of microorganisms to study gene regulation. Then the reporter gene expression can be easily measured under different conditions to gain insight into the regulation of the particular locus of interest. We have used transposon mutagenesis as a tool to generate insertional mutations with a modified Tn7 transposon containing the reporter gene URA3 (Tn7-URA3) to study subtelomeric silencing in the opportunistic fungal pathogen Candida glabrata. This method consists of two major steps: an in vitro Tn7-URA3 mutagenesis of a plasmid containing the desired subtelomeric region to be analyzed, followed by homologous recombination into the target region of the C. glabrata genome. As an alternative, a fusion PCR protocol can also be used in which the URA3 reporter gene can be "fused" together with the 5' and 3' regions of the desired insertion point by a two step PCR protocol. This fusion product can be introduced into the C. glabrata genome by homologous recombination after transformation in the same way as the Tn7-URA3 mutagenesis products. Once the URA3 reporter gene has been introduced in the desired locus in the C. glabrata genome, a simple plate growth assay is performed to assess the expression of the reporter gene.

Candida glabrata , a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the EPA genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the EPA family, we have assessed the importance of C. glabrata orthologues of known regulators of subtelomeric silencing in Saccharomyces cerevisiae. To this end, we used a series of strains containing insertions of the reporter URA3 gene within different intergenic regions throughout four telomeres of C. glabrata . Using these reporter strains, we have assessed the roles of SIR2 , SIR3 , SIR4 , HDF1 (yKu70), HDF2 (yKu80), and RIF1 in mediating silencing at four C. glabrata telomeres. We found that, whereas the SIR proteins are absolutely required for silencing of the reporter genes and the native subtelomeric EPA genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a cis element adjacent to the EPA3 locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the C. glabrata telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements.

In this work we assume that we have two given optical media with constant refraction indexes, which are separated by an arbitrary refracting surface. In one of the optical media we place a point light source at an arbitrary position. The aim of this work is to use a particular complete integral of the eikonal equation and Huygens' principle to obtain the refraction and reflection laws. We remark that this complete integral associates a new point light source with each light ray that arrives at the refracting surface. This means that by using only this complete integral it is not possible to determine the direction of propagation of the refracted light rays; the direction of propagation is obtained by imposing two extra conditions on the complete integral which are equivalent to Huygens' principle (in two dimensions, only one condition is needed). Finally, we establish the connection between the complete integral used here and that derived by using the k-function procedure introduced by Stavroudis, which works with plane wavefronts instead of spherical ones.