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The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a β-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78 kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

Aims: Clostridium thermoc ellum is a thermophilic, anaerobic bacterium that ferments cellulose and produces ethanol. While many of the predicted cellulases have been characterized from C. thermocellum , characte rization of additional members is still needed to unravel the function of these enzymes. Study Design : This report describes the first cloning, expression, characterization, and evaluation in cellulose degradation of Clostridium thermocellum Cel5L as well as its relationship to other cellulases. Results: Cel5L, a 526 amin o acid protein that shows low homology to other cellulases of C. thermocellum , possesses both endo and exo activity on  - glucan, carboxymethylcellulose, and cellooligosaccharides; the enzyme does not hydrolyze xylan, xyloglucan or glucomannan. When

We have applied a rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, in order to define an efficient enzyme cocktail to hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and ��-glucosidase (��G; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), ��-glucosidase (L��G; GH family 1), endoxylanases (LX1 and LX2; GH family 10) and ��-xylosidase (L��X; GH family 52) from multiple sources were cloned, expressed and purified as well. Enzymatic hydrolysis for various enzyme combinations was performed on Ammonia Fiber Expansion (AFEX) treated corn stover at various total protein loadings (30, 15 and 10 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary for obtaining high glucan and xylan yields was determined using a suitable mixture design of experiments. The optimal hybrid enzyme mixtures contain fungal cellulases (78% of total protein loading), which include CBH I (loading ranging between 9-51% of total enzyme load), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LXl (13%) and L��X (9%). The hybrid mixture works effectively at 50 ��C and pH 4.5 to efficiently saccharify AFEX treated corn stover giving as high as 95% glucan and 65% xylan conversion, respectively. This strategy of screening enzyme mixtures on pretreated lignocellulose will ultimately help develop enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq(™) Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62 pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles.

Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal-ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling.

Wnt signaling has been implicated in many patterning processes in a vertebrate embryo including morphogenetic cell rearrangements during gastrulation. Pipetail (wnt5) and silberblick (wnt11) zebrafish mutants undergo abnormal gastrulation with an undulated notochord ( pipetail) and cyclopic embryos ( silberblick) at later stages of development (Hammerschmidt et al., 1996; Heisenberg et al., 1996; Heisenberg et al., 2000; Rauch et al., 1997). Wnt proteins are known to transmit signals via the Frizzled seven-pass transmembrane receptor family (Bhanot et al., 1996). We have isolated cDNA encoding a new Frizzled family protein, zebrafish Frizzled-2 (Fz2). Zebrafish frizzled-2 is a likely ortholog of human frizzled-2 by the sequence homology alignments and mapping data analysis. Fz2 is expressed zygotically within the notochord, somitic and posterior paraxial mesoderm. For protein knockdown studies, we utilized morpholino phosphorodiamidate antisense oligonucleotides (morpholinos, MOs). Injection of two different morpholinos into early zebrafish embryos targeted to fz2 caused similar developmental defects. Fz2-morpholino injected embryos (morphants) are shorter than controls and display an undulating or kinked notochord, neural tube, and hypochord. Similar, although weaker, undulations of axial structures have been observed in pipetail mutant embryos, raising possibility that fz2 may function as a receptor in the wnt5 pathway regulating gastrulation movements. We isolated a novel zebrafish frizzled homolog from gastrula stage cDNA library by the polymerase chain reaction (PCR). The isolated gene encodes a putative Frizzled protein with highest homology to the frizzled-2 subfamily as evident from BLAST and CLUSTAL alignments (Fig. 1a, b). Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz2 on linkage group 3, linked to the marker Z22516 (distance 0.00cR, LOD score 22.2). To confirm that we isolated true frizzled-2 ortholog from zebrafish, we performed synteny analysis between corresponding zebrafish and human chromosome regions. Zebrafish fz2 maps to the marker Z22516, which is located between the hoxb9a and dlx8 genes on linkage group 3 in the LN54 panel (Hukriede et al., 1999; marker list at http://zfin.org/ZFIN/). This region of zebrafish linkage group 3 is syntenic to the human chromosome 17 region. Human fz2 maps by FISH to 17q21.1 (Zhao et al., 1995), human hoxB9 maps to 17q21-q22 (Acampora et al., 1989; Apiou et al., 1996; HUGO database), and human Dlx4, an ortholog of zebrafish Dlx8, maps to 17q21.33 (Nakamura et al., 1996; Stock et al., 1996). Thus, human frizzled-2 is likely to be the true ortholog of the zebrafish frizzled-2 gene. Using Northern blotting, we detected a single, zygotically expressed transcript of fz2, with maximal expression level approximately at the five-somite stage (Fig. 1c). In situ hybridization revealed weak and ubiquitous expression starting from the shield stage (data not shown). During early somitogenesis, fz2 RNA is detected within the forming somites as well as in two longitudinal stripes within the paraxial posterior mesoderm (Fig. 1, d–g). There is also weak expression within notochordal cells anterior to fz2 expression in the forming somites. At the 20-somite stage, fz2 expression is detected in the posterior part of the notochord and in the posterior somitic mesoderm (Fig. 1h, i). At 26 hpf, fz2 is localized within the tailbud mesoderm (Fig. 1k). Injecting 100–500 pg fz2 RNA into fish embryos at the 1–2 cell stage caused severe hyperdorsalization in .90% of experimental embryos (data not shown), as noted previously for other Frizzled proteins (Nasevicius et al., 1998). A number of fz2-overexpressing embryos displayed secondary axes (data not shown). This illustrates that fz2 is capable of activating dorsoventral axis induction pathway when overexpressed in zebrafish embryos. To study fz2 function, we utilized an antisense morpholino oligonucleotide approach (Nasevicius and Ekker, 2000; Summerton, 1999). Two nonoverlapping morpholinos were designed against the fz2 sequence. Injecting either morpholino against fz2 produced similar phenotypes, indicating a likely high specificity of targeting to fz2 transcripts. The resulting embryos (fz2 morphants) display characteristic defects not commonly seen with other unrelated morpholinos. At 70% epiboly, fz2 morphants have an oblong shape compared with

Members of the frizzled gene family encode seven-pass transmembrane proteins that function in the interpretation and reception of Wnt-mediated cell-cell communication events. To investigate frizzled function in early zebrafish development, we isolated the maternally contributed frizzled 10 (fz10) gene and localized it to linkage group 8 using radiation hybrid mapping. The cloned zebrafish fz10 is closely related to the fz10 group from other organisms. Zygotic expression of fz10 is observed in the posterior tail mesenchyme, dorsal neural tube, and different parts of the brain.

The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidum does not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.

Contains fulltext : 87463.pdf (Publisher’s version ) (Closed access) Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia, and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown; however, the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor. Here, we show that two different missense mutations in WNT5A, which result in amino acid substitutions of highly conserved cysteines, are associated with autosomal dominant Robinow syndrome. One mutation has been found in all living affected members of the original family described by Meinhard Robinow and another in a second unrelated patient. These missense mutations result in decreased WNT5A activity in functional assays of zebrafish and Xenopus development. This work suggests that a WNT5A/ROR2 signal transduction pathway is important in human craniofacial and skeletal development and that proper formation and growth of these structures is sensitive to variations in WNT5A function. 01 januari 2010

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

Background Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. Results We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. Conclusions This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.

Publisher Summary This chapter focuses on the sleeping beauty transposon for efficient gene delivery. The core use of the Sleeping Beauty (SB) transposon in zebrafish has been previously published. The advantages of using SB over DNA injection methods include a higher rate of transgenesis, single-copy integrations with reproducible and long-term Mendelian expression of the transgene cassette, an increase in the total number of chromosomes modified within a particular founder animal strain, and an enzymatically precise engineering of the resulting host chromosome. SB is a potent and reliable gene transfer and expression method for zebrafish. SB is used as a two-component system in zebrafish embryos: a transposon DNA vector and transposase-encoding mRNA. The transposon consists of DNA cargo of choice flanked by palindromic sequences of inverted and direct repeats (IR/DRs). The transposase is delivered as synthetic mRNA, and the embryo's potent early translational machinery processes the mRNA into the active transposase enzyme. Transposon vectors are injected along with transposase mRNA to achieve germline integration. The transposon DNA (8.3 ng/μl) and transposase mRNA (100 ng/μl) are mixed together on ice in an eppendorf tube just prior to injection. Combined with the capacity of the zebrafish model to accommodate forward genetic mutagenesis screens, the pu.1 transgenic lines described in the chapter can be very useful in identifying genes that influence myeloid-erythroid and lymphoid/progenitor cell development as well as the differentiation of other myeloid cell types, including granulocyte and monocyte/macrophage lineages.

We used a morpholino-based gene-targeting screen to identify a novel protein essential for vascular development using the zebrafish, Danio rerio. We show that syndecan-2, a cell-surface heparan sulfate proteoglycan, is essential for angiogenic sprouting during embryogenesis. The vascular function of syndecan-2 is likely conserved, as zebrafish and mouse syndecan-2 show similar expression patterns around major trunk vessels, and human syndecan-2 can restore angiogenic sprouting in syndecan-2 morphants. In contrast, forced expression of a truncated form of syndecan-2 results in embryos with defects in angiogenesis, indicating that the highly conserved cytoplasmic tail is important for the vascular function of syndecan-2. We further show that vascular endothelial growth factor (VEGF) and syndecan-2 genetically interact in vivo using both gain-of-function and loss-of-function studies in zebrafish. VEGF-mediated ectopic signaling is compromised in syndecan-2 morphants, and ectopic syndecan-2 potentiates ectopic VEGF signaling. Syndecan-2 as a novel angiogenic factor is a potential candidate for use in the development of angiogenesis-based therapies.

We have isolated and mapped a new wnt receptor frizzled family member, zebrafish frizzled 7a. Fz7a and a previously reported zebrafish fz7 (El-Messaoudi and Renucci, 2001) make an orthologous gene pair, however, they display distinct expression patterns. Fz7a shows strong maternal as well as zygotic expression. Fz7a transcript is enriched dorsally starting with the shield stage. At the end of gastrulation, Fz7a is abundantly expressed within anterior neuroectoderm and expressed more weakly within lateral mesoderm. Fz7a is detected during somitogenesis within the central nervous system, somatic and posterior lateral mesoderm. At 24 hpf, fz7a is expressed in migrating lateral line primordium. At 48 hpf, fz7a is detected in the ear, pectoral fin bud, and within neuromasts, which had originated from the lateral line primordium. Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz7a on linkage group 6, linked to the marker fi11h08 (distance 0.00cR, LOD score 14.1). To prove that fz7 and fz7a are indeed different genes we mapped fz7 as well using the same LN54 panel. Fz7 mapped to linkage group 9 with a LOD of 12.5, 27.31 cR from wnt 10a in between markers IBD2759 and fb50e04.

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.