Дисертації з теми "Vertebrate DNA"

Comparisons between vertebrate genome sequences, from mammals to fishes, have revealed thousands of conserved non-coding elements (CNEs) that are associated with developmental genes. Interestingly, the vast majority of these CNEs cannot be found in invertebrate genomes by sequence homology. As many CNEs have been demonstrated to act as enhancers in-vivo, it has been postulated that CNEs represent gene regulatory elements with crucial roles in aspects of development that are shared between vertebrates. To trace the evolution of CNE sequences in vertebrates, a preliminary search for CNEs in the lamprey genome was conducted using the draft lamprey genome sequence. This thesis documents how the CNEs identified in lamprey have been used as a guide to ask questions about the function and evolution of CNEs in the vertebrate lineage. Through the combined use of comparative genomics and developmental biology techniques, including a newly developed reporter assay for sea lamprey embryos, crucial first steps have been taken toward systematically de-coding these ancient gene regulatory elements. Special attention is paid toward utilising the low sequence identity of lamprey CNEs for „phylogenetic footprinting‟, an approach which uncovers striking enrichment of CNEs for a set of motifs that are characteristic of Hox-regulated elements. These findings help to establish CNEs within a developmental and evolutionary context.

Genome duplication is an essential task our cells have to achieve prior to cell division, and requires a highly specialized replication machinery to ensure it is performed in an accurate and complete manner. DNA primase and polymerases are essential components of the replisome. Primases initiate DNA replication by synthesising short RNA primers that are then elongated by faithful and processive replicative DNA polymerases. However, both exogenous and endogenous agents can damage DNA and hinder progression of the replicative machinery. Translesion synthesis DNA polymerases assist in bypassing these DNA lesions in a process called DNA damage tolerance that enables chromosomal replication to proceed in in spite of damaged templates. This thesis details the characterisation of a novel eukaryotic DNA primase, coiled-coil domain containing protein (CCDC111), a member of the Archaeo Eukaryotic Primase (AEP) superfamily. Preliminary in vitro characterisation of CCDC111 demonstrated that the recombinant protein is capable of both DNA-dependant priming and polymerase activities, which is unprecedented for a eukaryotic polymerase, and it was therefore renamed Primase-polymerase (PrimPol). The aim of this thesis was to provide one of the first cellular characterisations of PrimPol by generating a knockout of the gene in avian DT40 cells and also depleting the protein in human cells using RNAi. In vivo evidence supports the involvement of this novel polymerase in replication fork progression following replicative stress, such as exposure to UV light, but also during unperturbed DNA replication. Work in this thesis also indicates a role for PrimPol in mitochondrial DNA maintenance. Together, the data presented here establish a role for PrimPol in DNA damage tolerance in avian and human cells.

Non-human DNA (e.g. animal DNA) has become an increasingly useful tool to forensic science, aiding in linking suspects, victims, and crime scenes (24). However, due to many complexities (eg. the potential for non-human DNA to be from any species and the challenges of distinguishing DNA mixtures) and limited resources, this kind of DNA often remains unanalysed in investigations. Presumptive identification of blood is not always human specific and current DNA testing usually stops when DNA samples fail human DNA testing protocols. An efficient way to routinely detect and confirm the presence of animal DNA within samples is therefore needed. This research outlines the framework and development of a universal Vertebrate Class assay for the detection of Mammalian (mammal), Aves (bird), Fish, and Reptilia (reptile) DNA. The assay functions as a real-time PCR TaqMan multiplex assay, with an internal positive control. Primers and probes have been designed with Geneious Prime, targeting the mitochondrial genome, specifically regions of the cytochrome b gene (Cyt b) and cytochrome c oxidise subunit 1 gene (COX1) (4, 25-29). Multistage testing was conducted (eg. primer and probe screening and selection, SYBR green testing and TaqMan multiplex testing). Varied success was reported for the identification of Mammalian, Aves and Fish DNA samples across all multistage laboratory testing processes. The Reptilia probe was not able to be tested at this time due to unforeseen delays. With necessary further testing and validation, this will be the first Vertebrate Class assay design with potential capabilities of detecting Mammalian, Aves, Fish, and Reptilia classes simultaneously, providing both universal and specific detection in one convenient and rapid assay.

Master of Science<br>Department of Diagnostic Medicine/Pathobiology<br>Roman Reddy R. Ganta<br>Ehrlichia and Anaplasma species are rickettsial organisms which infect a variety of mammalian species. The organisms are transmitted from ticks and are maintained in reservoir hosts. Several pathogens have been identified in recent years as the causative agents for emerging infections in people. One of the primary reservoir hosts for the pathogens is the white tailed deer. In this study, 147 deer blood samples and 37 ticks were evaluated for the prevalence of Ehrlichia/Anaplasma species by TaqMan-based real time amplification assay and DNA sequence analysis. One hundred and thirteen (74%) samples tested positive with the Ehrlichia/Anaplasma genera-specific probe. Further analysis of the samples with the probes specific for human ehrlichiosis agents, E. chaffeensis and E. ewingii identified 4 (2.7%) and 7 (4.7%) positives, respectively. Test positives from 24 randomly selected samples were further evaluated by sequence analysis targeting to a 450 bp segment of 16S rRNA gene. All 24 samples were confirmed as positive for the Ehrlichia GA isolate # 4 (GenBank #U27104.1). DNAs from 37 pools of ticks collected from the white tailed deer were also evaluated. The TaqMan-based real time PCR assay with Anaplasma/Ehrlichia common probe identified 29 (78%) tick pools as positives whereas E. chaffeensis- and E. ewingii-specific probes identified three (8%) and one (3%) positives, respectively. The PCR and sequence analysis of tick samples identified Gram-negative bacteria species which included one endosymbiont of Rickettsia species (one tick pool), one Alcaligenes faecalis strain (three tick pools), five different Pseudomonas species (9 tick pools) and five different uncultured bacteria organisms (7 tick pools). Although the pathogenic potential of the white-tailed deer isolates of Anaplasma and Ehrlichia agents remains to be established, their high prevalence and the presence of human ehrlichiosis pathogens in white-tailed deer is similar to earlier findings. The high prevalence of the deer isolates of Anaplasma and Ehrlichia species demonstrates the need for further assessment of the pathogenic potential of these organisms to people and domestic animals.

Management of invasive species is notoriously difficult and often expensive. The aim of this study was to inform feral pig management practises in far-north Queensland by utilising molecular markers and geographic information systems to evaluate the affect of landscape features on feral pig population structure. This thesis evaluated landscape features at multiple spatial scales to identify landscape features that are a barrier or facilitator of feral pig movement and makes recommendations for future management strategies.

Genetic variation in natural populations of four species of swans (Cygnus bewickii, Cygnus olor, Cygnus buccinator and Cygnus cygnus) has been investigated by examining minisatellite loci using human DNA fingerprinting probes pSPT19.6 and pSPT18.15. It has been found that swan minisatellites are highly variable. However, the degree of variation depends on the population structure and species. Bewick's Swans at Slimbridge have the highest degree of minisatellite variation, Whooper Swans at Caerlaverock come second, and then Mute Swans, and Trumpeter Swans in Montana. Comparative study of DNA fingerprints among populations and among species suggested that swan minisatellites are subject to specific as well as population differentiation, although the function of minisatellites remains an unsolved mystery. Hypervariable minisatellites of swans that are detected by DNA fingerprinting are stably inherited as codominant markers. DNA fingerprinting has been used to study mating behaviour of Mute and Whooper Swans in the wild The results showed that the Whooper swans were almost strictly monogamous and Mute Swans exhibited an adaptable reproductive system. A genomic library from Cygnus olor was constructed and dozens of minisatellites were isolated. Most of the cloned swan minisatellites were variable, some showed specific variation, and one (pcoMS6.1) detected RFLPs in PstI digests of Trumpeter Swans.

The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

Le diadenosine tetraphosphate (ap**(4)a) est le principal produit de la reaction d'aminoacylation catalysee par certaines aminoacyl-trna synthetases. L'ap**(4)a est une molecule "signal" s'accumulant a l'interphase g1/5 du cycle cellulaire des cellules eucarydes declenchant ainsi la synthese du dna precedant la division cellulaire. Quantification du contenu cellulaire en ap**(4)a et en atp apres synchronisation des cellules (hepertome de rat, fibroblaste de souris) en culture par l'aphidicoline agent bloquant les cellules en phase s et par privation de serum qui arrete la croissance en mi-phase g. Mise au point d'un modele de reparation, dans les ovocytes ou les oeufs non fecondes de xenopus laevis, du plasmute pbr322 modifie par l'acetylaminofluorene. L'effet de l'ap**(4)a sur la reparation est etudie

Chez les vertebres, l'adn mitochondrial est dna bicatenaire (15 kb). Un intermediaire de replication possedant dune boucle de deplacement (boucle d) contient un brin h neosynthetise de taille fixe et est trouve en proportion variable dans les mitochondries. Le quart du genome mitochondrial de xenopus laevis a ete sequence: la region boucle d, 4 genes d'arn de transfert, le gene de l'apocytochrome b et celui de l'arn 125. La sequence nucleotique indique que l'organisation genetique de cette region est identique a celles des mammiferes. La cartographie au nucleotide pres du brin h de la boucle d a mis en evidence 2 familles de brin h ayant une extremite 3' commune, des structures secondaires sont potentiellement inapliquees dans la terminaison des brins h de la boucle d. Une proteine de 21,5 k da a ete isolee, se fixe a l'adn dans la region origine de replication et semble jouer un role regulateur dans la replication ou l'expression de l'adn mitochondrial. La replication du brin h peut etre observee dans un systeme de synthese d'adn "in vitro" (adn polymerase indispensable)

La regulation de l'expression du gene ngf a ete etudiee dans la lignee de fibroblastes nurins l-929 a l'aide d'une sonde de cdna permettant de quantifier le rna messager ngf. L'abondance du ngf transcript est dependante des conditions de culture: serum, hormones thyroidiennes et acide retinoique stimulent le taux de transcription du gene ngf alors que la dexamethasone la diminue. Le gene ngf du poulet et le dna complementaire de la chaine lourde des neurofilaments ont ete clones et sequences. Les sondes moleculaires rendent possibles l'etude de la coexpression au cours de l'embryogenese des genes codant pour des facteurs trophiques (ngf) et des marqueurs neuronaux (neurofilament)

Preparation et caracterisation de porphyrines cationiques dont le nombre et la position des charges varient. Cytotoxicite vis-a-vis des cellules l1210 et de bacteries sensibles aux intercalants et deficients ou non dans leurs systemes de reparation de l'adn

The Atlantic salmon (Salmo salar) is an iconic species that dominates the global finfish production sector with increasing market demand. The Scottish industry and government alone aspires for expansion of the sector to 210,000 t by 2020 with 154, 000 t produced in 2013. As such, there are pressures to improve sustainable development in particular to minimise the genetic impact of escapees on wild populations and reduce sea lice infection which are required for the granting of “green licenses” in Norway. The use of triploidy has been tested in the 1980’s with little success owing to suboptimal rearing conditions leading to elevated mortalities, poorer growth and a higher prevalence of deformities, in particular of the skeleton. Collectively: recent success of triploid trout farming, expansion to the salmon production sector and potential resulting pressure on wild stocks through escapee increases have reinstated interest to implement artificially induced triploid Atlantic salmon in commercial production. As diploid Atlantic salmon have undertaken extensive domestication to achieve the high quality production and welfare standards observed to date, triploid conspecifics too require husbandry optimisation to realise potential. In particular, industrialisation requires that higher observations of deformities and inconsistent growth trajectories during seawater ongrowing be resolved through optimisation of rearing regimes and subsequent standardization of husbandry protocols. Triploids possess additional genomic material and increased cell size yet reduced frequency that reflects known differences in physiology and supports that, in effect, triploids should be considered as a new species relative to diploid conspecifics. Therefore, this doctoral thesis aimed to study nutrition and temperature effects on triploid Atlantic salmon traits throughout the production cycle from ‘egg to plate’. Nutrition trials aimed to improve growth potential and mitigate skeletal deformities both in freshwater (FW) and saltwater (SW) whilst attempts were made to define a window of smoltification to ensure optimal ongrowing performance. Finally, impacts of embryonic temperature regimes that are known to impact long term performance and deformity development in triploids, were examined in relation to DNA regulation and yolk composition in an attempt to underpin potential mechanisms for the environmental impact of temperature on developmental phenotype. One of the main restrictions to triploid Atlantic salmon implementation is the increased prevalence and severity of skeletal deformities, particularly after the maring phase. The work performed in this thesis first demonstrated that protein and/or phosphorous (P) supplementation throughout SW ongrowing not only reduced the level of severely deformed (≥ 10 deformed vertebrae observable by x-radiography) individuals by 30 % but also sustained 6.8 % faster growth and improved harvest grade compared to triploids fed a standard grower diet (chapter 2). Comparison of x-radiography and severely deformed individuals between harvest and sea transfer highlighted that protein and P supplementation arrested deformity development whereas prevalence increased in triploids fed a standard grower diet. This implied that severe deformities were of FW origin and strongly suggest requirement for improved nutrition in FW to optimise SW performance. Therefore investigation of higher dietary P inclusion in FW was investigated and results showed significantly reduced number of deformed vertebrae and no severely deformed individuals in those fed 19.7 g total P Kg-1 compared with those fed 13.0 & 16.7 g total P Kg-1 (chapter 3). Most deformities were localised in the central (vertebrae 27 – 31) and caudal (vertebrae 52 – 57) regions for all treatments. However, triploids fed lower dietary P displayed a particular increase in prevalence within the tail region (vertebrae 32- 47) which is consistent with SW ongrowing reports and results from chapter 2, further highlighting FW origin of higher vertebral deformities reported in SW ongrowing in triploids. Higher P supplementation in FW also significantly improved growth in triploid parr compared to diploids and lower supplementation. However, this effect did not transpire in later FW smolt stages where weights were significantly higher in triploids fed lower compared to higher P supplementation. Expression of target genes involved in osteogenesis and bone P homeostasis in vertebrates were then analysed and a ploidy effect of osteogenic genes alp, igf1r and opn as well as a dietary effect on P homeostasis gene fgf23 was apparent in the parr stages but not smolt. In addition, stronger ploidy-diet effects were also observed in parr stages for whole body mineral concentrations. Collectively, growth, gene expression and whole body mineral content results indicate these earlier parr life stages may be more sensitive to P supplementation. This pronounced effect may be a consequence of seasonal accelerated growth associated with this period, where higher temperatures were also observed. The potential for shorter P supplementation windows in commercial production was addressed in chapter 4 with hope to cut economic cost to raw mineral inclusion in feed and also mitigate potential anthropogenic eutrophication on the environment that may be induced by P leached through uneaten feed and faeces. Triploids were fed higher dietary P (17.4 g total P Kg-1) until either early (5 g) or later (20 g) parr stages, or smolt (83 g) and monitored for performance throughout freshwater (FW) development. During later parr development (30 g), x-radiography assessment demonstrated that increased dietary P reduced the number of deformities and severely deformed individuals with no indication that feeding P for shorter windows improved skeletal integrity. Hence, P supplementation may be required throughout FW development for optimal skeletal performance. In addition, no differences in deformities were observed between triploid treatments at smolt. An effect of dietary P supplementation on whole body mineral concentration was observed in the early and later parr stages that was not as pronounced as smolt, which is consistent with results in chapter 3. Together, these results indicate that skeletal assessment during early developmental stages may not reflect smolt performance most likely as a consequence of seasonal effects of improved linear growth in the cooler winter temperatures prior to smolt where reversible deformities observed at parr may also be alleviated. In the same study (chapter 4), the inclusion of the probiotic Pediococcus acidilactici (Bactocell™) was also tested as a means to enhance gut assimilation as suggested in previous studies and therefore reduce the levels of P supplementation. Results clearly indicate superior skeletal performance in parr (30 g) as well as significantly less deformed vertebrae and no severely deformed individuals. However, at smolt (~83g), no effects of the dietary probiotic treatment were observed which may also be attributed to seasonal effects. Overall, nutritional research clearly indicate triploids require higher dietary P for optimal growth and skeletal development, which although is not consistent between life stages, is ultimately required throughout FW for optimal skeletal development at smolt. The use of probiotics offer a promising avenue for reduced P requirement in FW feed and further research should verify results and assess long-term performance. Timing of SW transfer according to correct parr-smolt transformation (PST) is essential for survival and growth performance in ongrowing where feeding and growth rate accelerate post-transfer. So far, SW transfer regimes and in particular the smoltification ‘window’ remains loosely defined in triploid Atlantic salmon and it is crucial that this be addressed to ensure optimal ongrowing survival and performance.

Cette etude concerne les regions d'adn delimitant le domaine des genes alpha globine du poulet. Dans les cellules de lignee erythroide, ce domaine est limite de part et d'autre par des regions en interaction permanente avec la matrice nucleaire. Les regions internes au domaine sont associees a la matrice pendant la transcription des genes. Les segments delimitant ce domaine se localisent a l'interieur ou a proximite de regions riches en aet t, comprenant aussi des sequences repetees. Une sequence riche en at contenant 4 sites homologues au consensus definis pour la topoisomerase ii, et un element repetifif appartenant a la famille cr1 a ete identifie dans la sequence du segment delimitant le domaine du cote 3'. Un element cr1 a l'interieur du gene epsilon globine de poulet a ete caracterise, cette structure permet d'envisager l'existence d'au moins deux mecanismes moleculaires alternatifs de dispersion de cette famille de sequence dans le genome du poulet. La sequence cr1 contient un site d'interaction pour un facteur nucleaire present dans les cellules erythroides

La myopathie de duchenne (dmd), maladie hereditaire grave liee au chromosome y. Isolement d'un point d'acces au locus dmd, par une marche genomique, les auteurs ont isole une region de 230 kb de ce gene gigantesque. Une etude structurale comparative entre deux especes eloignees (homme et poulet) a permis d'identifier une region de la proteine soumise a tres forte pression selective. Chez la souris, le mutant "max" pourrait correspondre a l'homologue de la mutation dmd et constituer a ce titre un modele animal de choix pour l'etude de certains aspects de la pathologie humaine

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.

Encapsulation d'adn dans des liposomes contenant du lactosylceramide dont le sucre terminal est reconnu specifiquement par des recepteurs presents sur la membrane plasmique des cellules visees, c. A. D. , les hepatocytes et les cellules endotheliales du foie et egalement les lymphocytes de la rate. Injection par voie intraveineuse des liposomes. Role de l'endocytose, dans leur internalisation. Modele genetique constitue du gene de la preproinsuline i de rat insere dans des vecteurs retroviraux permettant l'expression du gene dans des celules non insulogenes

Le papovavirus de hamster (hapv) possede un tropisme restreint in vivo vis a vis des keratinocytes et des lymphocytes. Il se replique dans les tumeurs cutanes qui apparaissent chez des hamsters syriens, et induit egalement des lymphomes chez le hamster. L'organisation genetique du hapv deduite de sa sequence a montre qu'il appartient a la famille des polyomavirus. Le hapv est present dans les lymphomes sous forme de multiples copies libres possedant toujours une deletion localisee dans la meme region du genome. Les signaux de transcription precoce du hapv semblent etre actives par la region precoce de ce virus

by Yam Kwok Fai.<br>Year shown on spine: 1997.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.<br>Includes bibliographical references (leaves 141-149).<br>Acknowledgments --- p.i<br>List of Contents --- p.ii<br>List of Figures --- p.viii<br>List of Tables --- p.xii<br>List of Primers --- p.xiii<br>Abbreviations --- p.xiv<br>Chapter Chapter 1 --- Introduction<br>Chapter 1.1 --- Growth Hormone (GH) --- p.1<br>Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3<br>Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4<br>Chapter 1.2.2 --- Biosynthesis and Degradation of GHR --- p.6<br>Chapter 1.2.3 --- Regulation of GHR Level --- p.7<br>Chapter 1.2.4 --- The Structure of GHR --- p.9<br>Chapter 1.2.5 --- The Structure of GHR Gene --- p.13<br>Chapter 1.2.6 --- Growth Hormone Binding Protein (GHBP) --- p.14<br>Chapter 1.2.7 --- The GH/Prolactin/Cytokine/Erythropoietin Receptor Superfamily --- p.15<br>Chapter 1.2.8 --- Proposed Signal Transduction Pathway --- p.17<br>Chapter 1.2.9 --- GHR Related Dwarfism --- p.22<br>Chapter i). --- Substitution of certain amino acid residues in the extracellular domain --- p.22<br>Chapter ii). --- Deletion of the extracellular domain --- p.23<br>Chapter a). --- deletion of a small portion of the binding protein<br>Chapter b). --- deletion of a large portion of the binding protein<br>Chapter c). --- deletion of a large portion of the binding domain and the whole transmembrane domain<br>Chapter iii). --- Associated with normal GHBP --- p.24<br>Chapter 1.3 --- Objectives of Cloning Vertebrate GHR cDNAs --- p.24<br>Chapter Chapter 2 --- General Experimental Methods<br>Chapter 2.1 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.26<br>Chapter 2.2 --- Isolation of Total RNA --- p.26<br>Chapter 2.3 --- Isolation of mRNA --- p.26<br>Chapter a). --- directly from tissue<br>Chapter b). --- from isolated total RNA<br>Chapter 2.4 --- Spectrophotometric Quantification and Qualification of DNA and RNA --- p.29<br>Chapter 2.5 --- First Strand cDNA Synthesis --- p.29<br>Chapter 2.6 --- Polymerase Chain Reaction (PCR) --- p.30<br>Chapter 2.7 --- Agarose Gel Electrophoresis --- p.31<br>Chapter 2.8 --- Formaldehyde Agarose Gel Electrophoresis of RNA --- p.31<br>Chapter 2.9 --- Capillary Transfer of DNA/RNA to a Nylon Membrane (Southern/Northern Blotting) --- p.32<br>Chapter a). --- DNA denaturing<br>Chapter b). --- Capillary transfer<br>Chapter 2.10 --- DNA Radiolabelling --- p.33<br>Chapter a). --- By random primer translation<br>Chapter b). --- By nick translation<br>Chapter 2.11 --- Spuncolumn Chromatography --- p.34<br>Chapter 2.12 --- Hybridization of Southern/Northern Blot --- p.35<br>Chapter 2.13 --- Autoradiography --- p.35<br>Chapter 2.14 --- Linearization and Dephosphorylation of Plasmid DNA --- p.36<br>Chapter 2.15 --- Restriction Digestion of DNA --- p.36<br>Chapter 2.16 --- Purification of DNA from Agarose Gel using GENECLEAN® Kit --- p.36<br>Chapter 2.17 --- 3' End Modification of PCR Amplified DNA --- p.37<br>Chapter 2.18 --- Ligation of DNA Fragments to Linearized Vector --- p.37<br>Chapter 2.19 --- Preparation of Escherichia coli Competent Cells --- p.38<br>Chapter 2.20 --- Transformation of the Escherichia coli Strain DH5a --- p.38<br>Chapter 2.21 --- Minipreparation of Plasmid DNA --- p.39<br>Chapter 2.22 --- DNA Purification by Phenol/Chloroform Extraction --- p.39<br>Chapter 2.23 --- Ethanol Precipitation of DNA and RNA --- p.40<br>Chapter 2.24 --- Preparation of Plasmid DNA using Wizard´ёØ Minipreps DNA Purification Kit from Promega --- p.40<br>Chapter 2.25 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.41<br>Chapter 2.26 --- DNA Sequencing --- p.42<br>Chapter 2.26.1 --- DNA Sequencing Reaction<br>Chapter a). --- T7 sequencing<br>Chapter b). --- PCR sequencing<br>Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.44<br>Chapter i). --- Preparation of 8% polyacrylamide gel solution<br>Chapter ii). --- Casting the gel<br>Chapter iii). --- Electrophoresis<br>Chapter Chapter 3 --- Molecular Cloning of Golden Hamster (Mesocricetus auratus) GHR cDNA<br>Chapter 3.1 --- Introduction --- p.46<br>Chapter 3.2 --- Experimental Methods<br>Chapter 3.2.1 --- Animals and Tissues --- p.47<br>Chapter 3.2.2 --- PCR Cloning of GHR cDNA Fragments in the Cytoplasmic Domain --- p.47<br>Chapter 3.2.2.1 --- Primer design and PCR strategy --- p.47<br>Chapter 3.2.2.2 --- PCR studies on the hamster liver and kidney first strand cDNA --- p.49<br>Chapter 3.2.2.3 --- Southern analysis of the PCR products --- p.50<br>Chapter 3.2.2.4 --- Subcloning and sequencing of PCR amplified cDNA fragments --- p.50<br>Chapter 3.2.3 --- Screening of a Hamster Liver cDNA Library --- p.51<br>Chapter 3.2.3.1 --- Preparation of the plating bacteria --- p.51<br>Chapter 3.2.3.2 --- Phage titering of the λ ZAP library --- p.51<br>Chapter 3.2.3.3 --- Primary screening of the amplified hamster liver cDNA library --- p.52<br>Chapter 3.2.3.4 --- Plaque uplifting and hybridization with hamster GHR cDNA fragment --- p.52<br>Chapter 3.2.3.5 --- Purification of putative clones from primary screening --- p.53<br>Chapter 3.2.3.6 --- Checking the size of the DNA insert --- p.53<br>Chapter 3.2.3.7 --- In vitro excision to release phagemid from the phage vector --- p.54<br>Chapter 3.2.3.8 --- Plasmid minipreparation of the putative clones --- p.56<br>Chapter 3.2.3.9 --- Nucleotide sequencing of the DNA inserts of different clones --- p.56<br>Chapter 3.2.4 --- Tissue Distribution of GHR in Hamster Tissues and the Relative Expression Level of GHR mRNAin these tissues --- p.58<br>Chapter 3.2.5 --- Cloning of the Full-length GHR cDNA into a Mammalian Vector --- p.59<br>Chapter 3.2.5.1 --- PCR amplification of the full-length hamster GHR cDNA --- p.59<br>Chapter 3.2.5.2 --- Preparation of the hamster GHR cDNA insert for ligation --- p.60<br>Chapter 3.2.5.3 --- Linearization of pRc/CMV expression vector --- p.60<br>Chapter 3.2.5.4 --- Ligation of the linearized expression vector with the full-length hamster GHR cDNA --- p.61<br>Chapter 3.3 --- Results<br>Chapter 3.3.1 --- PCR Amplification of Hamster GHR cDNA Fragments --- p.61<br>Chapter 3.3.1.1 --- RT-PCR --- p.61<br>Chapter 3.3.1.2 --- Southern blot analysis --- p.62<br>Chapter 3.3.1.3 --- Subcloning and nucleotide sequencing of PCR amplified hamster GHR cDNA fragments --- p.64<br>Chapter 3.3.2 --- Screening of an Amplified λZAP Hamster Liver cDNA Library --- p.70<br>Chapter 3.3.2.1 --- Preparation of the cDNA probe and phage titering --- p.70<br>Chapter 3.3.2.2 --- Screening of the cDNA library --- p.70<br>Chapter 3.3.2.3 --- PCR study of the 5' and 3' regions of the DNA insert of the clones selected for secondary screening --- p.72<br>Chapter 3.2.3.4 --- Nucleotide sequencing of the full-length hamster GHR cDNA --- p.73<br>Chapter 3.2.3.5 --- Tissue distribution of GHR in hamster and the relative expression level of the GHR mRNA in these tissues --- p.73<br>Chapter 3.2.3.6 --- Cloning of the full-length hamster GHR cDNA into a mammalian expression vector --- p.79<br>Chapter 3.4 --- Discussion<br>Chapter 3.4.1 --- Cloning of the Full-length hamster GHR cDNA --- p.81<br>Chapter 3.4.2 --- Comparison of the Nucleotide and the Predicted Amino Acid Sequences of the Hamster GHR with other Cloned GHRs --- p.82<br>Chapter 3.4.3 --- Tissue Distribution of GHR in Hamster and the Relative Expression Level of the GHR mRNA in these Tissues --- p.89<br>Chapter 3.4.4 --- Further Studies on Hamster GHR --- p.90<br>Chapter Chapter 4 --- Molecular Cloning of Chinese Bullfrog (Rana tigria rigulosa) GHR cDNA from Adult Frog Liver<br>Chapter 4.1 --- Introduction --- p.92<br>Chapter 4.2 --- Experimental Methods<br>Chapter 4.2.1 --- Animal and Tissues --- p.93<br>Chapter 4.2.2 --- Cloning of the Cytoplasmic Domain of Frog GHR cDNA by PCR --- p.93<br>Chapter 4.2.2.1 --- RT-PCR --- p.93<br>Chapter 4.2.2.2 --- Southern blot analysis of PCR amplified products --- p.95<br>Chapter 4.2.2.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.95<br>Chapter 4.2.2.4 --- Restriction analysis of GHR cDNA fragment between GHR p1 and GHR p2 --- p.95<br>Chapter 4.2.2.5 --- PCR cloning of other portions of frog GHR cDNA --- p.96<br>Chapter 4.2.2.6 --- Subcloning and sequencing of PCR amplified GHR cDNA fragment using primers other than GHR p1 and GHR p2 --- p.97<br>Chapter 4.3 --- Results<br>Chapter 4.3.1 --- Cloning of the Intracellular Domain of Frog GHR cDNA by RT-PCR --- p.97<br>Chapter 4.3.1.1 --- RT-PCR --- p.97<br>Chapter 4.3.1.2 --- Southern blot analysis --- p.98<br>Chapter 4.3.1.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.98<br>Chapter 4.3.1.4 --- Restriction enzyme analysis of GHR cDNA fragments --- p.102<br>Chapter 4.3.1.5 --- PCR cloning of other portions of frog GHR cDNA --- p.103<br>Chapter 4.3.1.6 --- Subcloning and sequencing of PCR products from other portions of frog GHR cDNA --- p.103<br>Chapter 4.4 --- Discussion<br>Chapter 4.4.1 --- Cloning of the Full-length frog GHR cDNA --- p.109<br>Chapter 4.4.2 --- Further Studies on Frog GHR --- p.117<br>Chapter Chapter 5 --- Attempts on the Molecular Cloning of Teleost GHR cDNA<br>Chapter 5.1 --- Introduction --- p.119<br>Chapter 5.2 --- Experimental Methods<br>Chapter 5.2.1 --- Animals and Tissues --- p.120<br>Chapter 5.2.2 --- PCR Cloning of Teleost GHR cDNA fragments --- p.120<br>Chapter 5.2.2.1 --- Design of PCR primers --- p.120<br>Chapter 5.2.2.2 --- Preparation of mRNA and synthesis of first strand cDNA --- p.122<br>Chapter 5.2.2.3 --- PCR studies on dace and snakehead fish liver first strand cDNA --- p.122<br>Chapter 5.2.2.3.1 --- PCR studies on dace liver first strand cDNA --- p.122<br>Chapter 5.2.2.3.2 --- PCR studies on snakehead fish liver first strand cDNA --- p.122<br>Chapter 5.2.3 --- "Northern Analysis on Dace, Snakehead fish and Eel mRNA" --- p.123<br>Chapter 5.3 --- Results<br>Chapter 5.3.1 --- Molecular Studies on Dace GHR cDNA --- p.123<br>Chapter 5.3.1.1 --- PCR studies on dace first strand cDNA --- p.123<br>Chapter 5.3.2 --- PCR Studies on Teleost First Strand cDNA --- p.128<br>Chapter 5.3.3 --- Northern Analysis on Teleost mRNA --- p.128<br>Chapter 5.4 --- Discussion --- p.130<br>Chapter 5.4.1 --- PCR Studies on Teleost GHR cDNA --- p.130<br>Chapter 5.4.2 --- Northern Analysis on Teleost mRNA --- p.131<br>Chapter Chapter 6 --- General Discussion<br>Chapter 6.1 --- Achievement of this Project --- p.134<br>Chapter 6.1.1 --- Hamster GHR --- p.134<br>Chapter 6.1.2 --- Frog GHR --- p.135<br>Chapter 6.1.3 --- Teleost GHR --- p.136<br>Chapter 6.2 --- Postulation on Cloned GHRs at the Molecular Level --- p.136<br>Bibliography --- p.141<br>Appendices --- p.150

abstract: Telomerase is a special reverse transcriptase that extends the linear chromosome termini in eukaryotes. Telomerase is also a unique ribonucleoprotein complex which is composed of the protein component called Telomerase Reverse Transcriptase (TERT) and a telomerase RNA component (TR). The enzyme from most vertebrate species is able to utilize a short template sequence within TR to synthesize a long stretch of telomeric DNA, an ability termed "repeat addition processivity". By using human telomerase reconstituted both in vitro (Rabbit Reticulocyte Lysate) and in vivo (293FT cells), I have demonstrated that a conserved motif in the reverse transcriptase domain of the telomerase protein is crucial for telomerase repeat addition processivity and rate. Furthermore, I have designed a "template-free" telomerase to show that RNA/DNA duplex binding is a critical step for telomere repeat synthesis. In an attempt to expand the understanding of vertebrate telomerase, I have studied RNA-protein interactions of telomerase from teleost fish. The teleost fish telomerase RNA (TR) is by far the smallest vertebrate TR identified, providing a valuable model for structural research.<br>Dissertation/Thesis<br>Ph.D. Biochemistry 2010

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2009<br>Disponível no documento<br>Fundação para a Ciência e Tecnologia, BD/18745/2004 POCI 2010