GAL Genes Research Articles

Correlations in Chromatin Movement in Diploid Yeast Revealed by Two-Color Three-Dimensional Single-Particle Tracking using the Double-Helix Point Spread Function (DH-PSF) Microscope

It is known that gene regulation in eukaryotes is intimately tied to the three-dimensional (3D) organization of chromatin within the nucleus, and the exact structure and dynamics of this organization is a current topic of intense investigation. In order to examine whether the activation of two alleles in a diploid genome is coordinated we performed fast (10 Hz), 3D, simultaneous tracking of the two alleles of the GAL locus in diploid budding yeast cells. We did so by labeling one copy of the locus with LacO/LacI-GFP and the other with TetO/TetR-mCherry and recording the resulting fluorescence images in parallel using the DH-PSF microscope, which enabled precise sub-diffraction 3D localization in each channel ( = [14,13,27] nm for GFP and [25,29,46] nm for mCherry). The simultaneously recorded tracks were then registered to within sub-diffraction distances. We then examined the statistics of allele velocity cross-correlation and colocalization in both the transcriptionally active (cells grown in galactose) and repressed (grown in dextrose) states. As a control we labeled and tracked a single allele with both colors of fluorescent proteins integrated ∼12-18 kb apart. The dual-allele cases showed significant subpopulations of positive correlations that were larger than the 90th percentile value of the single-allele case (0.11), comprising 51% (26/51) in dextrose and 40% (34/86) in galactose. The presence of positive correlations indicates that gene copies are often closely associated in space and time, which may be consistent, for instance, with intertwined polymers or a mutual association with external protein machinery. Interestingly, we find both correlated and uncorrelated examples which appear peripherally confined, as the GAL genes are known to preferentially associate with the nuclear periphery upon activation.

Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break.

In budding yeast, a single double-strand break (DSB) triggers extensive ATM (Tel1) and ATR (Mec1)-dependent phosphorylation of histone H2A (γ-H2AX) around the DSB. We describe Mec1- and Tel1-dependent phosphorylation of γ-H2B at H2B-T129. γ-H2B formation is impaired by γ-H2AX and its binding partner, Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL genes are turned off, γ-H2AX is restored within 5 min, in a Mec1 dependent manner; when these genes are again induced, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other centromeric regions, again depending on Mec1. Taken together, our data provide new insights on the function and establishment of the phosphorylation events occurring onto chromatin after DSB induction.

Long Noncoding RNAs Promote Transcriptional Poising of Inducible Genes

Author SummaryLong noncoding RNAs (lncRNAs) are a recently identified class of molecules that regulate the expression of protein-coding genes through a number of mechanisms, some of them poorly characterized. The GAL gene cluster of the yeast Saccharomyces cerevisiae encodes a series of three inducible genes that are turned on or off by the presence or absence of specific carbon sources in the environment. Previous studies have documented the presence of two lncRNAs—GAL10 and GAL10s—encoded by genes that overlap the GAL cluster. We have now uncovered a role for both these lncRNAs in promoting the activation of the GAL genes when they are released from repressive conditions. This activation occurs at the kinetic level, through more rapid recruitment of RNA polymerase II and decreased association of the co-repressor, Cyc8. Under normal conditions, but also especially when they are stabilized and their levels are up-regulated, these GAL lncRNAs promote faster GAL gene activation. We suggest that these lncRNA molecules poise inducible genes for quick response to extracellular cues, triggering a faster switch in transcriptional programs.

GAL regulon ofSaccharomyces cerevisiaeperforms optimally to maximize growth on galactose

The GAL regulon in Saccharomyces cerevisiae is a well-characterized genetic network that is utilized for the metabolism of galactose as an energy source. The network contains a transcriptional activator, Gal4p, which binds to its cognate-binding site to express GAL genes. Further, Gal80p and Gal3p are the repressor and galactose sensor, respectively, which are also under the regulation of GAL regulon. It is shown that the wild-type strain produces only about 80% of the maximum expression feasible from the regulon, which is observed in a mutant strain lacking Gal80p. This raises a fundamental question regarding the optimality of expression from the GAL regulon in S.cerevisiae. To address this issue, we evaluated the burden on growth due to the synthesis of GAL proteins in S.cerevisiae. The analysis demonstrated that both the media type and the extent of enzyme synthesized play a role in determining the burden on growth. We show that the burden can be quantified by relating to a parameter, β, the ratio of enzyme activity to the initial substrate concentration. The analysis demonstrated that the GAL regulon of the wild-type strain performed effectively to optimize growth on galactose.

The Nuclear Pore Regulates GAL1 Gene Transcription by Controlling the Localization of the SUMO Protease Ulp1

Transcription activation of some yeast genes correlates with their repositioning to the nuclear pore complex (NPC). The NPC-bound Mlp1 and Mlp2 proteins have been shown to associate with the GAL1 gene promoter and to maintain Ulp1, a key SUMO protease, at the NPC. Here, we show that the release of Ulp1 from the NPC increases the kinetics of GAL1 derepression, whereas artificial NPC anchoring of Ulp1 in the Δmlp1/2 strain restores normal GAL1 regulation. Moreover, artificial tethering of the Ulp1 catalytic domain to the GAL1 locus enhances the derepression kinetics. Our results also indicate that Ulp1 modulates the sumoylation state of Tup1 and Ssn6, two regulators of glucose-repressed genes, and that a loss of Ssn6 sumoylation correlates with an increase in GAL1 derepression kinetics. Altogether, our data highlight a role for the NPC-associated SUMO protease Ulp1 in regulating the sumoylation of gene-bound transcription regulators, positively affecting transcription kinetics in the context of the NPC.

Self-Association of the Gal4 Inhibitor Protein Gal80 Is Impaired by Gal3: Evidence for a New Mechanism in the GAL Gene Switch

The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.

Regulation of a Mammalian Gene Bearing a CpG Island Promoter and a Distal Enhancer

A quantitative nucleosome occupancy assay revealed rules for nucleosome disposition in yeast and showed how disposition affects regulation of the GAL genes. Here, we show how those findings apply to the control of Kit, a mammalian gene. The Kit promoter lies in a CpG island, and its enhancer (active in mast cells) lies some 150 kb upstream. Nucleosomes form with especially high avidities at the Kit promoter, a reaction that, we surmise, ensures extremely low basal expression. In mast cells, transcriptional activators displace nucleosomes that are less tightly formed at the Kit enhancer. In turn, the active enhancer replaces a single Kit promoter nucleosome with the transcriptional machinery, thereby inducing transcription over 1,000-fold. As at the yeast GAL genes, the inhibitory effects of nucleosomes facilitate high factors of induction by mammalian activators working in the absence of specific repressors.

Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae

Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.

An experimental approach to identify dynamical models of transcriptional regulation in living cells

We describe an innovative experimental approach, and a proof of principle investigation, for the application of System Identification techniques to derive quantitative dynamical models of transcriptional regulation in living cells. Specifically, we constructed an experimental platform for System Identification based on a microfluidic device, a time-lapse microscope, and a set of automated syringes all controlled by a computer. The platform allows delivering a time-varying concentration of any molecule of interest to the cells trapped in the microfluidics device (input) and real-time monitoring of a fluorescent reporter protein (output) at a high sampling rate. We tested this platform on the GAL1 promoter in the yeast Saccharomyces cerevisiae driving expression of a green fluorescent protein (Gfp) fused to the GAL1 gene. We demonstrated that the System Identification platform enables accurate measurements of the input (sugars concentrations in the medium) and output (Gfp fluorescence intensity) signals, thus making it possible to apply System Identification techniques to obtain a quantitative dynamical model of the promoter. We explored and compared linear and nonlinear model structures in order to select the most appropriate to derive a quantitative model of the promoter dynamics. Our platform can be used to quickly obtain quantitative models of eukaryotic promoters, currently a complex and time-consuming process.

Abstract 2635: Galectin-1 modulates glioblastoma cell migration via interaction with FAK.

Abstract Malignant glioblastomas (GBM) are the most common brain tumors and exhibit excessive growth, cell cycle regulation, neovascularization, angiogenesis, migration, immune escape and resistance to apoptosis. Galectin-1 (Gal-1) is a 14.5-kDa β-galactoside-binding protein, which displays intracellular (i.e., protein-protein interactions) and extracellular (i.e., protein-oligosaccharide interactions) functions with marked pro-angiogenic and pro-migratory effects in gliomas. Although glioma is considered one of the deadliest cancers with median survival ranging from nine to twelve months, therapeutic treatments that target Gal-1 may prove effective since Gal-1 is one of the genes that cause aggressive behaviors of human GBM such as migration and invasion. Gal-1 controls glioma cell migration and invasion by modifying the actin cytoskeleton and expression of small GTPases. In the present study, immunohistochemical, Real Time PCR and Western blot experiments performed with clinical samples demonstrated that Gal-1 is highly expressed in human GBM tissue as compared to healthy brain tissue. Matrigel invasion assays confirmed that treatment of glioma cell lines U251 and 5310 with human umbilical cord blood stem cells (hUCBSC) almost completely blocked invasion of GBM cells. We also observed that treatment of GBM cells with hUCBSC decreased the expression of both Gal-1 and FAK. Additionally, silencing either the Gal-1 or FAK gene using shRNA decreased migration and invasion of glioblastoma cells. Furthermore, using immunocytochemistry and Western blot analyses, we observed that Gal-1 and FAK interact with each other in glioblastoma. Taken together, our results suggest that Gal-1 and FAK might have a direct protein-protein interaction for their function in the tumorigenesis of glioblastoma. Citation Format: Kiran Kumar Velpula, Arnima Bhasin, Jane S. Zhang, Andrew J. Tsung, Krishna Kumar Veeravalli, Jasti S. Rao, Venkata Ramesh Dasari. Galectin-1 modulates glioblastoma cell migration via interaction with FAK. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2013-2635

RT-qPCR analysis of dexB and galE gene expression of Streptococcus alactolyticus in Astragalus membranaceus fermentation

Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A. membranaceus by S. alactolyticus had some similar properties to those from A. membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A. membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) during A. membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S. alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A. membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S. alactolyticus strain FGM were involved in the regulation of A. membranaceus fermentation and they might play some roles in the increase of polysaccharides.

Functional Analysis of Bre1p, an E3 Ligase for Histone H2B Ubiquitylation, in Regulation of RNA Polymerase II Association with Active Genes and Transcription in Vivo

H2B ubiquitylation is carried out by Bre1p, an E3 ligase, along with an E2 conjugase, Rad6p. H2B ubiquitylation has been previously implicated in promoting the association of RNA polymerase II with the coding sequence of the active GAL1 gene, and hence transcriptional elongation. Intriguingly, we find here that the association of RNA polymerase II with the active GAL1 coding sequence is not decreased in Δbre1, although it is required for H2B ubiquitylation. In contrast, the loss of Rad6p significantly impairs the association of RNA polymerase II with GAL1. Likewise, the point mutation of lysine 123 (ubiquitylation site) to arginine of H2B (H2B-K123R) also lowers the association of RNA polymerase II with GAL1, consistent with the role of H2B ubiquitylation in promoting RNA polymerase II association. Surprisingly, unlike the Δrad6 and H2B-K123R strains, complete deletion of BRE1 does not impair the association of RNA polymerase II with GAL1. However, deletion of the RING domain of Bre1p (that is essential for H2B ubiquitylation) impairs RNA polymerase II association with GAL1. These results imply that a non-RING domain of Bre1p counteracts the stimulatory role of the RING domain in regulating the association of RNA polymerase II with GAL1, and hence RNA polymerase II occupancy is not impaired in Δbre1. Consistently, GAL1 transcription is impaired in the absence of the RING domain of Bre1p, but not in Δbre1. Similar results are also obtained at other genes. Collectively, our results implicate both the stimulatory and repressive roles of Bre1p in regulation of RNA polymerase II association with active genes (and hence transcription) in vivo.

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver-directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi-functional targeting molecule, which maintains targeting, endosome-escaping, and DNA-binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full-length cDNA encoding anti-hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50-Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC-C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv-Mel-Gal4 triple fusion protein (C1MG) was purified with a Ni(2+) chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni(2+) column purification. Western blot and immunohistochemistry showed the antigen-binding ability of C1MG to the cell surface of the liver-derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane-disrupting activity. DNA-binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome-escaping and DNA-binding capacity, which makes it probable to further study its liver-specific DNA delivery efficacy in vivo.

Vps Factors Are Required for Efficient Transcription Elongation in Budding Yeast

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.

Upregulation of galactose metabolic pathway by N-acetylglucosamine induced endogenous synthesis of galactose in Candida albicans

N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-13C1-glucosamine confirmed the flow of 13C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.

Functional Analysis of Rad14p, a DNA Damage Recognition Factor in Nucleotide Excision Repair, in Regulation of Transcription in Vivo

Rad14p is a DNA damage recognition factor in nucleotide excision repair. Intriguingly, we show here that Rad14p associates with the promoter of a galactose-inducible GAL1 gene after transcriptional induction in the absence of DNA lesion. Such an association of Rad14p facilitates the recruitment of TBP, TFIIH, and RNA polymerase II to the GAL1 promoter. Furthermore, the association of RNA polymerase II with the GAL1 promoter is significantly decreased in the absence of Rad14p, when the coding sequence was deleted. These results support the role of Rad14p in transcriptional initiation. Consistently, the level of GAL1 mRNA is significantly decreased in the absence of Rad14p. Similar results are also obtained at other galactose-inducible GAL genes such as GAL7 and GAL10. Likewise, Rad14p promotes transcription of other non-GAL genes such as CUP1, CTT1, and STL1 after transcriptional induction. However, the effect of Rad14p on the steady-state levels of transcription of GAL genes or constitutively active genes such as ADH1, PGK1, PYK1, and RPS5 is not observed. Thus, Rad14p promotes initial transcription but does not appear to regulate the steady-state level. Collectively, our results unveil a new role of Rad14p in stimulating transcription in addition to its well-known function in nucleotide excision repair.

Physiological genomics analysis for Alzheimer′s disease

Alzheimer's disease is a common kind of dementia. This disorder can be detected in all countries around the world. This neurological disorder affects millions of population and becomes an important concern in modern neurology. There are many researches on the pathogenesis of Alzheimer's disease. Although it has been determined for a long time, there is no clear-cut that this is a case with genetic disorder or not. A physiological genomics is a new application that is useful for track function to genes within the human genome and can be applied for answering the problem of underlying pathobiology of complex diseases. The physiogenomics can be helpful for study of systemic approach on the pathophysiology, and genomics might provide useful information to better understand the pathogenesis of Alzheimer's disease. The present advent in genomics technique makes it possible to trace for the underlying genomics of disease. In this work, physiological genomics analysis for Alzheimer's disease was performed. The standard published technique is used for assessment. According to this work, there are 20 identified physiogenomics relationship on several chromosomes. Considering the results, the HADH2 gene on chromosome X, APBA1 gene on chromosome 9, AGER gene on chromosome 6, GSK3B gene on chromosome 3, CDKHR1 gene on chromosome 17, APPBP1 gene on chromosome 16, APBA2 gene on chromosome 15, GAL gene on chromosome 11, and APLP2 gene on chromosome 11 have the highest physiogenomics score (9.26) while the CASP3 gene on chromosome 4 and the SNCA gene on chromosome 4 have the lowest physiogenomics score (7.44). The results from this study confirm that Alzheimer's disease has a polygenomic origin.

Computer assisted evaluation of retinal vessels tortuosity in Fabry disease

Fabry Disease (FD) is a rare X-linked metabolic disorder characterized by diffuse deposition of sphingolipids in many tissues. Retinal vessel tortuosity is a common ocular manifestation in FD and may represent a useful marker for the disease. Unfortunately its clinical evaluation is poorly reproducibile and alternative means of evaluation may be of interest. We tested a new semi-automatic software measuring retinal vessel tortuosity from eye fundus digital images in a group of FD patients. Observational case-control study evaluating four mathematical parameters describing tortuosity (relative length, sum of angle metric [SOAM], product of angle distance [PAD], triangular index) obtained from fundus pictures of 35 FD patients and 35 age-matched controls. Only the right eye was considered in order to reduce bias. Mann-Whitney test was used to compare the FD group versus the control group, males versus females and patients with versus without clinically identified retinal vessels tortuosity in the FD group. Linear regression analysis was performed on a subgroup of patients to evaluate the possible association of retinal vessels tortuosity parameters with age and with markers of systemic disease's progression. Three parameters (SOAM, PAD and triangular index) were significantly higher in FD patients in comparison with the controls (p < 0.0001, p = 0.001, p = 0.002 respectively). In the FD group the same three parameters showed higher values in hemizygous males than in heterozygous females ((p < 0.0001, p = 0.002, p < 0.0001 respectively). A computer assisted analysis of retinal vasculature demonstrated an increased vessels tortuosity in FD patients. The technique might be useful to establish disease severity and monitor its progression.

Gal4-based Enhancer-Trapping in the Malaria MosquitoAnopheles stephensi

Transposon-based forward and reverse genetic technologies will contribute greatly to ongoing efforts to study mosquito functional genomics. A piggyBac transposon-based enhancer-trap system was developed that functions efficiently in the human malaria vector, Anopheles stephensi. The system consists of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location; six lines with a single piggyBac-UAStdTomato element; and two lines, each with a single Minos element containing the piggyBac-transposase gene under the regulatory control of the hsp70 promoter from Drosophila melanogaster. Enhancer detection depended upon the efficient remobilization of piggyBac-Gal4 transposons, which contain the yeast transcription factor gene Gal4 under the regulatory control of a basal promoter. Gal4 expression was detected through the expression of the fluorescent protein gene tdTomato under the regulatory control of a promoter with Gal4-binding UAS elements. From five genetic screens for larval- and adult-specific enhancers, 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5- to 3-fold higher in female germ lines compared with male germ lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut, and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.

Rad26p, a Transcription-Coupled Repair Factor, Promotes the Eviction and Prevents the Reassociation of Histone H2A–H2B Dimer during Transcriptional Elongation in Vivo

We have recently demonstrated the formation of an atypical histone H2A-H2B dimer-enriched chromatin at the coding sequence of the active gene in the absence of Rad26p in vivo. However, the mechanisms for such a surprising observation remain unknown. Here, using a ChIP assay, we demonstrate that Rad26p promotes the eviction of histone H2A-H2B dimer and prevents the reassociation of the dimer with naked DNA in the wake of elongating RNA polymerase II at the coding sequence of the active GAL1 gene. Thus, the absence of Rad26p leads to the generation of an atypical histone H2A-H2B dimer-enriched chromatin at the active coding sequence in vivo.