No description available.

No description available.

In 1871, a unique substance was isolated from the white blood cells of pus. This substance, which later became known as chromatin, was shown to be a nucleoprotein complex which encompasses the majority of genomic DNA in all eukaryotes. Although chromatin was once viewed as primarily a structural component of the nucleus, it is now accepted that it also plays an important role in the modulation of transcription of individual genes. In this study, the 5S rRNA genes in Xenopus laevis were used as a system to investigate potential roles for chromatin structures in transcription regulation. X. laevis produces two major classes of 5S rRNA: the somatic type is present in most cells whereas the oocyte type is produced only during oogenesis and the early stages of embryogenesis. These two gene families share a very similar coding region and employ identical transcription machinery, leading researchers to believe that it is how these genes are packed into chromatin which is responsible for the differential developmental regulation. Initially, this study focused on the binding constraints placed on the RNA polymerase III basal transcription factor, transcription factor IIIA (TFIIIA), by a histone octamer. Five overlapping fragments of the X. laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes and it was shown that each fragment positions a histone octamer at unique translational sites. Using these nucleosomes it was demonstrated that nucleosome translational positioning is the major determinant of the binding of TFIIIA to the 5S rRNA genes. The relationship between core histone acetylation and transcription of the X. laevis 5S rRNA genes was also investigated. By immunopreciptitating chromatin fragments from a X. laevis kidney cell line with an antibody specific for hyperacetylated histone H4, it was shown that the oocyte 5S rRNA genes are packaged with hypoacetylated histone H4 when transcriptionally repressed.This taken together with the results of others, suggests a link between histone acetylation and RNA polymerase III transcription. However this study was unable to shed light on the basis for this relationship as it was found that histone acetylation did not affect the binding of TFIIIA to nucleosomal DNA. In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. It was shown that despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin and TFIIIA from Xenopus laevis. Finally, it has long been proposed that selective binding of histone H1 is, in part, responsible for the differential developmental regulation of the oocyte and somatic 5S rRNA genes in Xenopus laevis. In this study it was shown that histone H1 bound both oocyte and somatic genes equally after reconstitution into mononucleosomes or oligonucleosome arrays. Furthermore it was shown that the binding of histone H1 selectively repressed only oocyte gene transcription, and that a RNA polymerase III selectively repressed only oocyte gene transcription, and that a RNA polymerase III transcription complex was able to initiate transcription of nucleosomal somatic templates regardless of whether histone H1 was present. These results support a model in which the differential regulation of the 5S rRNA genes is not due simply to the prevention of histone HI binding by transcription complexes on the somatic genes, but rather a difference in the interaction of histone HI with the somatic and oocyte genes. / Graduate

A Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine University of the Witwatersrand Parktown, Johannesburg, South Africa June 2017 / Atrazine, an extensively used herbicide is amongst the commonly detected herbicides in groundwater. Atrazine concentrations as low as 0.01μg/l have been implicated to affect frog populations, thus much attention has been placed on its use and safety. Several studies have examined atrazine effects on reproductive organs, immune systems and population fitness of adult Xenopus laevis species and we found no studies on the effects of atrazine on the liver and kidney. This study investigated biochemical and histopathological effects of chronic exposure to atrazine on livers and kidneys of adult Xenopus laevis frogs, post metamorphosis. Forty male frogs were randomly divided into four groups (A -D) of 10 frogs each, housed in stainless silver tanks with 60L of water and atrazine concentration of 0μg/l A: control, B: 0.01μg/l, C: 200μg/l and D: 500μg/l respectively, for 90 days. Liver (ALT, ALKp and AST) and kidney (urea, creatinine) biomarkers, malondialdehyde, an indicator of lipid peroxidation, histopathology, melanomacrophage percentage area and fibrosis were examined. Significant increases of ALT and creatinine were observed at 200 and 500μg/l (P<0.05). Malondialdehyde was significantly increased at 500μg/l (P<0.05). Histopathologically, the liver showed disorganization in the arrangement of hepatic cords, hypertrophied hepatocytes, hepatocyte vacuolization, vascular congestion and dilation, infiltration of inflammatory cells and apoptosis and/or necrosis, with the highest atrazine concentration causing the most adverse effects. The kidney showed glomerular atrophy and degeneration, tubular lumen dilation, vacuolization and degeneration of thick loop of Henle tubule epithelial cells. Melanomacrophage percentage areas were significantly decreased at 0.01μg/l and 500μg/l and significantly increased at 200μg/l (P<0.05). No significant fibrosis was observed in all treated groups. The results suggest that very low and high environmentally relevant doses of atrazine have the ability to adversely affect organs of amphibian species and potentially related aquatic organisms. / MT2017

No description available.

非洲爪蛙(Xenopus laevis)和熱帶爪蛙(Xenopus tropicalis)是經典的模式動物，廣泛應用於胚胎學，發育生物學和人類疾病模型研究領域。然而，由於缺乏有效地同源重組技術和胚胎幹細胞誘導技術，在上述兩種模式動物中很難通過定點突變研究特定基因的功能。這些技術瓶頸制約其在遺傳學研究方面的應用。近來，通過鋅指核酸酶(ZFNs)和類轉錄激活因子效應物核酸酶(TALENs)介導的特異性基因突變技術成功地應用於各種模式動物模型，包括：線蟲、斑馬魚和大鼠。鋅指核酸酶和TALENs核酸酶都是由可編碼設計的DNA結合功能域和非特異性FokI核酸酶的功能域組成的人工構建DNA核酸酶。結合到相鄰DNA位點的鋅指核酸酶或TALENs核酸酶的單體通過FokI功能域結合形成二聚體，從而啟動其核酸酶活性，在預設靶點產生DNA雙鏈斷裂 (double-strand breaks)。通常，非同源末端連接(NHEJ)在修復DNA雙鏈斷裂過程中會導致缺失或者插入突變(indel)。在此研究中，我們在世界上首次利用TALENs核酸酶在爪蛙胚胎中高效誘導產生體細胞突變。我們優化了Golden Gate TALENs核酸酶的拼接方法，使其便於體外RNA轉錄和顯微注射到爪蛙胚胎中。我們還利用基於聚合酶鏈式反應(PCR)的檢測方法，用於檢測基因突變效率。我們設計八對TALENs核酸酶，它們分別靶向識別爪蛙中八個基因。試驗結果表明它們全部都能夠在爪蛙胚胎中誘導基因突變，突變率最高達百分之九十五點七(95.7%)。我們進一步證明，TALENs核酸酶誘導產生的突變可以通過生殖細胞高效地傳遞給F1代的爪蛙。不僅如此，我們還嘗試運用最新的RNA介導的基因組編輯方法(CRISPR)在小鼠誘導多功能幹細胞(iPSCs)模型中研究基因修正。初步結果顯示, 我們設計的TALENs核酸酶和CRISPR可以在小鼠誘導多功能幹細胞中有效地產生基因突變。 / 綜上所述，我們在世界上首次在爪蛙中報導了運用TALENs核酸酶進行反向遺傳學研究。利用TALENs核酸酶誘導突變的方法簡單但高效。我們的實驗結果表明TALENs核酸酶是一種在爪蛙中進行基因編輯或者基因敲除的有效工具。 / Xenopus laevis and Xenopus tropicalis are classical and powerful animal models widely used in the study of embryonic development and human disease modeling. However, due to the lack of methodologies for homologous recombination and embryonic stem cell derivation, it is difficult to perform specific gene targeting inthese two models, which has impeded their use in genetic studies for decades. Recently, site-specific gene targeting by using either zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) has been successfully applied in various animal models including C. elegans, zebrafish, and rat. Both ZFNs and TALENs are engineered DNA nucleases that consist of a custom-designed DNA-binding domain and a nonspecific nuclease domain derived from FokI endonuclease. Binding of adjacent ZFNs or TALENs allows dimerization of the endonuclease domains, leading to double-strand breaks at the predetermined site. These double-strand DNA breaks are frequently repaired through non-omologous end joining (NHEJ), resulting in deletion or insertion (indel) mutations. Here we reported that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germline transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for in vitro RNA transcription and microinjection into Xenopus embryos, and designed a reliable PCR-based assay for the evaluation of gene disruption efficiency. Totally, eight pairs of TALENs were constructed to target eight different Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci according to our PCR-based assay. Furthermore, mutations induced by TALENs were highly efficiently passed through the germline to F1 frogs. Moreover, we tried to employ newly published RNA-mediated genome editing tool, clustered regularly interspaced palindromic repeat (CRISPR), to study gene correction in mice induced pluripotent stem cells (iPSCs) model. Our preliminary data showed that TALENs and CRISPR we constructed can efficiently introduce mutations at predetermined sites in mice iPSCs. / So far, our result is the first report to perform specific reverse genetic via TALENs in Xenopus. Together with simple and reliable approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lei, Yong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 146-171). / Abstracts also in Chinese. / Abstract --- p.I / Acknowledgements --- p.V / Statement --- p.VI / Abbreviation --- p.VII / Contents --- p.IX / Chapter 1. --- Research Background --- p.1 / Chapter 1.1 --- Introduction of reverse genetics --- p.1 / Chapter 1.2 --- Introduction of Xenopus tropicalis --- p.3 / Chapter 1.3 --- Xenopus tropicalis as an animal model for reverse genetics --- p.6 / Chapter 1.4 --- Zinc finger nucleases (ZFNs) --- p.10 / Chapter 1.5 --- Transcription activator-like effector nucleases (TALENs) --- p.29 / Chapter 1.6 --- Clustered regularly interspaced short palindromic repeats (CRISPR) --- p.40 / Chapter 2. --- Objective --- p.52 / Chapter 3. --- Methods and Materials --- p.54 / Chapter 3.1 --- Materials --- p.54 / Chapter 3.1.1 --- Reagents --- p.54 / Chapter 3.1.2 --- Vectors --- p.55 / Chapter 3.1.3 --- Primers --- p.57 / Chapter 3.2 --- Molecular Biology --- p.59 / Chapter 3.2.1 --- Preparation of chemical competent E.coli. --- p.59 / Chapter 3.2.2 --- Transformation --- p.60 / Chapter 3.2.3 --- Mini-preparation of plasmid --- p.61 / Chapter 3.2.4 --- Midi-preparation of plasmid --- p.62 / Chapter 3.2.5 --- Tissue RNA extraction and purification --- p.63 / Chapter 3.2.6 --- Reverse-transcription polymerase chain reaction --- p.64 / Chapter 3.2.7 --- Polymerase chain reaction (PCR) --- p.64 / Chapter 3.2.8 --- PCR/Gel extraction --- p.65 / Chapter 3.2.9 --- Synthesis of mRNA for microinjection --- p.65 / Chapter 3.2.10 --- Synthesis of DIG-labeled anti-sense RNA probe --- p.65 / Chapter 3.2.11 --- Subcloning --- p.66 / Chapter 3.2.12 --- TA cloning --- p.67 / Chapter 3.3 --- Xenopus embryo manipulation --- p.67 / Chapter 3.3.1 --- Xenopus maintenance and handling --- p.67 / Chapter 3.3.2 --- Embryos collection and handing --- p.68 / Chapter 3.3.3 --- Microinjection --- p.69 / Chapter 3.3.4 --- lacZ staining --- p.70 / Chapter 3.3.5 --- Whole-mount in situ hybridization (WHISH) --- p.70 / Chapter 3.4 --- Gene disruption via TALENs --- p.76 / Chapter 3.4.1 --- Extraction and normalization of TALEN assembly vectors --- p.76 / Chapter 3.4.2 --- TALENs assembly with Golden Gate method. --- p.78 / Chapter 3.4.3 --- Synthesize TALEN mRNAs in vitro. --- p.84 / Chapter 3.4.4 --- Microinjection of TALENs into Xenopus embryos and evaluation of gene disruption efficiency --- p.84 / Chapter 3.5 --- CRISPR gRNA synthesis --- p.90 / Chapter 3.6 --- T7E1 assay for mutagenesis detection --- p.94 / Chapter 3.7 --- Tissue culture of mouse induced pluripotent stem cells (iPSCs). --- p.94 / Chapter 3.7.1 --- STO feeder cell collection --- p.94 / Chapter 3.7.2 --- iPSCs passage --- p.95 / Chapter 3.7.3 --- List of tissue culture medium --- p.96 / Chapter 4. --- Results --- p.97 / Chapter 4.1 --- TALENs induce targeted gene disruption in Xenopus embryos --- p.97 / Chapter 4.2 --- Phenotypes of somatic mutations induced by TALENs in X. tropicalis --- p.113 / Chapter 4.3 --- Mutations induced by TALENs were heritable in X. tropicalis --- p.117 / Chapter 4.4 --- TALENs and CRISPR-Cas induced gene disruption in mouse induced pluripotent stem cells (iPSCs) --- p.119 / Chapter 4.5 --- The application of TALE-based regulator --- p.124 / Chapter 4.6 --- List of TALENs, TALE-activator, and CRISPR-Cas vectors --- p.125 / Chapter 5. --- Discussion --- p.131 / Chapter 5.1 --- TALENs induce somatic and heritable mutagenesis in Xenopus --- p.131 / Chapter 5.2 --- Engineered endonucleases are valuable tools in the study of reverse genetics --- p.136 / Chapter 5.3 --- Potential applications of ZFPs and TALEs in genetic manipulation --- p.139 / Chapter 5.4 --- Prospects of genome editing approaches in the therapies of human diseases --- p.141 / Chapter 6. --- References --- p.146 / Chapter 7. --- Publications --- p.172

Early embryonic development proceeds without zygotic transcription. Genetic control is executed by maternally inherited mRNAs that are expressed in a temporally regulated manner. To set up the body plan, it is pivotal to both exert translational regulation of maternal mRNAs and to integrate maternal signals that drive cell fate determination. Xenopus nanos1, dead end (dnd) and VegT are maternal messages critical for the germline and somatic development. nanos1 and dnd localize to the germ plasm at the vegetal cortex of oocytes, while VegT occupies a cortical region overlapping with, but broader than, that of the germ plasm. In this dissertation, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. We find that a RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level for eukaryotes that is essential for normal development. Further studies showed that co-injection of dnd and nanos1 into oocytes resulted in nanos1 activation. Consistent with Dnd being the activator, Nanos1 expression was attenuated in the absence of dnd activity. Recombinant Dnd interacted directly with nanos1 RNA in vitro and possessed ATPase activity. Our findings suggested that Dnd is the potential activator for nanos1 translation by directly disrupting the repressive structural element. We previously showed that VegT, the maternal transcription factor, is normally inherited by the primordial germ cells (PGCs). VegT is the endoderm determinant and must be silenced in PGCs to preserve the germline. We showed that a classic Pumilio Binding Element (PBE) within the VegT 3’UTR mediated translational repression of a fluorescent reporter in the germline. Accordingly, a direct interaction between Xenopus Pumilio1 RNA binding domain and the VegT PBE was demonstrated in a band shift assay. The Pumilio protein belongs to the Pum-FBF family that functions in translational repression. We show that Pumilio represses VegT in vivo. Our results suggest that the germline stays unresponsive to the somatic determinant VegT in part by Pumilio- and Nanos-mediated translational repression.

Insects detect specific chemicals in the environment with olfactory receptors (ORs), which represent a novel class of ligand-gated ion channel. Insect ORs are comprised of at least one common subunit (OR83b in Drosophila) and at least one odorant-binding subunit. However, the molecular details of insect OR architecture, such as how they bind odorants, are unknown. This lack of knowledge hinders the development of compounds that may modulate OR function and potentially control insects involved in disease propagation and agricultural damage. The intent of this project is to investigate the structure and function of insect ORs. To this end, the utility of the Xenopus oocyte heterologous expression system was explored. Assay optimization, accuracy, and investigations on functional requirements were first performed using the Drosophila OR (DmOR) 35a/83b. The utility of the assay system was also demonstrated by identification of the honey bee (Apis mellifera) OR 11/2 as a receptor for the queen pheromone, 9-oxo-2-decenoic acid. A series of DmORs was cloned and expressed in Xenopus oocytes and individual receptors were selected for further study. DmOR85a/83b was shown to possess an incredibly high degree of enantioselectivity for the odorant ethyl 3-hydroxybutyrate. The receptive range of DmOR67a/83b was explored and observations were made on potential features of the odorant-binding site and a ligand odorophore. DmORs were also used to investigate the contributions of individual subunits toward the odorant-binding site and pore structure. Also, evidence for receptor antagonism by odorants was revealed. DmORs were screened with methanethiosulfonate reagents and the substituted cysteine accessibility method to identify residues 146-150 of DmOR85b as functionally important in receptor activation. This region, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2, was shown to be physically adjacent to the odorant-binding site itself. Finally, residues within the extracellular half of TMS3 in DmOR85b were implicated in odorant-induced activation by screening DmOR85b mutants for altered ligand preferences. Therefore, this project provides the first identification of insect OR subunit components involved in odorant recognition, and represents an important starting point for detailed analysis of the molecular basis for insect OR activation by odorants.

Thesis (M.S.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Karen Katula; submitted to the Dept. of Biology. Includes bibliographical references (p. 70-77).

Integrins are a large family of transmembrane cell adhesion receptors that are found on the surface of eukaryotic cells. Integrins act predominantly as cell surface receptors for extracellular matrix (ECM) proteins, but also have bidirectional signaling properties that allow them to play fundamental roles in development and cancer metastasis. It has become clear that during gastrulation – a period during which cells participate in morphogenetic movements that lead to the generation of a tripoblastic embryo – the integrin repertoire of each cell is in constant flux. This change in cell surface receptors is mediated through intracellular pathways, which in turn, are regulated by associations with cytoplasmic proteins. One such molecule, GIPC (GAIP-interacting protein, C-terminus), is thought to have a role in regulating α5β1 integrin surface expression, as well as integrin-mediated inside-out and outside-in signaling pathways by mediating the integrin’s ability to interact with the ECM protein, fibronectin (FN). I use Xenopus laevis as my experimental model system to study GIPC-regulated integrin function. Xenopus provides a useful model system for regulation of integrin function as α5β1-FN interactions are spatially and temporally regulated. Additionally Xenopus embryos are amenable to molecular manipulations in vivo, and the tissue can be excised from embryos and cultured in vitro. I have investigated the function of GIPC using site-directed mutagenesis to alter the PDZ domain site in Xenopus GIPC (XGIPC). Expression of dominant negative XGIPC results in the interruption of gastrulation movements in the early embryo. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that XGIPC physically interacts with the cytoplasmic domain of the α5 and α6 integrin subunit. Furthermore, I have determined that the interaction of XGIPC with α5β1 is required for assembly of a FN matrix. Cell migration and convergent extension assays demonstrate that XGIPC likely plays other undefined roles in modulating α5β1 function. XGIPC was found to be required for efficient trafficking of α5β1, as determined by α5β1 internalization assays in A6 cells. Together, my data indicate a critical role for XGIPC in modulating α5β1 integrin function during early embryonic morphogenesis.