Thesis (PhD)--University of Stellenbosch, 2002 / ENGLISH ABSTRACT: Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae, undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashion to elongated cells that bud in a unipolar fashion. The daughter cells stay attached to the mother cells, resulting in chains of cells referred to as pseudohyphae. These filaments can grow invasively into the growth substrate (haploid), or away from the colony (diploid), and are hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrich substrates. This filamentous growth response to nutrient limitation was shown to be dependent on the expression of, amongst others, the MUC1 gene. MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchored threonine/serine-rich protein that bears structural resemblance to mammalian mucins and to the yeast flocculins. Deletion and overexpression studies demonstrated that it is critical for pseudohyphal differentiation and invasive growth, and that overexpression of the gene also results in strongly flocculating yeast strains. The upstream regulatory region of MUC1 comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream of the translational start site have been shown to confer regulation on MUC1 expression. The large promoter region is not unique to MUC1, however, since it is almost identical to that of the functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3 genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilise starch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing end of the starch molecule, thereby making it accessible to yeast cells. The high identity between the promoters of MUC1 and STA1-3 suggests that the two genes are co-regulated. In addition, several transcription factors that regulate the transcriptional levels of both MUC1 and STA2 have been identified and include Msn1p and the previously uncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levels of MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth, pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genes are indeed co-regulated. The main objective of this study was to characterise Mss11p and its role in the co-regulation of MUC1 and STA2 (as a representative member of the STA gene family). A detailed expression analysis, using Northern blots and Lacl reporter gene expression studies in different media, confirmed that these genes are indeed co-regulated to a large extent. MUC1 and STA2 are also regulated by the same transcriptional regulators, which include not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the mating pheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator of the flocculation genes. Overexpression of the genes encoding these factors results in elevated expression levels of both MUC1 and STA2 in most nutritional conditions and enhances the filamentous growth phenotypes of the strain, as well as the ability to degrade starch. On the other hand, the deletion thereof results in severe reductions in the transcription levels of MUC1 and STA2, with equally severe reductions in filamentous growth and the ability to hydrolyse starch. These expression studies also showed that the repressive effect of STA10, a previously uncharacterised negative regulator of STA2, is actually a phenotype conferred by a FLOB mutation in some laboratory strains of S. cerevisiae. The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeast genome. By sequencing the upstream areas of STA2 and STA3 and comparing them to the sequence of MUC 1, it was shown that these upstream areas are 99.7%identical over more than 3 900 base pairs (bp) upstream of the translational start. With the exception of a few minor substitutions, the only significant difference between the MUC1 and STA2 promoters is the presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in the promoters of any of the STA1-3 genes. Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exert their control over the transcription of MUC1 and STA2 from an 90-bp sequence located at -1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence also mediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1. Despite the similarities in the expression patterns of MUC1 and STA2, some discrepancies also exist. The most significant difference is that, in wild-type cells and under all nutritional conditions tested, MUC1 transcription is reduced significantly if compared to the transcription levels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that are present in the promoter region of MUC1, but absent from that of STA2. To place the transcriptional regulators of MUC1 and STA2 in the context of known signal transduction pathways, an epistasis analysis was conducted between MSN1, MSS11 and components of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKA pathway that were shown to be required for the filamentous growth response. This analysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transduction pathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. the cAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways. However, Mss11p seems to function downstream of all three the identified pathways. This suggestsa critical and central role for Mss11p in determining the transcription levels of MUC1 and STA2. To further characterise Mss11p and its role in the transcriptional regulation of MUC1 and STA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shown to harbour two distinct activation domains required for the activation of MUC1 and STA2, but also able to activate a reporter gene expressed from under the GALl promoter. The more prominent of the activation domains of Mss11p was shown to be one of the domains with homology to Flo8p, designated H2. The H2 domain has significant homology to a number of proteins of unknown function from a range of different organisms. A multi-sequence alignment allowed the identification of conserved amino acids in this domain. Mutations in two of the four conserved amino acid pairs in the H2 domain completely eliminated the activation function of Mss11p. The poly-glutamine and poly-asparagine domains of Mss11p are not required for its activation function. The deletion of these domains has no impact on the ability of Mss11p to activate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter gene expressed from under the GAL7 promoter. Gal4p fusions of either of these domains were also unable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neither of these domains performs a function in the role of Mss11p as a transcriptional activator. We also demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for the transcriptional activation function of Mss11p. In an attempt to identify other target genes of Mss11p, the use of micro-arrays was employed to assessthe impact of the overexpression and deletion of MSS11 on the total yeast transcriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15 genetic background that are significantly (more than 15-fold) enhanced by the overexpression of MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2 transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C, YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11 is deleted. To integrate all the results, three possible models for the activation of MUC1 and STA2 transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptional mediator, possibly in a protein complex, to convey information from upstream regulatory elements to the transcription machinery assembledat the core promoters of MUC1 and STA2; (ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcription factor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as part of a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2 promoters in responseto specific nutritional signals. / AFRIKAANSE OPSOMMING: Wanneer voedingstowwe beperkend raak, ondergaan selle van die botselvormende gis, Saccharomyces cerevisiae, fn transformasie vanaf ronde selle, wat in fn aksiale (haploïede) of bipolêre (diploïede) patroon bot, tot verlengde selle, wat slegs op een punt bot. Die dogterselle blyaan die moederselle geheg, sodat kettings van selle, wat as pseudohifes bekend staan, gevorm word. Hierdie filamente kan fn groeisubstraat binnedring (haploïede) of vanaf die kolonie weggroei (diptoïede), en is moontlik fn aanpassing van die gisselle wat hulle in staat stelom na meer voedingstofryke substrate te groei. Die vermoë om filamente in respons tot voedingstoftekorte te vorm, is onderhewig aan die uitdrukking van, onder meer, die MUC1-geen. MUC1 (ook bekend as FL011) kodeer vir fn selwand-geassosieerde treonien/serien-ryke proteten met fn GPI-anker wat strukturele verwantskappe met die mukiene van soogdiere en die flokkuliene van giste toon. Delesie- en ooruitdrukkingstudies het bewys dat dit krities is vir die ontwikkeling van pseudohifes en penetrerende groei, terwyl die ooruitdrukking daarvan ook tot sterk flokkulerende gisrasse lei. Die stroom-op regulatoriese area van MUC1 vorm die grootste promotor wat tot dusver in gis geïdentifiseer is, en daar is bewys dat areas so ver as 2.4 kb stroom-op van die translasie-inisiëringsetel die regulering van MUC1 beïnvloed. Hierdie groot promotor is egter nie uniek tot MUC1 nie, aangesien fn amper identiese promotor die regulering van die funksioneelonverwante STA2-geen beheer. Die STA2-geen, asook die identiese STA1- en STA3-gene, kodeer vir ekstrasellulêre glukoamilase isosieme wat die gis in staat stelom stysel as koolstofbron te benut. Dit bevry glukosemolekules vanaf die nie-reduserende punt van die styselmolekuul en stel dit sodoende aan gisselle beskikbaar. Die hoë vlak van eendersheid tussen dié twee promotors veronderstel dat die twee gene op soortgelyke wyse gereguleer word. Verskeie transkripsiefaktore wat die transkripsievlakke van beide MUC1 en STA2 beheer, is ook geïdentifiseer, Dit sluit Msn1p en die tot dusver ongekarakteriseerde Mss11p in. Ooruitdrukking van Msn1p of Mss11p lei tot verhoogde vlakke van MUC1 en STA2 se transkripsie en fn dramatiese toename in flokkulasie, asook die vermoë om penetrerend te groei, pseudohifes te vorm en stysel te benut. Dit bevestig dat die twee gene wel tot fn groot mate op dieselfde wyse gereguleer word. Die hoofdoel van hierdie studie was om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 te karakteriseer. Gedetailleerde uitdrukkingsanalises met behulp van die Northern-kladtegniek en facZverklikkergeeneksperimente in verskillende media het bevestig dat die gene wel tot fn groot mate op dieselfde wyse gereguleer word. Transkripsie van MUC1 en STA2 word ook deur dieselfde transkripsionele reguleerders beheer, wat nie net Msn1pen Mss11p insluit nie, maar ook Ste12p, die transkripsiefaktor van die paringsferomoon/filamentagtige groei seintransduksiekaskade, en Fl08p, fn transkripsionele aktiveerder van die flokkulasiegene. Ooruitdrukking van die gene wat vir hierdie faktore kodeer, veroorsaak verhoogde uitdrukkingsvlakke van beide MUC1 en STA2 onder die meeste groeitoestande en verbeter die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Andersyds veroorsaak delesies van die gene 'n dramatiese afname in die transkripsievlakke van MUC1 en STA2, met vergelykbare afnames in die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Hierdie uitdrukkingstudies het ook bewys dat die onderdrukkingseffek van STA10, 'n tot dusver ongekarakteriseerde, negatiewe reguleerder van STA2, aan 'n mutasie in FLOB in sekere laboratoriumrasse van S. cerevisiae toegeskryf kan word. Die stroom-op regulatoriese areas van MUC1 en STA2 is die grootste promotors in die gis se genoom. Deur die nukleotiedvolgordes van die ver stroom-op areas van STA2 en STA3 te bepaal en hulle met dié van MUC1 te vergelyk, is daar vasgestel dat die stroom-op areas van die gene 99.7% identies is oor meer as 3 900 basispare (bp) stroom-op van die beginsetel van translasie. Met die uitsondering van enkele basispaarverskille, is die enigste noemenswaardige verskil tussen die promotors van MUC1 en STA2 die teenwoordigheid van 'n 20 bp- en 'n 64 hp-fragment wat in die MUC1-promotor aangetref word, maar nie in die promotors van die STA1-3 gene nie. Deur 'n promotordelesie-analise kon daar bewys word dat Mss11p, Msn1p en Flo8p beheer uitoefen oor die transkripsie van MUC1 en STA2 vanaf 'n 90-bp-fragment, wat by posisie -1 160 tot -1 070 in die STA2-promotor en posisie -1 210 tot -1 130 in die MUC1-promotor aangetref word. Koolstofkatabolietonderdrukking van MUC1 en STA2 se transkripsie geskied ook deur middel van hierdie fragment. Ten spyte van die ooreenkomste in die uitdrukkingspatrone van MUC1 en STA2, kom daar tog ook verskille voor. Die mees opvallende verskil is dat, in wilde-tipe selle en onder alle toestande tot dusver getoets, die transkripsievlakke van MUC1 aansienlik laer is as dié van STA2. Dit word toegeskryf aan die teenwoordigheid van die 20 bp- en 64 bp-fragmente, wat in die promotor van MUC1 teenwoordig is, maar in die promotor van STA2 afwesig is. Om die transkripsionele reguleerders van MUC1 en STA2 in die konteks van bekende seintransduksieweë te plaas, is 'n epistase-analise gedoen tussen MSN1, MSS11 en komponente van die paringsferomoon/filamentagtige groei MAP-kinasekaskade en die cAMPPKA- weg wat uitgewys het dat dit 'n rol in die filamentagtige groeirespons speel. Hierdie analise het onthul dat Msn1p in 'n derde, tot dusver onbeskryfde, seintransduksieweg funksioneer, wat ook stroom-af van Ras2p is, maar wat onafhanklik funksioneer van die twee bekende weë, die cAMP-PKA-weg en die paringsferomoon/filamentagtige groei MAPkinasekaskade. Mss11p blyk egter stroom-af van al drie dié weë te funksioneer. Dit wys dat Mss11p 'n kritiese en sentrale rol in die bepaling van MUC1 en STA2 se transkripsievlakke speel. Om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 se transkripsie verder te karakteriseer, is dit aan 'n volledige delesie- en mutasie-analise onderwerp. Dit het gewys dat Mss11p twee verskillende aktiveringsdomeine bevat wat vir die transkripsionele aktivering van STA2 en MUC1 benodig word, maar wat ook 'n verklikkergeen kon aktiveer wat onder die GAL7-promotor uitgedruk word. Die prominentste van die twee aktiveringsdomeine van Mss11p is een van die domeine wat homologie toon met 'n soortgelyke domein van Flo8p, die sogenaamde H2-domein. Die H2-domein toon hornologie met 'n verskeidenheid van organismesse proteïene, waarvan die funksie onbekend is. 'n Vergelyking van al die relevante aminosuurvolgordes uit dié proteïene het gehelp om 'n aantal gekonserveerde aminosure te identifiseer. Mutasies van twee van die vier gekonserveerde aminosuurpare het die vermoë van Mss11p om transkripsie te aktiveer, heeltemal geëlimineer. Die poliglutamien- en poliasparagiendomeine van Mss11p word nie vir die aktiveringsfunksie benodig nie. Die delesie van die domeine het geen impak gehad op die vermoë van Mss11p om die transkripsie van MUC1 en STA2 te aktiveer nie, of op die vermoë van die Gal4p-Mss11p fusie om die lacZ-verklikkergeen onder regulering van die GAL7-promotor te aktiveer nie. Gal4p-fusies met enige van die domeine was ook nie in staat om die PGAL7-lacZverklikkergeen te aktiveer nie. Daar kan dus afgelei word dat nie een van die twee domeine 'n funksie in die rol van Mss11p as transkripsionele aktiveerder het nie. Soortgelyke eksperimente het bewys dat die moontlike ATP/GTP-bindingsdomein (P-lus) nie vir die transkripsionele aktiveringsfunksie van Mss11p benodig word nie. In 'n poging om ander teikengene van Mss11p te identifiseer, is mikro-ekspressieroosters gebruik om die impak van die ooruitdrukking en delesie van MSS11 op die totale transkriptoom van die gis te bepaal. Dié resultate het gewys dat MUC1 en STA2 die enigste twee gene in die ISP15genetiese agtergrond is waarvan transkripsie noemenswaardig (meer as 15-voudig) deur die ooruitdrukking van MSS11 verhoog word. Dit wil dus voorkom asof Mss11p 'n baie spesifieke rol in die transkripsie van MUC1 en STA2 speel. Hierdie analise het ook verskeie gene (DBP2, ROM2, YPLOBOC,YGR053C, YNL179C, YGR066C) geïdentifiseer wat deur die ooruitdrukking van MSS11 onderdruk word en deur die delesie van MSS11 geaktiveer word. Ten einde al die resultate te integreer, word drie moontlike modelle vir die aktivering van MUC1- en STA2-transkripsie deur Mss11p voorgestel: (i) Mss11p vervul die rol van 'n transkripsionele tussenganger, moontlik as deel van 'n proteïenkompleks, om die inligting van die stroom-op regulatoriese elemente aan die transkripsiemasjinerie wat oor die kernpromotor van MUC1 en STA2 gebind is, oor te dra; (ii) Mss11p speel 'n meer direkte rol in transkripsionele aktivering, moontlik as 'n transkripsiefaktor self; en (iii) Mss11p maak die transkripsie van MUC1 en STA2 moontlik as deel van 'n groter kompleks wat die chromatienblokkade oor die promotors van STA2 en MUC1 in respons tot spesifieke seine verslap of verwyder.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

Phytate is the major storage form of phosphorus in crop seeds, but is not well digested by humans and non-ruminant animals. In addition, phytate chelates several essential micronutrients which are also excreted contributing to phosphorus pollution in the environment. This research was aimed at the biochemical and molecular characterization of two low phytate pea mutant lines, 1-150-81 and 1-2347-144 developed at the Crop Development Centre, University of Saskatchewan in collaboration with Dr. Victor Raboy, USDA, Idaho. Low phytic acid (lpa) crops are low in phytic acid and high in inorganic phosphorus (Pi). In Study I, two lpa pea genotypes, 1-150-81, 1-2347-144, and their progenitor CDC Bronco were evaluated in field trials for two years. The lpa genotypes did not significantly differ from CDC Bronco in all agronomic traits assessed except for lower seed weight and grain yield. The concentration of IP6 at 14 DAF was not significantly different among CDC Bronco, 1-150-81 and 1-2347-144. However, the concentrations of IP6 among CDC Bronco, 1-150-81 and 1-2347-144 started to differ significantly from 21 DAF onwards. The lpa genotypes 1-150-81 and 1-2347-144 showed 65% and 60% reduction in IP6, respectively, when compared to their progenitor CDC Bronco at 49 DAF. The Pi concentrations between the lpa genotypes were similar and significantly higher than CDC Bronco from 21 DAF to 49 DAF. At 49 DAF, 1-150-81 and 1-2347-144 were 72 and 84% higher in Pi, respectively, than CDC Bronco. The total P concentration was similar in lpa genotypes and CDC Bronco throughout the seed development. This study elucidated the rate and accumulation of phosphorus compounds in lpa genotypes. In Study II, aiming at understanding the genetic basis of the lpa mutation in pea lines 1-150-81 and 1-2347-144, a 1530 bp open reading frame of myo-inositol phosphate synthase gene (MIPS) was amplified from CDC Bronco and the lpa genotypes. Sequencing results showed no difference in coding sequence in MIPS between CDC Bronco and lpa genotypes. Transcript levels of both MIPS and myo-inositol tetrakisphosphate1-kinase (ITPK1) were relatively lower at 49 DAF than at 14 DAF for CDC Bronco and lpa lines. There was no difference in expression level of both MIPS and ITPK1 between CDC Bronco and the lpa genotypes at 49 DAF. The data demonstrated that mutation in MIPS was not responsible for lpa trait in pea. Study III was aimed at developing a single nucleotide polymorphism (SNP) based genetic linkage map and mapping genomic regions associated with phytic acid-phosphorus (PA-P) concentration using PR-15 recombinant inbred lines (RILs) derived from a cross between a low phytate (lpa) pea genotype, 1-2347-144 and normal phytate pea cultivar CDC Meadow. A total of 163 RILs were genotyped using 1536 SNP markers in an Illumina GoldenGate array. Three hundred and sixty seven polymorphic SNP markers, ordered into 7 linkage groups (LGs), generated a linkage map with a total length of 437.2 cM. The phytic acid locus was mapped on to LG5. A quantitative trait locus (QTL) for iron bioavailability was mapped on to the same location in LG5 as phytic acid concentration. Potential benefits arising out of this research include improved bioavailability of phosphorus, iron and zinc in foods and feeds, less phosphorus excretion and environmental pollution and a saving in feed costs.

by Sham Lok To. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 143-171). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / List of figures --- p.ix / List of tables --- p.xi / Chapter Chapter One --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- About L. edodes --- p.3 / Chapter 1.1.2 --- Nutritional and medicinal values of L. edodes --- p.4 / Chapter 1.1.3 --- Life cycle of L. edodes --- p.6 / Chapter 1.1.4 --- Environmental factors affecting fruiting body formation in L. edodes --- p.6 / Chapter 1.2 --- Molecular mechanisms of fruiting body development in L. edodes --- p.8 / Chapter 1.2.1 --- Expression profiling and identification of differentially expressed genes during fruiting --- p.8 / Chapter 1.2.2 --- Changing in membrane structure --- p.11 / Chapter 1.2.3 --- The signal transduction cascade --- p.12 / Chapter 1.3 --- Transformation in L. edodes and in other fungi --- p.14 / Chapter 1.3.1 --- Transformation of L. edodes --- p.14 / Chapter 1.3.2 --- Transformation in other fungi --- p.17 / Chapter 1.4 --- Bioinformatics tools for comparative promoter analysis --- p.22 / Chapter 1.5 --- Objectives and significance --- p.26 / Chapter Chapter Two --- Promoter analysis of differentially expressed genes (DEGs) in the fruiting body development in L. edodes --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Strains and cultivation conditions --- p.29 / Chapter 2.2.2 --- Genome walking of the 5' flanking region of the DEGs --- p.29 / Chapter 2.2.3 --- Annealing Control Primed (ACP) PCR --- p.31 / Chapter 2.2.4 --- Construction of genomic DNA library --- p.36 / Chapter 2.2.5 --- Nested PCR to amplify the target sequences --- p.37 / Chapter 2.2.6 --- Cloning and sequencing of the 5' flanking region --- p.38 / Chapter 2.2.7 --- Determination of transcription start site by the Neural Network algorithm --- p.39 / Chapter 2.2.8 --- Identification of putative transcription factor binding sites --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Construction of adaptor linked template for genome walking --- p.41 / Chapter 2.3.2 --- Sequence analysis and quality control --- p.41 / Chapter 2.3.3 --- Comparison of various methods in genome walking --- p.42 / Chapter 2.3.4 --- Promoter analysis --- p.42 / Chapter 2.4 --- Discussion --- p.58 / Chapter Chapter Three --- In-silico analysis of transcription factor binding sites and identification transcription factors expressed in L. edodes --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Material and methods --- p.67 / Chapter 3.2.1 --- Sequence manipulation and extraction of homologous ESTs from C. cinereus --- p.67 / Chapter 3.2.2 --- Extraction of 5' flanking region of the corresponding ESTs and promoter prediction --- p.67 / Chapter 3.2.3 --- Positional cloning of mating type factor A --- p.68 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- Sequence extraction and manipulation --- p.70 / Chapter 3.3.2 --- In-silico analysis of transcription factor binding sites in C. cinereus . --- p.70 / Chapter 3.3.3 --- Comparison of putative TFBS between L. edodes and C. cinereus --- p.71 / Chapter 3.3.4 --- Identification of transcription factors in L. edodes by positional cloning --- p.71 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter Four --- Identification，expression profiling and promoter analysis of hydrophobin genes --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Material and methods --- p.92 / Chapter 4.2.1 --- Clustering and grouping of the hydrophobin ESTs --- p.92 / Chapter 4.2.2 --- Identification of the consensus sequences of the hydrophobin groups --- p.93 / Chapter 4.2.3 --- RNA Sources and Preparation --- p.93 / Chapter 4.2.4 --- Expression profiling of hydrophobin genes by RT-PCR --- p.95 / Chapter 4.2.5 --- Promoter cloning and analysis of hydrophobin genes --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Isolation and characterization of four newly found hydrophobin genes --- p.97 / Chapter 4.3.2 --- Expression levels of hydrophobins --- p.100 / Chapter 4.3.3 --- Promoter sequencing of the hydrophobins --- p.103 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter Five --- Transformation of L. edodes --- p.110 / Chapter 5.1 --- Introduction --- p.110 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Vectors and primers design --- p.112 / Chapter 5.2.2 --- Maxi-preparation of plasmids --- p.112 / Chapter 5.2.3 --- Cultural condition and optimization of protoplasts release --- p.114 / Chapter 5.2.4 --- PEG mediated transformation --- p.115 / Chapter 5.2.5 --- Electroporation mediated transformation --- p.116 / Chapter 5.2.6 --- PCR screening of regenerated transformant --- p.116 / Chapter 5.2.7 --- Particle bombardment --- p.117 / Chapter 5.3 --- Results --- p.121 / Chapter 5.4 --- Discussion --- p.128 / Chapter Chapter Six --- General discussions --- p.132 / References --- p.143

Lee Ming Tsung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 160-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutritional values --- p.2 / Chapter 1.3 --- Medicinal values --- p.3 / Chapter 1.3.1 --- Anti-tumor effect --- p.3 / Chapter 1.3.2 --- Anti-viral and anti-caries effect --- p.4 / Chapter 1.3.3 --- Immunopotentiating effect --- p.4 / Chapter 1.3.4 --- Hypocholesterolaemic effect --- p.5 / Chapter 1.4 --- Life cycle and morphology --- p.6 / Chapter 1.5 --- Growth requirements --- p.9 / Chapter 1.5.1 --- Nutritional factors --- p.9 / Chapter 1.5.2 --- Physical and chemical factors --- p.10 / Chapter 1.6 --- Application of L. edodes --- p.12 / Chapter 1.7 --- Endocytosis --- p.13 / Chapter 1.7.1 --- Different types of endocytosis --- p.13 / Chapter 1.7.1.1 --- Phagocytosis --- p.14 / Chapter 1.7.1.2 --- Pinocytosis --- p.15 / Chapter 1.7.1.3 --- Receptor-mediated endocytosis --- p.15 / Chapter 1.7.2 --- The Endocytic Pathway --- p.17 / Chapter 1.7.3 --- Endocytosis in fungi --- p.20 / Chapter 1.7.4 --- Rab GTPases --- p.21 / Chapter 1.7.4.1 --- Control of the active and inactive state of Rab proteins --- p.22 / Chapter 1.7.4.2 --- Regulation of docking and fusion of membrane in endosomal trafficking --- p.23 / Chapter 1.7.4.3 --- Rab7 GTPase --- p.26 / Chapter 1.8 --- Aims of the project --- p.28 / Chapter Chapter 2 --- Protein-protein Interaction Study of Le.Rab7 by in vivo and in vitro Interaction Assay --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Yeast two-hybrid screening --- p.36 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Rab7-pGBK.T7 --- p.36 / Chapter 2.2.1.1.1 --- Bacterial transformation --- p.36 / Chapter 2.2.1.1.2 --- PCR screening for positive transformants --- p.38 / Chapter 2.2.1.1.3 --- Plasmid preparation and confirmation of transformants --- p.38 / Chapter 2.2.1.1.4 --- Sequencing --- p.39 / Chapter 2.2.1.2 --- Confirmation of Le.Rab7 protein expression in yeast --- p.40 / Chapter 2.2.1.2.1 --- Yeast transformation --- p.40 / Chapter 2.2.1.2.2 --- Yeast protein extraction --- p.40 / Chapter 2.2.1.2.3 --- Western Blotting --- p.41 / Chapter 2.2.1.3 --- Yeast Two-hybrid screening by Yeast-mating --- p.42 / Chapter 2.2.1.4 --- Identification of Preys --- p.44 / Chapter 2.2.1.4.1 --- PCR screening for clones grown on plates --- p.44 / Chapter 2.2.1.4.2 --- Colony lift filter assay --- p.45 / Chapter 2.2.1.4.3 --- Sequencing --- p.47 / Chapter 2.2.1.5 --- Confirmation of interaction by Co-transformation assay --- p.47 / Chapter 2.2.1.5.1 --- Plasmid preparation of positive clones --- p.47 / Chapter 2.2.1.5.2 --- Transformation and bacterial plasmid preparation --- p.48 / Chapter 2.2.1.5.3 --- Yeast two-hybrid screening by co-transformation --- p.48 / Chapter 2.2.1.5.4 --- Colony lift filter assay --- p.50 / Chapter 2.2.2 --- Rapid Amplification of cDNA 5'ends --- p.51 / Chapter 2.2.2.1 --- RNA preparation --- p.51 / Chapter 2.2.2.1.1 --- Strains and culture conditions --- p.51 / Chapter 2.2.2.1.2 --- RNA extraction --- p.51 / Chapter 2.2.2.2 --- 5' RACE --- p.52 / Chapter 2.2.2.2.1 --- RNA processing --- p.52 / Chapter 2.2.2.2.2 --- Reverse transcription --- p.53 / Chapter 2.2.2.2.3 --- Nested PCR for 5'RLM-RACE --- p.53 / Chapter 2.2.2.3 --- "Gel analysis of products, TA cloning of RACE product and sequencing" --- p.54 / Chapter 2.2.2.4 --- Cloning of full-length Le.Rab5 --- p.54 / Chapter 2.2.3 --- In vitro protein-protein interaction assay --- p.55 / Chapter 2.2.3.1 --- Plasmid extraction from E.coli --- p.55 / Chapter 2.2.3.2 --- In vitro translation --- p.56 / Chapter 2.2.3.3 --- In vitro co-immunoprecipitation --- p.56 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Yeast two-hybrid analysis by yeast mating assay --- p.57 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Ra67-pGBKT7 --- p.57 / Chapter 2.3.1.1.1 --- PCR screening for positive transformants --- p.57 / Chapter 2.3.1.1.2 --- Plasmid preparation and confirmation of transformants --- p.58 / Chapter 2.3.1.1.3 --- Sequencing --- p.59 / Chapter 2.2.1.2 --- Confirmation of protein expression in yeast --- p.60 / Chapter 2.3.1.2.1 --- Yeast transformation --- p.60 / Chapter 2.3.1.2.2 --- SDS-PAGE and Western blotting of Le.Rab7 in yeast --- p.61 / Chapter 2.2.1.3 --- Yeast two-hybrid screening by yeast mating assay --- p.62 / Chapter 2.2.1.4 --- Identification of Preys --- p.63 / Chapter 2.3.1.4.1 --- PCR screening for clones grown on plates --- p.63 / Chapter 2.3.1.4.2 --- Colony lift assay --- p.65 / Chapter 2.3.1.4.3 --- Sequencing --- p.67 / Chapter 2.3.2 --- Confirmation of interactions by co-transformation assay --- p.70 / Chapter 2.2.2.1 --- Yeast two-hybrid analysis by co-transformation assay --- p.70 / Chapter 2.2.2.2 --- Colony lift filter assay --- p.70 / Chapter 2.2.2.3 --- Selection of prey plasmids for in vitro binding assay --- p.72 / Chapter 2.3.3 --- Rapid amplification of cDNA ends (RACE) --- p.76 / Chapter 2.2.3.1 --- TA cloning of RACE product and sequencing --- p.76 / Chapter 2.2.3.2 --- Cloning of full-length Le.Rab5 --- p.79 / Chapter 2.3.4 --- In vitro protein-protein interaction assay --- p.80 / Chapter 2.4 --- Discussion --- p.82 / Chapter Chapter 3 --- Temporal and Spatial expression of Le.Rab7，Le.Rab5 and Le.RACKl --- p.87 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Northern blot analysis --- p.93 / Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.93 / Chapter 3.2.1.2 --- Northern blotting --- p.94 / Chapter 3.2.1.2.1 --- Transfer of RNAs --- p.94 / Chapter 3.2.1.2.2 --- Probe preparation --- p.95 / Chapter 3.2.1.2.3 --- "Hybridization, Stringency washes and Signal detection" --- p.96 / Chapter 3.2.2 --- Quantitative RT-PCR --- p.97 / Chapter 3.2.2.1 --- cDNA synthesis from different developmental stages --- p.97 / Chapter 3.2.2.1.1 --- RNA preparation extraction --- p.97 / Chapter 3.2.2.1.2 --- DNase I treatment --- p.97 / Chapter 3.2.2.1.3 --- Reverse transcription --- p.98 / Chapter 3.2.2.2 --- Real time PCR --- p.98 / Chapter 3.2.2.2.1 --- Primer design and verification --- p.98 / Chapter 3.2.2.2.2 --- Real time PCR reaction and data analysis --- p.100 / Chapter 3.2.3 --- In situ RNA-RNA hybridization --- p.101 / Chapter 3.2.3.1 --- Preparation of samples and probes --- p.101 / Chapter 3.2.3.1.1 --- Tissue preparation --- p.101 / Chapter 3.2.3.1.2 --- RNA probe synthesis --- p.101 / Chapter 3.2.3.2 --- Hybridization and Signal development --- p.102 / Chapter 3.2.3.3 --- Image viewing --- p.103 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Northern blot analysis --- p.105 / Chapter 3.3.2 --- Quantitative RT-PCR assays --- p.109 / Chapter 3.3.3 --- In situ RNA-RNA hybridization --- p.113 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- Existence of endocytosis and Protein localization of Le.Rab7 in L. edodes --- p.123 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.2 --- Materials and Methods --- p.127 / Chapter 4.2.1 --- Tracing the endocytie pathway using FM4-64 dye --- p.127 / Chapter 4.2.1.1 --- Strains and culture conditions --- p.127 / Chapter 4.2.1.2 --- FM4-64 internalization in mycelium and gill tissue of L. edodes --- p.127 / Chapter 4.2.2 --- Drug treatment effect on the internalization of FM4-64 dye --- p.128 / Chapter 4.2.3 --- Double labeling with AM4-64 and anti-Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1 --- Synthesis of Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1.1 --- Customization of Le.Rab7 antiserum --- p.129 / Chapter 4.2.3.1.2 --- Validation of anti-Le.Rab7 polyclonal antiserum --- p.129 / Chapter 4.2.3.2 --- Double immunofluorescence labeling --- p.130 / Chapter 4.2.4 --- Immunohistochemistry of young and mature fruiting body --- p.131 / Chapter 4.2.4.1 --- Tissue preparation --- p.131 / Chapter 4.2.4.2 --- Immunohistochemical staining --- p.132 / Chapter 4.2.4.3 --- Image viewing --- p.133 / Chapter 4.3 --- Results --- p.134 / Chapter 4.3.1 --- Presence of endocytosis in L .edodes --- p.134 / Chapter 4.3.2 --- Validation of active transport of FM4-64 --- p.137 / Chapter 4.3.3 --- Dye internalization at specific structures in L. edodes --- p.138 / Chapter 4.3.4 --- Presence of Le.Rab7 protein in the endosomal structures along the endocytic pathway --- p.142 / Chapter 4.3.5 --- Presence of Le.Rab7 protein in the pre- and hymenophore of fruiting body --- p.145 / Chapter 4.4 --- Discussion --- p.148 / Chapter Chapter 5 --- General discussion --- p.152 / References --- p.160

Orientador: Claudia Marcia Aparecida Carareto / Banca: Gustavo Campos e Silva Kuhn / Banca: Luis Gustavo da Conceição Galego / Resumo: Este estudo teve por objetivo investigar se os retrotransposons sem LTRs (NLTRs) Helena e BS estiveram envolvidos em eventos de transferência horizontal (HT) durante a diversificação concomitante pela qual passaram as espécies do subgrupo melanogaster, do grupo melanogaster de Drosophila, e do subgênero Zaprionus, na África Tropical. O modo de transposição dos elementos NLTRs, sem a formação de um DNA intermediário, é utilizado para explicar a escassez de relatos de HT desse tipo de elemento. Estudos anteriores evidenciaram alta taxa de HT de retrotransposons com LTRs em espécies dos dois grupos citados, o que fundamentou a hipótese que essas espécies passaram por uma fase permissiva à ocorrência de HT em decorrência da sobreposição geográfica e temporal que sofreram durante sua diversificação. Para testar essa hipótese, uma região do gene da transcriptase reversa dos retrotransposons sem LTRs Helena e BS foi utilizada para investigar a ocorrência de HT entre as espécies. As sequências foram obtidas de linhagens de Zaprionus, via sequenciamento, após amplificação e clonagem, e via análises in silico nos genomas de Drosophila disponíveis nos bancos de dados, ou via sequenciamento de genomas de espécies de Drosophila e de Zaprionus. Foram realizadas análises evolutivas (análises de divergência e estimativa dos tempos de divergência entre as sequências) e filogenéticas (inferência bayesiana e verossimilhança máxima), bem como análises de network para estimar as relações de... / Abstract: The aim of this study was to investigate whether the retrotransposons without LTRs (NLTRs) Helena and BS were involved in horizontal transfer (HT) events during the concomitant diversification of the species of the melanogaster subgroup of the Drosophila melanogaster group, and the subgenus Zaprionus, in Tropical Africa. The mode of transposition of the NLTRs, without the formation of a DNA intermediate, is used to explain the scarcity of HT reports of this type of element. Previous studies evidenced a high HT rate of LTRs retrotransposons in species of the two groups above cited, which supported the hypothesis that these species went through a permissive phase to the occurrence of HT, as a result of the geographical and temporal overlap that they suffered during their diversification. To investigate this hypothesis, a region of the reverse transcriptase gene of the NLTR retrotransposons Helena and BS was used to investigate the occurrence of HT between the species. Sequences were obtained from Zaprionus strains, via sequencing, after amplification and cloning, and via in silico analyzes in the Drosophila genomes available in the databases, or via sequencing of Drosophila and Zaprionus genomes. Evolutionary (divergence analyses and estimation of divergence times between sequences) and phylogenetic (Bayesian inference and maximum likelihood) analyses were performed, as well as network analyses to estimate the evolutionary relationships between the sequences and, thus, to test the hypothesis of HT. The results showed that there is (i) irregular distribution of the two elements in the genera Zaprionus and Drosophila, (ii) high similarity between the sequences of the melanogaster complex and the Zaprionus subgenus, (iii) incongruities between the phylogenies of the elements and host species, and iv) times of divergence between the sequences of the two species elements of the ... / Mestre

by Vong Puinga Queenie Maria. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 176-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.