lacZ Reporter Gene Research Articles

Mesenchymal bone marrow stem cells within polyglycolic acid tube observed in vivo after six weeks enhance facial nerve regeneration

Autografting is the gold-standard method for facial nerve repair with tissue loss. Its association with high-quality scaffolds and cell implants has disclosed distinct experimental outcomes. The aim of this study was to evaluate the functional and histological effects of bone marrow stem cells (BMSC) combined with polyglycolic acid tube (PGAt) in autografted rat facial nerves. After neurotmesis of the mandibular branch of the rat facial nerve, surgical repair consisted of nerve autografting (groups A–E) contained in pGAT (groups B–E), filled with basement membrane matrix (groups C–E) with undifferentiated BMSC (group D) or Schwann-like cells that had differentiated from BMSC (group E). Axon morphometrics and an objective compound muscle action potentials (CMAP) analysis were conducted. Immunofluorescence assays were carried out with Schwann cell marker S100 and anti-β-galactosidase to label exogenous cells. Six weeks after surgery, animals from either cell-containing group had mean CMAP amplitudes significantly higher than control groups. Differently from other groups, facial nerves with Schwann-like cell implants had mean axonal densities within reference values. This same group had the highest mean axonal diameter in distal segments. We observed expression of the reporter gene lacZ in nerve cells in the graft and distally from it in groups D and E. Group-E cells had lacZ coexpressed with S100. In conclusion, regeneration of the facial nerve was improved by BMSC within PGAt in rats, yet Schwann-like cells were associated with superior effects. Accordingly, groups D and E had BMSC integrated in neural tissue with maintenance of former cell phenotype for six weeks.

Charting the regulatory landscape of the mouse genome in vivo

Background Over the past years it has become apparent that a substantial part of mammalian gene regulation is achieved through the concerted action of long-range elements. Technical advances have greatly improved our ability to identify such elements, but still little is known about how they regulate gene expression mechanistically. It is particularly unclear to what extent genomic context influences the activity of regulatory elements, and therefore how authentically in vitro or transgene assays represent the actual activity of such elements. To characterise the regulatory architecture of genomic loci in vivo, we have developed GROMIT (Genome Regulatory Organisation Mapping with Integrated Transposons) [1]. GROMIT relies on the controlled mobilisation of a Sleeping Beauty transposon to distribute a lacZ reporter gene, acting as a regulatory sensor, throughout the mouse genome. The sensor integrates into the genome, without disrupting endogenous gene expression, and the precise location of insertions can be mapped.

Infant satiety depends on transient expression of cholecystokinin‐1 receptors on ependymal cells lining the third ventricle in mice

Cholecystokinin (CCK) is a hypothetical controller for suckling and infancy body weight, although the underlying mechanisms remain unclear. Therefore, the present study analysed the mechanisms using mice lacking the CCK-1 receptor (CCK1R-/-). Although CCK1R-/- mice displayed normal weights at birth and adulthood, CCK1R-/- pups had enlarged adipocytes and were overweight from the first to second week after birth, regardless of maternal genotype. The lacZ reporter gene assay and/or calcium imaging analysis demonstrated that CCK-1 receptors were abundant in satiety-controlling regions such as the hypothalamus, brainstem, nodose ganglion and pylorus in adults, whereas these signals were few to lacking at pre-weanling stages. At postnatal day (PD) 6, the increase in cFos expression in the medullary nucleus tractus solitarius was similarly triggered by gastrointestinal milk- or saline filling in both genotypes, further indicating immature CCK-1 receptor function in an ascending satiety-controlling system during infancy. Conversely, third ventricle ependymal tanycyte-like cells expressed CCK-1 receptors with expression peaking at PD6. At PD6, wild-type but not CCK1R-/- mice had increased cFos immunoreactivity in ependymal cells following gastrointestinal milk filling whereas the response became negligible at PD12. In addition, ependymal cFos was not increased by saline filling, indicating that these responses are dependent on CCK-1 receptors, developmental stage and nutrients. Furthermore, body weights of wild-type pups were transiently increased by blocking ependymal CCK receptor function with microinjection of a CCK-1 antagonist, but not a CCK-2 antagonist. Hence, we demonstrate de novo functions of ependymal CCK-1 receptors and reveal a new aspect of infant satiety-controlling mechanisms.

Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in Pseudoplusia includens larvae

Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrosis virus were quantified in penultimate instar Pseudoplusia includens (soybean looper) larvae using a recombinant encoding the lacZ reporter gene (AcMNPV-hsp70/lacZ). Larvae inoculated orally with AcMNPV-hsp70/lacZ occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were susceptible to fatal infection (LD50=40.8 OB; 13.8 BV plaque forming units). Pseudoplusia includens displayed increased developmental resistance as larvae were orally inoculated with OB at later times during the penultimate instar. The optical brightener M2R, an inhibitor of the apoptotic processes that drive developmental resistance, did not significantly affect the virulence of AcMNPV-hsp70/lacZ OB. To study pathogenesis, newly molted penultimate instar P. includens were orally inoculated with 60 OB and examined from 0.5 to 96h post inoculation (h.p.i). Infection in the midgut was first detected at 4h.p.i in 32% of the larvae and was apparent in cells of the tracheal system at 8h.p.i. LacZ-positive (LacZ+) hemocytes were first observed 2h later. At 18h.p.i, a low proportion of the hemocytes were LacZ+ (5.6%). However, flow cytometry analysis of cell surface expression of the viral protein GP64 showed that 84.5% of the hemocytes collected at 18h.p.i were infected with AcMNPV, suggesting that flow cytometry may be a more sensitive method for identifying AcMNPV-infected cells. Because P. includens display no physiological barriers to AcMNPV OB and is permissive to fatal infection, this species could be controlled in organic cropping systems using naturally occurring strains of AcMNPV.

A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export

The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine–glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

Expression pattern of Protein Kinase C ε during mouse embryogenesis

BackgroundProtein kinase C epsilon (PKCϵ) belongs to the novel PKC subfamily, which consists of diacylglycerol dependent- and calcium independent-PKCs. Previous studies have shown that PKCϵ is important in different contexts, such as wound healing or cancer. In this study, we contribute to expand the knowledge on PKCϵ by reporting its expression pattern during murine midgestation using the LacZ reporter gene and immunostaining procedures.ResultsSites showing highest PKCϵ expression were heart at ealier stages, and ganglia in older embryos. Other stained domains included somites, bone, stomach, kidney, and blood vessels.ConclusionsThe seemingly strong expression of PKCϵ in heart and ganglia shown in this study suggests a important role of this isoform in the vascular and nervous systems during mouse development. However, functional redundancy with other PKCs during midgestation within these domains and others reported here possibly exists since PKCϵ deficient mice do not display obvious embryonic developmental defects.

Tyrosine hydroxylase gene transfections to different sites of striatum in the rat model of Parkinson’s disease

The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the outcome of gene delivery or on the physiological state of target tissues have not been analyzed as much and systematically. Therefore, we have infused adenovirus vectors expressing either a therapeutic tyrosine hydroxylase (TH) gene or a lacZ reporter gene into striatum in a rat model of PD. The experimental procedures were tested using the Ad lacZ vector in order to optimize concentrations, volumes, infusion speeds and transfection times. The expression of Ad lacZ vector was lower and declined earlier in the lesioned than unlesioned striatum suggesting that the lesion affects on the transfection efficiency and outcome of gene transfection. The effect of three different approaches of Ad TH vector transfection was compared: 1) the delivery of Ad TH gene vector alone into one single site of striatum, 2) the delivery of Ad TH gene vector alone into multiple sites of striatum, and 3) the delivery of Ad TH gene vector into one site of striatum followed by a continuous infusion of tetrahydrobiopterin (BH4) cofactor with a mini pump. There was a small and transient unsignificant decrease in the turning behavior when the Ad TH vector was delivered into one site of the striatum. Simultaneous infusion into several sites or together with BH4 cofactor did not improve more the effect of gene delivery. Thus, although the effects were unsignificant, the Ad TH transfection seemed to decrease the turning behavior in the rat model of PD and the optimal effect was seen at some specific doses and time points. Furthermore, the outcome of gene therapy could depend in addition to the amount and efficacy of gene vectors also on the physiological state and experimental strategies.

The Molecular Chaperone Hsp90 is required for Actin Dynamics during Sperm Individualization in Drosophila

Heat shock proteins are known not only to function in stress response but also to participate in various developmental activities of the cell. Hsp90 of Drosophila has been shown to be required for spermatogenesis, since a hypomorphic allele of the hsp83 gene, hsp8308445, is male sterile with all stages requiring microtubule functions being affected. But the role of Hsp90 in actin dynamics during male germ cell development has not been investigated so far. We have extended the studies on the hsp83 08445 allele, and show that the sperm flagella are fragile and loosely packed in this allele, and the actin cone assembly and progression during individualization of sperms are badly affected. Further, sperm nuclei are misshapen and not embedded in the F-actin extensions of the head cyst cells, resulting in loss of their alignment and retention at the base of the testis. Thus, our data indicates a critical role for Hsp90 in actin dynamics during sperm individualization. We also provide a comprehensive account of the LacZ reporter gene activity associated with the hsp8308445 allele in comparison to the wild type Hsp90 distribution in various larval and adult tissues. Furthermore, we report an important observation that the chromatin of somatic cyst cells of testis is structured in a characteristic fashion as visualized by DAPI staining, which can be conveniently used as a specific marker for this cell type, eliminating the need for a marker antibody.

Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes

As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

332 COMBINATION OF ZINC FINGER NUCLEASES AND MODIFIED BACTERIAL ARTIFICIAL CHROMOSOMES REVEALS TREMENDOUS RATES OF HOMOLOGOUS RECOMBINATION

DNA-based vectors have been used for decades to modify the genomes of mammalian cells by homologous recombination in a specific and site-directed way. Even though various modifications of the procedure have been presented, efficiency is relatively low for many target sites and novel projects still have an unforeseeable outcome. This is in particularly true for site-directed mutagenesis in primary cells intended for use in the generation of large animal models because of their impaired predisposition for homologous recombination compared with stem cells. The recent development of site-specific nucleases is based on a completely different principle: they do not necessarily involve recombination between DNA strands, but rather make use of the inefficient correction of double-strand breaks in the genomic DNA by the cellular DNA repair machinery after such a double-strand break has been introduced by a synthetic enzyme that directed nuclease activity to a defined site in the genome. Here, we intended to evaluate the potential of zinc finger nucleases (ZFN) to introduce a lacZ reporter gene into the CFTR locus. Initially, the efficiency of 3 different ZFN pairs was examined under different conditions revealing modification efficiencies between 0 and 38%. An optimized protocol was used to combine the most efficient ZFN pair with either a bacterial artificial chromosome (BAC) vector or a conventional targeting vector carrying the desired modification. Although the conventional vector failed to introduce the reporter gene in any of more than 200 clones examined, the BAC correctly modified the target site in 32 of 75 clones in a heterozygous way and in 10 out of 75 clones in a homozygous way. However, the introduction of small vector fragments into the CFTR locus in rare cases indicated that the ZFN caused a double-strand break but the vector was not able to act as a recombination donor. On the other hand, transfection of the BAC alone only resulted in 1 modified clone out of 98 and, thus, our data strongly support the hypothesis that the forced introduction of double-strand breaks dramatically increases the rate of homologous recombination, but they also provide indication that the design of the targeting vector has a profound influence on the efficiency.

Abstract 18700: Accelerated Endothelial Regeneration and Survival Attenuate Vein Graft Atherosclerosis via Terahydrobiopterin-Dependent eNOS Coupling

As a result of surgical trauma, ischemia and exposure to arterial pressure the majority of endothelium in vein grafts (VG) undergo necrosis or apoptosis. We hypothesized that increased eNOS coupling could protect endothelial viability after VG surgery. To investigate this we utilized a transgenic mouse with endothelial-targeted overexpression of the essential eNOS coupling cofactor BH4. We investigated the effect of BH4 on endothelial cell survival and vascular remodeling in vitro and in vivo using ApoE-KO mice with endothelial-targeted overexpression of the rate limiting enzyme of BH4 synthesis GTP cyclohydrolase 1 (GCH) and LacZ reporter gene. BH4 levels were increased 10-fold in primary endothelium and 5-fold in aorta from GCH/ApoE-KO mice compared with ApoE-KO, without concomitant increases in eNOS protein. Despite no differences at baseline, following 4 days of culture, total endothelial cell number, colony size and number from GCH/ApoE-KO mice were increased (5-fold P=0.05; 2-fold P=0.02; 2-fold P=0.06 respectively, n=6) as were putative EPC levels from bone-marrow mononuclear cells determined by culture (15%,P=0.04) and FACS (26%, P=0.03). Vascular injury as a result of VG surgery in ApoE-KO mice led to significant neointima that was augmented by 61% (P=0.002) in GCH/ApoE-KO mice. Furthermore, endothelial repopulation was accelerated by 21% (P=0.01) and endothelial survival 44% (P=0.03) in these animals. O2- production was significantly attenuated in GCH/ApoE-KO mice measured on both aortic sections using oxidative microtopography and whole aorta by lucigenin chemiluminescence. eNOS inhibition by L-NAME reversed these effects indicating that under basal conditions GCH/ApoE-KO mice had preserved eNOS coupling promoting NO synthesis. In keeping with these findings in 219 patients with coronary artery disease, analysis of saphenous vein and internal mammary artery identified that high vascular BH4 levels were associated with greater vasorelaxations to acetylcholine (P<0.05) and reduced total L-NAME inhibitable O2- suggesting improved eNOS coupling. Availability of BH4 within the endothelium critically modulates the response to vascular injury by maintaining eNOS coupling and accelerating endothelial regeneration.

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ-based plasmids with a choice of three inducible promoters, P(GAL1) , P(CUP1) and P(ADH2) . These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA-mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid-based and integrated chromosomal cassettes, and compared to that of the glycolytic P(PGK1) promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P(PGK1) , P(TEF1) and P(HXT7-391) .

Precision of Hunchback Expression in the Drosophila Embryo

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected "stripe" enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. Here, we employ BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. We propose that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient.

Arsenic stress elicits cytosolic Ca2+ bursts and Crz1 activation in Saccharomyces cerevisiae

Although arsenic is notoriously poisonous to life, its utilization in therapeutics brings many benefits to human health, so it is therefore essential to discover the molecular mechanisms underlying arsenic stress responses in eukaryotic cells. Aiming to determine the contribution of Ca(2+) signalling pathways to arsenic stress responses, we took advantage of the use of Saccharomyces cerevisiae as a model organism. Here we show that Ca(2+) enhances the tolerance of the wild-type and arsenic-sensitive yap1 strains to arsenic stress in a Crz1-dependent manner, thus providing the first evidence that Ca(2+) signalling cascades are involved in arsenic stress responses. Moreover, our results indicate that arsenic shock elicits a cytosolic Ca(2+) burst in these strains, without the addition of exogenous Ca(2+) sources, strongly supporting the notion that Ca(2+) homeostasis is disrupted by arsenic stress. In response to an arsenite-induced increase of Ca(2+) in the cytosol, Crz1 is dephosphorylated and translocated to the nucleus, and stimulates CDRE-driven expression of the lacZ reporter gene in a Cnb1-dependent manner. The activation of Crz1 by arsenite culminates in the induction of the endogenous genes PMR1, PMC1 and GSC2. Taken together, these data establish that activation of Ca(2+) signalling pathways and the downstream activation of the Crz1 transcription factor contribute to arsenic tolerance in the eukaryotic model organism S. cerevisiae.

The regulatory factor PcRFX1 controls the expression of the three genes of β-lactam biosynthesis in Penicillium chrysogenum

Penicillin biosynthesis is subjected to a complex regulatory network of signalling molecules that may serve as model for other secondary metabolites. The information provided by the new “omics” era about Penicillium chrysogenum and the advances in the knowledge of molecular mechanisms responsible for improved productivity, make this fungus an excellent model to decipher the mechanisms controlling the penicillin biosynthetic pathway. In this work, we have characterized a novel transcription factor PcRFX1, which is an ortholog of the Acremonium chrysogenum CPCR1 and Penicillium marneffei RfxA regulatory proteins. PcRFX1 DNA binding sequences were found in the promoter region of the pcbAB, pcbC and penDE genes. We show in this article that these motifs control the expression of the β-galactosidase lacZ reporter gene, indicating that they may direct the PcRFX1-mediated regulation of the penicillin biosynthetic genes. By means of Pcrfx1 gene knock-down and overexpression techniques we confirmed that PcRFX1 controls penicillin biosynthesis through the regulation of the pcbAB, pcbC and penDE transcription. Morphology and development seemed not to be controlled by this transcription factor under the conditions studied and only sporulation was slightly reduced after the silencing of the Pcrfx1 gene. A genome-wide analysis of processes putatively regulated by this transcription factor was carried out in P. chrysogenum. Results suggested that PcRFX1, in addition to regulate penicillin biosynthesis, is also involved in the control of several pathways of primary metabolism.

Yap1-dependent oxidative stress response provides a link to riboflavin production in Ashbya gossypii

Ashbya gossypii is a natural overproducer of riboflavin. Overproduction of riboflavin can be induced by environmental stress, e.g. nutritional or oxidative stress. The Yap-protein family has a well-documented role in stress response. Particularly, Yap1 has a major role in directing the oxidative stress responses. The A. gossypii YAP-family consists of only three genes in contrast to its closest relative Eremothecium cymbalariae, which has four YAP-homologs. Gene order at Eremothecium YAP-loci is conserved with the reconstructed yeast ancestor. AgYap1p is unique amongst Yap-homologs as it lacks the cysteine-rich domains (CRDs). AgYAP1 expression is inducible and GFP-AgYap1 localizes to the nucleus. Agyap1 mutants displayed higher sensitivity against oxidative stress – H2O2 and menadione - and are strongly reduced in riboflavin production. High levels of cAMP, which also reduce riboflavin production, show a synergistic effect on this sensitivity. AgYAP1 and a chimera of AgYAP1 (with the DNA-binding domain) and ScYAP1 (with the CRDs) can both complement the Scyap1 oxidative stress sensitivity. This suggests that the DNA-binding sites of ScYap1 are conserved in A. gossypii. Expression of AgRIB4, which contains three putative Yap1-binding sites, assayed via a lacZ-reporter gene was strongly reduced in an Agyap1 mutant suggesting a direct involvement of AgYap1 in riboflavin production. Furthermore, our data show that application of H2O2 stress leads to an increase in riboflavin production in a Yap1-dependent manner.

Functional Characterization of the HumanRPGRProximal Promoter

Mutations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene account for more than 70% of X-linked RP cases. This study aims to characterize the proximal promoter region of the human RPGR gene.The 5'-flanking region (5 kb) of human RPGR was cloned and sequenced. A potential transcription start site and transcription factor binding motifs were identified by bioinformatic analysis. Constructs containing the putative human RPGR promoter region upstream of a luciferase reporter gene were generated and analyzed by transient transfection and luciferase assays. Transgenic mouse lines carrying a 3-kb human RPGR promoter sequence fused to lacZ were generated and RPGR proximal promoter activity was analyzed by X-gal staining.Bioinformatic analyses of the human RPGR 5'-flanking region uncovered potential transcription factor binding sites and a CpG island. Transient transfection assays with RPGR promoter/luciferase reporter constructs revealed a 980-bp fragment (-952 to +28) that produced higher levels of luciferase activity. Mutagenesis identified a putative Sp1 binding site that was critical for regulating transcriptional activity. We generated transgenic mice in which a lacZ reporter gene was controlled by the 3-kb upstream region of RPGR. β-galactosidase expression was predominantly found in mouse retina, brain, and kidney. In the retina, the photoreceptor cell layer showed the strongest β-galactosidase staining.Our study defined the human RPGR proximal promoter region in which a 3-kb fragment contained sufficient regulatory elements to control RPGR expression in mouse retina and other tissues. Characterization of the RPGR promoter will facilitate understanding of the functional role of RPGR in the retina and gene therapy of X-linked RP.

Isolation and characterization of two ABRE-binding proteins: EABF and EABF1 from the oil palm

Abscisic acid (ABA) is an important phytohormone involved in the abiotic stress resistance in plants. The ABA-responsive element (ABRE) binding factors play significant roles in the plant development and response to abiotic stresses, but none so far have been isolated and characterized from the oil palm. Two ABA-responsive cDNA clones, named EABF and EABF1, were isolated from the oil palm fruits using yeast one-hybrid system. The EABF had a conserved AP2/EREBP DNA-binding domain (DNA-BD) and a potential nuclear localization sequence (NLS). No previously known DNA-BD was identified from the EABF1 sequence. The EABF and EABF1 proteins were classified as DREB/CBF and bZIP family members based on the multiple sequence alignment and phylogenetic analysis. Both proteins showed ABRE-binding and transcriptional activation properties in yeast. Furthermore, both proteins were able to trans-activate the down-stream expression of the LacZ reporter gene in yeast. An electrophoretic mobility shift assay revealed that in addition to the ABRE sequence, both proteins could bind to the DRE sequence as well. Transcriptional analysis revealed that the expression of EABF was induced in response to the ABA in the oil palm fruits and leaves, but not in roots, while the EABF1 was constitutively induced in all tissues. The expressions of both genes were strongly induced in fruits in response to the ABA, ethylene, methyl jasmonate, drought, cold and high-salinity treatments, indicating that the EABF and EABF1 might act as connectors among different stress signal transduction pathways. Our results indicate that the EABF and EABF1 are novel stress-responsive transcription factors, which are involved in the abiotic stress response and ABA signaling in the oil palm and could be used for production of stress-tolerant transgenic crops.

γ-Heptalactone is an endogenously produced quorum-sensing molecule regulating growth and secondary metabolite production by Aspergillus nidulans

Microbes monitor their population density through a mechanism termed quorum sensing. It is believed that quorum-sensing molecules diffuse from the microbial cells and circulate in the surrounding environment as a function of cell density. When these molecules reach a threshold concentration, the gene expression of the entire population is altered in a coordinated manner. This work provides evidence that Aspergillus nidulans produces at least one small diffusible molecule during its growth cycle which accumulates at high cell density and alters the organism's behaviour. When added to low-density cell cultures, ethyl acetate extracts from stationary phase culture supernatants of A. nidulans resulted in the abolition of the lag phase, induced an earlier deceleration phase with 16.3% decrease in the final cell dry weight and resulted in a 37.8% increase in the expression of ipnA::lacZ reporter gene construct, which was used as a marker for penicillin production compared to non-treated controls. The bioactive molecule present in the stationary phase extract was purified to homogeneity and was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy to be γ-heptalactone. This study provides the first evidence that A. nidulans produces γ-heptalactone at a high cell density and it can alter the organism's behaviour at a low cell density. γ-Heptalactone hence acts as a quorum-sensing molecule in the producing strain.