μL Of DNA Research Articles

Erratum to: NACP-Rep1 relates to Beck Depression Inventory Scores in Healthy Humans

A reanalysis of the genotypes in our publication NACP-Rep1 relates to Beck Depression Inventory scores in healthy humans (Lenz et al. 2011) revealed a deviation from the principles of the HardyWeinberg equilibrium (HWE, goodness-of-fit test [χ], p<10). As reported in literature, several factors might cause violations of HWE. For the polymorphism, a deviation from HWE is not uncommon. In their collaborative analysis, Maraganore et al. (2006) excluded genotype data from 3 of 18 study sites because the genotype frequencies deviated from HWE. Different reasons might account for the excess of homozygotes in our sample. Keeping in mind that the polymorphism represents a mixed sequence repeat (Xia et al. 1996), fragments with the same length in sequence analysis might represent heterozygous alleles, possibly resulting in an overestimation of homozygous individuals. However, the strong deviation from HWE in our study signaled a genotyping artifact. Therefore, we regenotyped a subsample of individuals to validate our previous results (new primer pair: forward 5‘-TAT TTT GAC CTT TGT TTT GGC-3‘, reverse 5‘FAM-TTT ATG TAT TTT ATT CCC TGG C-3‘; 0.25 μl forward primer, 0.25 μl reverse primer [each 20 μM], 5 μl HotStarTaq Master Mix [Qiagen, Hilden, Germany], 4 μl H2O and 0.5 μl DNA template; PCR conditions 1×95 °C 15 min, 40×95 °C 30 s, 50 °C 30 s, 72 °C 30 s, 1×72 °C 15 min). The results revealed initial misgenotyping primarily due to an overestimation of homozygous individuals. We recalculated the previously described statistical analyses using corrected genotype frequencies (Lenz et al. 2011). The corrected genotypes were in HWE (p=0.12). Both the non-parametric correlation analysis and the linear regression analysis showed significant positive associations between mean and the BDI score (n=213; Spearman’s ρ=0.136, p=0.048; B±SD, 0.424±0.195, beta=0.148, T=2.178, p=0.031; model summary: R=0.148, R=0.022, adjusted R=0.017; ANOVA, F=4.743, p=0.031). Subsequently, we divided the group into three subgroups according to the additive repeat length (Bonsch et al. 2005). Short: 265/267 (n=8), 265/269 (n=3), 267/267 (n=19); intermediate: 265/271 (n=3), 267/269 (n=74), The online version of the original article can be found at http://dx.doi.org/ 10.1007/s12031-011-9493-7. B. Lenz (*) : C. Sysk :N. Thuerauf :M. Clepce :K. Reich : H. Frieling : S. Bleich : J. Kornhuber Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6-10, 91054 Erlangen, Germany e-mail: bernd.lenz@uk-erlangen.de

R132C IDH1 Mutations Are Found in Spindle Cell Hemangiomas and Not in Other Vascular Tumors or Malformations

Spindle cell hemangioma (SCH) is a rare, benign vascular tumor of the dermis and subcutis. The lesions can be multifocal and are overrepresented in Maffucci syndrome, in which patients also have multiple enchondromas. Somatic mosaic R132C IDH1 hotspot mutations were recently identified in Maffucci syndrome. We evaluated the presence of mutations in solitary and multiple SCHs in patients without multiple enchondromas and tested a range of other vascular lesions that enter into the differential diagnosis. The R132C IDH1 mutation was identified by hydrolysis probes assay and confirmed by Sanger sequencing in 18 of 28 (64%) SCHs; of the 10 negative cases, 2 harbored a mutation in IDH2 (R172T and R172M) by Sanger sequencing. None of 154 other vascular malformations and tumors harbored an IDH1 R132C mutation, and R132H IDH1 mutations were absent in all 182 cases. All 16 SCHs examined by immunohistochemistry were negative for expression of HIF-1α. In conclusion, 20 of 28 (71%) SCHs harbored mutations in exon 4 of IDH1 or IDH2. Given that mutations were absent in 154 other vascular lesions, the mutation seems to be highly specific for SCH. The mutation does not induce expression of HIF-1α in SCH, and therefore the exact mechanism by which mutations in IDH1 or IDH2 lead to vascular tumorigenesis remains to be established.

Non-sporulating molds in clinical specimens: agents of infections?

Non-sporulating molds (NSMs) or mycelia sterilia isolated from clinical specimens are assumed to be environmental contaminants and their identification is not usually pursued. Knowing the identity of these isolates may help to determine their importance as pathogens. In this study, internal transcribed spacer (ITS) sequences were used to identify 15 non-sporulating fungi isolated as pure cultures from clinical specimens such as skin, nail, blood and other fluids. Fungal DNA was directly extracted from Whatman FTA® cards inoculated with fungal suspension, amplified using ITS 1 and 2 primers and sequenced. Sequences were matched using BLAST search, with published fungal identities. Of 15 NSMs, one isolate from blood taken from a lymphoma patient was identified as Schizophylum commune, a Basidiomycete fungus which has been reported as a human pathogen causing systemic disease. Four isolates were identified as plant pathogens; Penicillium ochrochloron from bronchoalveolar fluid, Phlebia sp. from pleural fluid, Phoma multirostrata and Inonotus rickii from skin specimens. Two others were identified as saprophytes or endophytes; Xylaria feejeensis from nail and Fomitopsis cf. meliae from skin. All other isolates, including three from blood and two from peritoneal & endotracheal fluid could not be conclusively identified based on the ITS sequences alone. To determine the presence of possible virulence factors in NSMs, the Phlebia sp. isolate was evaluated for keratinase activity in vitro, on human nail and was shown to have high keratin degrading activity which indicates potential pathogenicity. In conclusion, this study has found that non-sporulating fungi may have a role in causing infections in humans; further studies are needed to establish their involvement. Introduction When non-sporulating molds (NSM) are isolated as pure cultures from patients, it raises a question on their ability to cause infection. Although often dismissed as insignificant, environmental organisms, DNA based identification has revealed potential pathogens among NSMs (Pounder et al. 2007). Molecular methods for fungal identification are well established, with the ribosomal DNA internal transcribed spacer (ITS) sequence utilised for species determination. Pathogenic fungi are known to produce proteolytic enzymes, such as keratinase in dermatophytes, which is important for fungal invasion of cutaneous sites (Viani et al. 2001). The keratinase activity level correlates with severity of infection caused. This study aimed to identify clinical NSM isolates and determine their pathogenic capability. Results Figure 1 : NSM isolates identified based on ITS sequences ReferenceS Pounder, J.I., Simmon, K.E., Barton, C.A., Hohmann, S.L., Brandt, M.E. & Petti, C.A. 2007. Discovering Potential Pathogens among Fungi Identified as Nonsporulating Molds. Journal of Clinical Microbiology 45(2): 568-571. Viani, F.C., Santos, J.I.D., Paula, C.R., Larson, C.E. & Gambale, W. 2001. Production of extracellular enzymes by Microsporum canis and their role in its virulence. Medical Mycology 39: 463-468. Dentinger, B.T.M., Margaritescu, S. & Moncalvo J.M. 2010. Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms. Molecular Ecology Resources. 10: 628-633. METHODOLOGY UZ 177/10 Schizophylum commune UZ 517 Penicillium ochrochloron UZ 529/10 Phoma multirostrata UZ 586/10 Phlebia sp. UZ 661/10 Fomitopsis cf. meliae UZ 365/08 Inonotus rickii UZ 634 Xylaria feejeensis Fungal Molecular Identification (Dentinger et al. 2010) Fungal suspension prepared from PDA inoculated onto Whatman FTA® card (200 μL) DNA directly extracted from FTA card punches using Sigma Extract-N-AmpTM Plant PCR Kit Extracted DNA amplified using ITS 1 and ITS 2 primers PCR product purified and sequenced; sequences matched with published data Keratinase Activity Determination (Viani et al. 2001) JADUAL 1. Nilai purata dan sisihan piawai bagi kawalan negatif, kawalan positif dan fungus ujian Fungal isolate Keratinase Unit SD F. solani 5.340 2.097 T. mentagrophytes 4.000 1.572 Non-sporulating mold (Phlebia sp.) 3.060 1.501 F. oxysporum 2.273 1.258 Table 1: Keratinase activity of pathogenic fungi and NSM CONCLUSION Of fifteen NSMs, seven isolates were identified; one (S.commune) as a known pathogen. The Phlebia sp. produced keratinase enzyme at levels similar to dermatophyte fungi. Non-sporulationg molds have pathogenic potential and may be the cause of infections in humans. Fungal suspension incubated with human nail powder and keratin azure in a mineral medium for 21 days. Keratinase activity determined by quantitatively measuring dye release from keratin azure in mineral medium using a spectrophotometre. An increase of 0.01 in the absorbance measurement at 595 nm is equivalent to 1 Keratinase Unit (KU) of enzyme activity.

Transfer of Intestinal Microbiota From Lean Donors Increases Insulin Sensitivity in Individuals With Metabolic Syndrome

Alterations in intestinal microbiota are associated with obesity and insulin resistance. We studied the effects of infusing intestinal microbiota from lean donors to male recipients with metabolic syndrome on the recipients' microbiota composition and glucose metabolism. Subjects were assigned randomly to groups that were given small intestinal infusions of allogenic or autologous microbiota. Six weeks after infusion of microbiota from lean donors, insulin sensitivity of recipients increased (median rate of glucose disappearance changed from 26.2 to 45.3 μmol/kg/min; P < .05) along with levels of butyrate-producing intestinal microbiota. Intestinal microbiota might be developed as therapeutic agents to increase insulin sensitivity in humans; www.trialregister.nl; registered at the Dutch Trial Register (NTR1776).

First report of association of a begomovirus with the leaf curl disease of a common weed, datura inoxia.

Begomovirus is one of the largest genus of the family Geminiviridae and the vector white fly (Bemisia tabaci) is prevalent in the tropical and subtropical regions of the world [4]. Weeds may infect crops and those weeds may serve as a reservoir for crop-infecting geminiviruses [1, 5]. In the present report we identified a begomovirus infecting Datura inoxia, a common hallucinogenic alkaloid containing toxic weed found in India annually. D. inoxia showing typical begomovirus symptoms i.e., leaf curling, decrease in leaf size, and growth stunting as well as healthy leaves were collected from the fields of Rajasthan, India (Fig. 1). The DNA was extracted from infected leaves using CTAB method and PCR was carried out by using universal coat protein primer AC 1048 and AV 494 [2]. A typical PCR contained about 2.5 μl DNA template, 2.5 μl Taq 10× buffer (10 mM Tris–HCl, pH 8.8, 50 mM KCl and 1 % [v/v]), 1.5 μl MgCl2, 2 μl of each dNTPs, 0.5 μl of Taq DNA Polymerase, and 1 μl of each primer. The PCR thermal profile were pre-PCR denaturation at 94 °C for 60 s followed by 35 cycles of denaturing at 94 °C for 60 s, annealing at 55 °C for 2 min and extension at 72 °C for 3 min, and a final extension at 72 °C for 5 min. The PCR product was partially sequenced ({type:entrez-nucleotide,attrs:{text:JN000702,term_id:334847691,term_text:JN000702}}JN000702) and 550 nucleotides sequence was used to construct a phylogenetic tree using the neighbor-joining method in DNASIS Pro ver. 2.6. In order to confirm the geminivirus infectivity, Southern blotting was performed according to the method of Kon et al. [3]. Cloned DNA-A (2.7 Kb) of Chilli leaf curl virus was used to develop the radio-labeled probes. Fig. 1 Datura inoxia showing leaf curl symptoms in Lakshmangarh, Rajasthan in 2011 Sequence analysis of the virus under study showed 82–83 % nucleotide sequence identity with the corresponding region of Chilli leaf curl virus ({type:entrez-nucleotide,attrs:{text:FM210476,term_id:199601437,term_text:FM210476}}FM210476), Tomato leaf curl virus ({type:entrez-nucleotide,attrs:{text:AJ810360,term_id:62815995,term_text:AJ810360}}AJ810360), and Croton yellow vein virus ({type:entrez-nucleotide,attrs:{text:FN543112,term_id:304939561,term_text:FN543112}}FN543112). In neighbor-joining tree, the coat protein gene of the virus in datura leaf curl formed a separate monophyletic cluster with a low bootstrap value (Fig. 2). To complement the information the amplified product was used for the Southern hybridization. The strong signal showed that the virus in D. inoxia had some homology with Chilli leaf curl virus. The result suggested that the virus in D. inoxia samples belonged to the genus begomovirus. To the best of our knowledge, this is the first report of begomovirus associated with leaf curl of D. inoxia. Fig. 2 Neighbor-Joining tree based on the partial sequence of coat protein gene ({type:entrez-nucleotide,attrs:{text:JN000702,term_id:334847691,term_text:JN000702}}JN000702), of the virus isolate obtained from leaf curl disease of Datura inoxia ...

Production of phagmid library of camelid single-domain VHH antibodies against UreC of H.pylori

should be developed. Methods: The extracted DNA from two colorectal cell lines (SW480 and HCT116) and normal blood were used for BAT26 LightCycler PCR. We have used a technique to detectMSI by LightCycler PCR andmelting point analysis using sequence-specific hybridization probes (HyProbes) labeled with LightCycler dyes. 2 μl of DNA (50–100 ng) wasmixed with 13 μl amplification solution, resulting in a final concentration of 3 mM MgCl2, 0.5 μM of each primers BAT26-U and BAT26-D primers, 0.15 μM BAT26donor-HyProbe and BAT26accept-HyProbe and 1X LightCycler DNA Master Hybridization Probes-Mix. Subsequent melting point analysis was done. Results: The Tm alterations within BAT26 marker was detected in DNA fromHCT116 cell line compared to SW480andnormal lymphocyte. Conclusion: Microsatellite melting point analysis allows a fast microsatellite analysis of the two most sensitive and specific markers for MSI detection in colorectal cancer.

Rare Case of Trichomonal Peritonitis

Rare Case of Trichomonal Peritonitis

P3-S1.12 High concordance of test results of the Chlamydia trachomatis detection and genotyping kit compared to the COBAS Amplicor CT/NG test

BackgroundImproving diagnostic methods for the detection of Chlamydia trachomatis (CT), including genotyping, can contribute to control of CT by acquiring knowledge on epidemiology, transmission, sexual networks and pathogenicity. In the...

Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Regulation of CaV1.2 channels in cardiac myocytes by the β-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the β-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation

The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species.

Optimization for ISSR-PCR reaction system of tea plant

The orthogonal design was used to optimize ISSR amplification system of tea plant in five factors (Taq polymerase, Mg2+, DNA templet, dNTP, primer) at four levels respectively. The best level of each factor were selected, a suitable ISSR reaction system was established, namely 20 μL reaction system containing 1.0 U/(20 μL) Taq DNA polymerase, 2.0 mmol/L Mg2+, 40 ng/(20 μL) DNA templet, 0.20 mmol/L dNTP, 0.25 μmol/L primer. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR, showing that the most suitable temperature of primer ISSR1, UBC881 was 52.4 ℃, 59.0 ℃.

Evaluation of Plasmodium vivax ELISA for the blood screen

Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood-related products in ROK. We evaluated a P.vivax-specific enzyme-linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P.vivax-infected patients, 39 samples from post-treatment patients upon follow-up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P.vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P.vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.

HNF1α and SREBP2 are important regulators of NPC1L1 in human liver

HNF1α and SREBP2 are important regulators of NPC1L1 in human liver

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Real-Time PCR for Diagnosis of Oculoglandular Tularemia

Real-Time PCR for Diagnosis of Oculoglandular Tularemia

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.

Tissue Sampling and Genomic DNA Purification from the Western Clawed Frog Xenopus tropicalis: Figure 1.

INTRODUCTIONThe extraction of genomic DNA from Western clawed frog (Xenopus tropicalis) tissue samples is an essential step for genotyping known mutations, identifying live animals carrying nonfluorescent transgenes, and reverse genetic screening techniques. We describe here a method for sampling tail tissue from X. tropicalis tadpoles at stage 48 and later. This method of tissue sampling allows a significant amount of genomic DNA to be obtained from tadpoles without killing them. In both Xenopus laevis and X. tropicalis, the tip of the tail is able to regenerate following surgery and its removal does not have a significant effect on the survival of the tadpole when performed carefully. Genomic DNA purification can be carried out in “deep-well” 96-well plates, making it amenable to high-throughput applications. Typically, the DNA yield is sufficient for more than 100 standard 50-μL polymerase chain reactions (PCRs) (using 1-5 μL of DNA per reaction), so it can be used for genetic screening and mapping studies.

GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

The transcription factors GATA-1 and GATA-2 have been implicated in promoting differentiation of eosinophilic leukocytes. In this study, we examined the roles of GATA-1 and GATA-2 in activating transcription of the secretory ribonuclease, the eosinophil-derived neurotoxin (EDN/RNase 2). Augmented expression of both GATA-1 and GATA-2 was detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Deletion or mutation of one or both of the two consensus GATA-binding sites in the extended 1000-bp 5' promoter of the EDN gene resulted in profound reduction in reporter gene activity. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicate that GATA-1 and GATA-2 proteins bind to both functional GATA consensus sequences in the EDN promoter. Interestingly, RNA silencing of GATA-1 alone had no impact on EDN expression; silencing of GATA-2 resulted in diminished expression of EDN, and also diminished expression of GATA-1 in both butyric acid-induced HL-60 clone 15 cells and in differentiating human eosinophils derived from CD34(+) hematopoietic progenitors. Likewise, overexpression of GATA-2 in uninduced HL-60 clone 15 cells resulted in augmented transcription of both EDN and GATA-1. Taken together, our data suggest that GATA-2 functions directly via interactions with the EDN promoter and also indirectly, via its ability to regulate the expression of GATA-1 in differentiating eosinophils and eosinophil cell lines.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding approximately 2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further approximately 40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.