Academic literature on the topic '<i>Coprinopsis cinerea<'

Targeted gene knockout in Coprinopsis cinerea, yeast in vivo bioconversion and in vitro assays elucidated the lagopodin biosynthetic pathway, including a complexity-generating network of oxidation steps.

Conocer las características de cepas fúngicas locales aumenta nuestra capacidad de poder hacer uso de sus propiedades e implementar, en consecuencia, nuevas y mejores formas de aprovechamiento de los recursos biológicos nativos. Con el fin de contribuir al conocimiento de estos organismos, en este trabajo se presentan descripciones detalladas y estudios de cultivo de 5 especies previamente reportadas como basidiomicetes endofíticos: Coprinopsis cinerea, Granulobasidium vellereum, Inonotus rickii, Phanerochaete chrysosporium y Trichosporon sporotrichoides, aisladas como endofitos de madera de plátanos de sombra (Platanus acerifolia) en la Ciudad de Buenos Aires, Argentina. Coprinopsis cinerea es descripta por primera vez en cultivo y se señalan las diferencias que presentan los cultivos de G. vellereum e I. rickii con reportes previos. Los caracteres de cultivo de P. chrysosporium y T. sporotrichoides no presentaron diferencias apreciables con descripciones previas

ABSTRACT Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

Previously, we observed an acid-induced short-term wall extension in Flammulina velutipes apical stipes during a 15 min period after a change from a neutral to an acidic pH. This acid-induced stipe wall extension was eliminated by heating and reconstituted by a snail expansin-like protein, although we failed to isolate any endogenous expansin-like protein from F. velutipes because of its limited 1 mm fast elongation region. In this study, we report that Coprinopsis cinerea stipes possess a 9 mm fast elongation apical region, which is suitable as a model material for wall extension studies. The elongating apical stipe showed two phases of acid-induced wall extension, an initial quick short-term wall extension during the first 15 min and a slower, gradually decaying long-term wall extension over the subsequent 2 h. After heating or protein inactivation pretreatment, apical stipes lost the long-term wall extension, retaining a slower short-term wall extension, which was reconstituted by an expansin-like snail protein. In contrast, the non-elongating basal stipes showed only a weaker short-term wall extension. We propose that the long-term wall extension is a protein-mediated process involved in stipe elongation, whereas the short-term wall extension is a non-protein mediated process not involved in stipe elongation.

Coprinopsis cinerea is extensively used as a model to study the development of homobasidiomycete fungi. Unraveling the molecular basis of fungal developmental processes would contribute to evolutionary studies, improve the knowledge about multicellularity development, and lead to improvement in the breeding and cultivation of edible or medical homobasidiomycete mushrooms.<br>I studied the fungal developmental processes in two aspects: 1) the hypothesis of a “developmental hourglass”, and 2) the existence of microRNA-like RNA (milRNA) genes in fungi.<br>The model of “developmental hourglass” suggests that middevelopment is the most conserved and the most resistant to evolutionary changes. Although extensively explored in animals and plants, such hourglass pattern has not been reported in fungi yet. I tested the hourglass model in C. cinerea using two complementary approaches, the transcriptome age index (TAI) and the transcriptome divergence index (TDI). Both the TAI and the TDI profiles displayed an hourglass pattern over the development of C. cinerea; the young fruiting body stage was the waist that expressed the evolutionarily oldest transcriptome (lowest TAI) and gave the strongest signal of purifying selection (lowest TDI). By cross-kingdom comparisons, it is found that all three kingdoms displayed high expression levels of genes in “information storage and processing” at the waist stage, while genes in “metabolism” became more active later; besides, genes at the waist stage were underrepresented in “signal transduction mechanisms”.<br>MicroRNA (miRNA) is a group of endogenous non-coding regulatory RNAs of ~22 nt that regulate gene expression in various biological processes such as cell differentiation, development regulation and heterochromatin formation. Past research work reported several simple filamentous fungi to contain milRNAs in their genomes; however, no milRNA has been reported in mushrooms so far. Through computational prediction, I identified 16 putative milRNA genes in C. cinerea. Besides, evolutionary analysis showed that C. cinerea contains Dicer-like proteins (DCLs), which may play roles in milRNA biogenesis.<br>Both the discovery of a “developmental hourglass” and milRNA genes laid a foundation for analysis of fruiting body formation in fungi, and for evolutionary analysis of multicellular development across kingdoms.<br>灰蓋鬼傘（Coprinopsis cinerea）作為一個典型的生物模型，被廣泛地運用在擔子菌生長發育的研究中。從分子層面上解析出真菌生長發育的過程，可以促進進化生物學的研究，提高對多細胞生物演化的認知，以及改善食用菌和藥用菌的育種與培養。<br>我從兩個角度去分析了真菌的生長發育過程：1）真菌的“發育沙漏”假說，2）真菌基因組裡類微RNA（microRNA-like RNA [milRNA]）的基因。<br>“發育沙漏”模型指出，發育中期在進化中是最保守的、最能抵抗進化帶來的改變。這個沙漏模型在動物與植物中被廣泛地研究與證實，但是迄今為止，並未有人對其在真菌中的存在進行過探究。我以C. cinerea作為研究模型，採用了兩種互補的方法，transcriptome age index (TAI) 和 transcriptome divergence index (TDI)，探究了發育沙漏在真菌中的存在。兩個指數均在C. cinerea發育過程中呈現出了沙漏的形狀；年輕的子實體（young fruiting body）階段是沙漏的腰身，表達出進化中最古老的基因並給出了最強的淨化選擇（purifying selection）的信號。我將三大真核生物進行了比較，發現在腰身階段時，在“信息儲存和處理”中發揮作用的基因呈現出了高表達量，而“代謝”基因的表達量在後期更高；而且在腰身階段表達的基因中，負責“信號傳遞機制”的基因偏少。<br>微RNA（microRNA [miRNA]）是一類長約22個核苷酸、不轉譯蛋白質的RNA分子。它們可以調節其他基因的表達，在多方面的生理過程中發生作用，比如細胞分化、發育調節、異染色質的形成等等。近幾年的研究表明，一些簡單的絲狀真菌的基因組中含有類微RNA（milRNA）的基因；但是，至今未有報導說蘑菇是否含有此類基因。我運用計算預測的方法在C. cinerea的基因組裡找到了16個可能的類微RNA基因。此外，從進化的角度分析，我發現C. cinerea含有類Dicer蛋白酶（Dicer-like proteins [DCLs]），而這些類Dicer蛋白酶可能在milRNA的產生過程中發揮作用。<br>這篇論文所報導的兩個發現──真菌的“發育漏斗“和類微RNA基因，均為之後子實體發育的研究及多細胞發育的研究奠定了基礎。<br>Cheng, Xuanjin.<br>Thesis Ph.D. Chinese University of Hong Kong 2015.<br>Includes bibliographical references.<br>Abstracts also in Chinese.<br>Title from PDF title page (viewed on 09, September, 2016).<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.

Coprinopsis cinerea, 亦稀灰蓋鬼傘，是研究擔子菌發育過程的模範生物。它的生命週期短，容易培養，亦有已建立的遺傳和分子生物研究技術。最近，它的完整基因組序列亦被發表。C. cinerea 的子實體萌生和發展是快速而複雜的過程。它受多種因素影響，如交配基因，營養消耗，光照和溫度。然而，我們對於C. cinerea 出菇的基本機制和涉及的分子途徑仍不清楚。在這項研究中，我採用NimbleGen 微陣列，以了解13,320 個C. cinerea 基因模型的表達。此微陣列覆蓋不同發育階段，包括雙核菌絲體，子實體初體，第2階段原基體，年青和成熟子實體。11,815個預測，在至少一個發育階段表達。707個基因模型在出菇的萌生過程有差異表達。我發現一些可能參與子實體的萌生和發展的份子。它們可能在檢測養分、形態、信號轉導和應激反應方面，擔當重要角色。此外，我亦分析了轉錄因子 、蛋白激酶組和細胞色素P450的基因表達模式。<br>C. cinerea 的子實體發展是與光暗週期同步。當子實體初體在沒有光的環境培植，使會形成dark stipe。101個基因在dark stipe 表達差異。它們可能參與原基體成熟的過程。此微陣列基因表達數據，對了解菇機制有價格的信息。<br>胞吞作用是真核細胞透過質膜內陷將外來物質攝取的過程。Rab5 和Rab7 分別控制早期和晚期的胞吞作用。C. cinerea 的胞吞作用是組織由110個基因組成。FM4-64 螢光顯示在C. cinerea 菌絲體的胞吞作用是依賴肌動蛋白和能源。從菌絲體到年青子實體，Cc. Rab5的表達維持相同水平，而Cc.Rab7 的表達則不斷增加，兩者在成熟子實體的表達都是最高，原位雜交體技術顯示 Cc.Rab5 和Cc.Rab7 的 mRNA在年青子實體的子實層以及整個子實層上菌摺表達。我在第2階段原基體進行RNA 乾擾，致使Cc.Rab5 和Cc.Rab7的基因表達敲落。這導致原基體的生長遲緩。及後形成的成熟子實體亦有異常形態。因此，我推測Cc. Rab5 和Cc.Rab7參與出菇過程，並影響擔孢子的形成。這些結果表明，胞吞作用在C.Cinerea 子實體發育過程中發揮定一定的作用。<br>Coprinopsis cinerea, is a model organism for studying developmental processes in basidiomycetous fungi. It has a short life cycle, easy to be cultivated in laboratory and can be accessed by various genetic and molecular techniques. Recently, its complete genome sequence was released. The fruiting body development in C. cinerea is a rapid yet complicated process. It is under the regulation of various factors such as mating type genes, nutrients depletion, light and temperature. However, the underlying mechanism and molecular events involved during fruiting body initiation and development remains unclear.<br>In this study, fruiting body developmental stages including mycelium, fruiting initials, stage 2 primordium, young and mature fruiting body, were analyzed with a comprehensive NimbleGen microarray. 11,815 out of 13,320 predicted gene models were expressed in at least one of the stages. 707 genes were differentially expressed during fruiting body initiation. Potential players involved in nutrients sensing, morphogenesis, signaling pathways and stress response were identified. In particular, expression patterns of all transcription factors, kinome and cytochrome P450s were analyzed.<br>The fruiting body development of C. cinerea is synchronized with the light/dark cycle. Differentially expressed genes were found in dark stipe produced by keeping fruiting initials in complete darkness. 101 genes, which are likely to be involved in maturation of primordium were identified.<br>Endocytosis is an essential process in eukaryotes through which cells take up extracellular substrates by membrane invaginations. Rab5 and Rab7 control the early and late stage of endocytosis respectively. The C. cinerea endocytic machinery composed of 110 genes models. The endocytic pathway was traced by FM4-64 and was found to be actin- and energy-dependent. Temporal and spatial expressions of Cc.Rab5 and Cc.Rab7 during fruiting body development were studied. Cc.Rab5 expressed constitutively from mycelium to young fruiting body stage, and reached the highest in the mature fruiting body. The expression of Cc.Rab7 increased continually from mycelium to mature fruiting body stage. From the in situ RNA-RNA hybridization results, both transcripts were localized at the hymenium layer in the young fruiting body and throughout the gill tissue of the mature cap. Knock-down of Cc.Rab5 and Cc.Rab7 by siRNA resulted in retarded growth of the stage 2 primordium and abnormal mature fruiting body. Cc.Rab5 and Cc.Rab7 may be involved in the formation of basidiospores. Endocytosis may play some roles during fruiting body development in C. cinerea.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Lee, Yung Yung.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 156-179).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.<br>Abstract --- p.i<br>論文摘要 --- p.iii<br>Abbreviations --- p.iv<br>Acknowledgements --- p.v<br>Table of Contents --- p.vi<br>List of Figures --- p.x<br>List of Tables --- p.xiii<br>Chapter Chapter 1 --- Literature Review --- p.1<br>Chapter 1.1 --- Importance of fruiting body (mushrooms) production --- p.1<br>Chapter 1.2 --- Introduction on Coprinopsis cinerea --- p.1<br>Chapter 1.2.1 --- General introduction --- p.1<br>Chapter 1.2.2 --- Life cycle and morphology --- p.2<br>Chapter 1.2.3 --- Growth conditions for C. cinerea --- p.4<br>Chapter 1.3 --- Regulation of fruiting development in C. cinerea --- p.5<br>Chapter 1.3.1 --- Regulation by mating types --- p.5<br>Chapter 1.3.2 --- Regulation by light-dark cycle --- p.6<br>Chapter 1.3.3 --- Regulation by physiological factors --- p.9<br>Chapter 1.4 --- Fruiting-specific genes --- p.9<br>Chapter 1.5 --- Genome project of C. cinerea --- p.10<br>Chapter 1.6 --- Transformation and gene silencing in C. cinerea --- p.11<br>Chapter 1.7 --- DNA microarray --- p.12<br>Chapter 1.8 --- Endocytosis --- p.13<br>Chapter 1.8.1 --- The endocytic pathway --- p.14<br>Chapter 1.8.2 --- Rab GTPase --- p.17<br>Chapter 1.8.2.1 --- Control of the active and inactive state of Rab proteins --- p.18<br>Chapter 1.8.2.2 --- Functions of Rab GTPases in vesicular transport --- p.19<br>Chapter 1.8.2.3 --- Rab5 and Rab7 GTPase --- p.20<br>Chapter 1.8.3 --- Endocytosis in fungi --- p.21<br>Chapter 1.9 --- Aims of project --- p.22<br>Chapter Chapter 2 --- Whole genome expression analysis during fruiting body development --- p.25<br>Chapter 2.1 --- Introduction --- p.25<br>Chapter 2.2 --- Materials and Methods --- p.26<br>Chapter 2.2.1 --- NimbleGen 12x135K gene expression microarray --- p.26<br>Chapter 2.2.1.1 --- Strains and culture conditions --- p.26<br>Chapter 2.2.1.2 --- RNA extraction --- p.27<br>Chapter 2.2.1.3 --- Overall design of the NimbleGen custom Microarray --- p.28<br>Chapter 2.2.1.4 --- Microarray hybridization, data extraction and normalization --- p.28<br>Chapter 2.2.2 --- Microarray data analysis, clustering and GO assignment --- p.29<br>Chapter 2.2.3 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.29<br>Chapter 2.2.3.1 --- cDNA synthesis --- p.29<br>Chapter 2.2.3.2 --- Primer design and verification --- p.30<br>Chapter 2.2.3.3 --- Real time PCR and data analysis --- p.32<br>Chapter 2.3 --- Results --- p.33<br>Chapter 2.3.1 --- Whole-genome expression during fruiting body development --- p.33<br>Chapter 2.3.2 --- Differentially expressed genes during fruiting body initiation --- p.48<br>Chapter 2.3.2.1 --- Fruiting body initiation-specific genes --- p.52<br>Chapter 2.3.3 --- Gene expression analysis during fruiting body development --- p.53<br>Chapter 2.3.4 --- The C. cinerea kinome --- p.55<br>Chapter 2.3.5 --- Transcription factors in C. cinerea --- p.60<br>Chapter 2.3.6 --- The cytochrome P450 family in C. cinerea --- p.65<br>Chapter 2.3.7 --- Validation of NimbleGen microarray data by quantitative real-time PCR --- p.68<br>Chapter 2.4 --- Discussion --- p.77<br>Chapter Chapter 3 --- Effect of light on gene expression of fruiting body development --- p.92<br>Chapter 3.1 --- Introduction --- p.92<br>Chapter 3.2 --- Materials and Methods --- p.93<br>Chapter 3.2.1 --- NimbleGen 12x135K gene expression microarray --- p.93<br>Chapter 3.2.2 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.93<br>Chapter 3.2.2.1 --- cDNA synthesis --- p.94<br>Chapter 3.2.2.2 --- Primer design and verification --- p.94<br>Chapter 3.3 --- Results --- p.95<br>Chapter 3.3.1 --- Differentially expressed genes in dark stipes --- p.95<br>Chapter 3.3.2 --- Validation of expression patterns of NimbleGen microarray by real-time PCR --- p.102<br>Chapter 3.4 --- Discussion --- p.107<br>Chapter Chapter 4 --- Endocytosis in C. cinerea and its role in fruiting body development --- p.111<br>Chapter 4.1 --- Introduction --- p.112<br>Chapter 4.2 --- Materials and Methods --- p.112<br>Chapter 4.2.1 --- The endosomal machinery of C. cinerea --- p.112<br>Chapter 4.2.2 --- Tracing the endocytic pathway sing FM-64 --- p.113<br>Chapter 4.2.2.1 --- Strains and culture conditions --- p.113<br>Chapter 4.2.2.2 --- FM4-64 internalization in mycelium of C. cinerea --- p.113<br>Chapter 4.2.2.3 --- Drug treatment effect on the internalization of FM4-64 dye --- p.114<br>Chapter 4.2.3 --- Temporal and spatial expression of Cc.Rab5 and Cc.Rab7 --- p.114<br>Chapter 4.2.3.1 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.114<br>Chapter 4.2.3.1.1 --- RNA extraction and cDNA synthesis --- p.114<br>Chapter 4.2.3.1.2 --- TA cloning of amplification products and bacterial transformation --- p.115<br>Chapter 4.2.3.1.3 --- PCR screening for positive transformants and sequencing --- p.115<br>Chapter 4.2.3.2 --- Quantitative real-time PCR --- p.116<br>Chapter 4.2.3.2.1 --- RNA extraction and cDNA synthesis --- p.116<br>Chapter 4.2.3.2.2 --- Primer design and verification --- p.116<br>Chapter 4.2.3.2.3 --- Real time PCR and data analysis --- p.117<br>Chapter 4.2.3.3 --- In situ RNA-RNA hybridization --- p.117<br>Chapter 4.2.3.3.1 --- Tissue preparation --- p.117<br>Chapter 4.2.3.3.2 --- RNA probe synthesis --- p.117<br>Chapter 4.2.3.3.3 --- Hybridization, signal development and image viewing --- p.118<br>Chapter 4.2.4 --- Knock-down of endogenous Cc.Rab5 and Cc.Rab7 by siRNA --- p.119<br>Chapter 4.2.4.1 --- Strains and culture conditions --- p.119<br>Chapter 4.2.4.2 --- Production of dsRNA of Cc.Rab5 and Cc.Rab7 --- p.119<br>Chapter 4.2.4.3 --- Digestion of dsRNA to give siRNA --- p.120<br>Chapter 4.2.4.4 --- Effects of Cc.Rab5 and Cc.Rab7 siRNA on fruiting body development --- p.120<br>Chapter 4.2.4.4.1 --- Application of siRNA to C. cinerea culture --- p.120<br>Chapter 4.2.4.4.2 --- Validation of the knock-down efficacy by real-time PCR --- p.121<br>Chapter 4.3 --- Results --- p.122<br>Chapter 4.3.1 --- The endosomal machinery of C. cinerea --- p.122<br>Chapter 4.3.2 --- The endocytic pathway of C. cinerea --- p.127<br>Chapter 4.3.2.1 --- Time-course of FM4-64 internalization --- p.127<br>Chapter 4.3.2.2 --- Validation of active transport of FM4-64 --- p.129<br>Chapter 4.3.3 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.131<br>Chapter 4.3.4 --- Temporal expression of Cc.Rab5 and Cc.Rab7 --- p.133<br>Chapter 4.3.5 --- Spatial expression of Cc.Rab5 and Cc.Rab7 --- p.136<br>Chapter 4.3.6 --- Effects of Cc.Rab5 and Cc.Rab7 knock-down by siRNA --- p.140<br>Chapter 4.3.6.1 --- Observation of effect of siRNA on fruiting body development --- p.140<br>Chapter 4.3.6.2 --- Validation of the efficacy of external application of siRNA --- p.143<br>Chapter 4.4 --- Discussion --- p.146<br>Chapter Chapter 5 --- Concluding remarks --- p.152<br>References --- p.156<br>Appendix --- p.180

Cheng, Chi Keung.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (leaves 144-160).<br>Abstracts in English and Chinese.<br>English Abstract --- p.ii<br>Chinese Abstract --- p.iv<br>Acknowledgements --- p.v<br>Abbreviations --- p.vi<br>Table of Contents --- p.vii<br>List of Figures --- p.x<br>List of Tables --- p.xii<br>Chapter Chapter 1 --- Literature Review<br>Chapter 1.1 --- Introduction & Taxonomy --- p.1<br>Chapter 1.2 --- Life cycle and morphology --- p.1<br>Chapter 1.3 --- Growth requirements --- p.4<br>Chapter 1.3.1 --- Nutritional requirements --- p.4<br>Chapter 1.3.2 --- Environment factors --- p.5<br>Chapter 1.4 --- Fruiting body development in Coprinopsis cinerea --- p.6<br>Chapter 1.4.1 --- Physiology of the fruiting process --- p.6<br>Chapter 1.4.2 --- Other studies related to the fruiting process --- p.7<br>Chapter 1.5 --- Other biological studies in Coprinopsis cinerea --- p.8<br>Chapter 1.5.1 --- Meiosis studies --- p.9<br>Chapter 1.5.2 --- Mating analyses --- p.10<br>Chapter 1.5.3 --- Peroxidase production --- p.11<br>Chapter 1.5.4 --- Transformation and gene silencing --- p.12<br>Chapter 1.5.5 --- Other studies --- p.13<br>Chapter 1.6 --- C. cinerea genome project --- p.13<br>Chapter 1.7 --- Transcriptome analyses --- p.14<br>Chapter 1.7.1 --- Serial Analysis of Gene Expression (SAGE) --- p.14<br>Chapter 1.7.2 --- Analyzing the 5´ةend of transcripts --- p.16<br>Chapter 1.7.3 --- Mapping of SAGE tags to the genome --- p.19<br>Chapter 1.8 --- High throughput sequencing --- p.20<br>Chapter 1.8.1 --- Pyrophosphate sequencing --- p.20<br>Chapter 1.8.2 --- Application of pyrosequencing --- p.21<br>Chapter 1.9 --- Aims of project --- p.22<br>Chapter Chapter 2 --- 5' Serial Analysis of Gene Expression (5' SAGE) from mycelial and primordial stages of C. cinerea<br>Chapter 2.1 --- Introduction --- p.24<br>Chapter 2.2 --- Materials and Methods --- p.29<br>Chapter 2.2.1 --- 5' SAGE libraries construction --- p.29<br>Chapter 2.2.1.1 --- Mushroom mycelium and primordium cultivation --- p.29<br>Chapter 2.2.1.2 --- RNA extraction --- p.29<br>Chapter 2.2.1.3 --- Isolation of mRNA --- p.30<br>Chapter 2.2.1.4 --- cDNA synthesis --- p.31<br>Chapter 2.2.1.5 --- Mmel digestion and Polyacrylamide gel electrophoresis --- p.32<br>Chapter 2.2.1.6 --- Formation and amplification of ditag --- p.33<br>Chapter 2.2.2 --- Identification of lOObp ditag --- p.34<br>Chapter 2.2.3 --- High throughput pyrosequencing --- p.35<br>Chapter 2.2.4 --- Tags extraction from ditags --- p.35<br>Chapter 2.2.5 --- Genome mapping and annotation --- p.36<br>Chapter 2.3 --- Results --- p.37<br>Chapter 2.3.1 --- 5´ةSAGE libraries construction --- p.37<br>Chapter 2.3.1.1 --- cDNA synthesis --- p.37<br>Chapter 2.3.1.2 --- Mmel digestion and ditag formation --- p.38<br>Chapter 2.3.2 --- Identification of lOObp ditags --- p.39<br>Chapter 2.3.3 --- High throughput pyrosequencing --- p.40<br>Chapter 2.3.4 --- Tags extraction from ditags --- p.41<br>Chapter 2.3.5 --- Genome mapping and annotation --- p.42<br>Chapter 2.4 --- Discussion --- p.46<br>Chapter 2.4.1 --- 5´ةSAGE libraries construction --- p.46<br>Chapter 2.4.2 --- Tags extraction and genome mapping --- p.46<br>Chapter 2.4.3 --- Observations based on the genome mapping data --- p.48<br>Chapter Chapter 3 --- Validation of expression patterns of 5' SAGE libraries and analysis of differentially expressed genes<br>Chapter 3.1 --- Introduction --- p.55<br>Chapter 3.2 --- Materials and Methods --- p.58<br>Chapter 3.2.1 --- Identification of housekeeping gene by Northern Blot analysis --- p.58<br>Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.58<br>Chapter 3.2.1.2 --- Transfer of RNAs --- p.58<br>Chapter 3.2.1.3 --- Probe preparation --- p.59<br>Chapter 3.2.1.4 --- "Hybridization, Stringency washes and signal detection" --- p.60<br>Chapter 3.2.2 --- Quantitative real-time PCR --- p.61<br>Chapter 3.2.2.1 --- cDNA synthesis from 2 developmental stages --- p.61<br>Chapter 3.2.2.2 --- Primer design and verification --- p.62<br>Chapter 3.2.2.3 --- Real time PCR reaction and data analysis --- p.65<br>Chapter 3.2.3 --- Gene expression level comparison --- p.65<br>Chapter 3.3 --- Results --- p.67<br>Chapter 3.3.1 --- Identification of housekeeping gene by Northern Blot analysis --- p.67<br>Chapter 3.3.2 --- Quantitative real-time PCR analysis --- p.71<br>Chapter 3.3.3 --- Gene expression level comparison --- p.78<br>Chapter 3.4 --- Discussion --- p.126<br>Chapter 3.4.1 --- Validation of 5´ة SAGE libraries --- p.126<br>Chapter 3.4.2 --- Analysis of highly and differentially expressed genes --- p.127<br>Chapter Chapter 4 --- General discussion --- p.135<br>References --- p.144<br>Appendix --- p.161