Speaker ：曹欣瑜 Advisor ：藍清隆 老師 Date ： Identification of genes expressed by Cryptococcus gattii during iron deprivation Brazilian Journal of Microbiology 45,3, (2014)

2 前言

3 Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii ) are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation.

前言 4 There are two main pathogenic species within the Cryptococcus genus, namely C. neoformans and C. gattii. C. neoformans var. grubii and C. neoformans var. neoformans have been isolated worldwide, and typically cause disease in hosts with impaired immunity.

前言 5 In mammalian hosts, the majority of iron is locked within iron-binding proteins, pathogens must therefore be equipped with competitive iron acquisition and uptake systems. C. neoformans possesses cell surface reductases that reduce ferric iron to its ferrous state (Cfo1), export reductants, such as 3-hydroxyanthranilic acid, and iron permease (Cft1) for transport into the cytosol as ferric ion. In addition, for C. neoformans, many studies have described genes related to iron regulation, including Cryptococcus iron regulator 1 (CIR1) and HAPX, genes that control the expression of iron-dependent genes.

前言 6 In C. gattii, little is known about iron metabolism and its implication in virulence. But there have observed a similar regulation of metabolic proteins in both the pathogenic yeasts, C. gattii and C. neoformans. However, certain proteins related to iron homeostasis in C. neoformans were not identified. In order to investigate the adaptive cellular responses when host-iron availability to pathogenic microorganisms is reduced, we attempted to identify the differential gene expression profile of the C. gattii reference strain R265 under conditions of iron deprivation, using representational difference analysis (RDA).

7 實驗目的

8 To identify the differential gene expression profile of the C. gattii reference strain R265 under conditions of iron deprivation, using representational difference analysis (RDA).

9 實驗項目

實驗項目 -2 個 table 10

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實驗項目 12 Table 1 - Summary of the computational analysis of the transcripts obtained from C. gattii grown in a low iron medium for 3 h at 37 °C Table 2 - Summary of the computational analysis of the transcripts obtained from C. gattii grown in a low iron medium for 12 h at 37 °C.

13 實驗流程

實驗流程 - 準備 Strain, culture conditions and RNA extraction 14 The C. gattii strain R265 was used for the RDA experiments and gene expression analysis. R265 was routinely grown in Yeast Peptone Dextrose (YPD) broth (yeast extract 1%, peptone 1% and glucose 2%) before cultivation in medium containing low levels of iron (limited-iron medium (LIM) and an iron-repleted medium (LIM+Fe, with the addition of 100MFeHEDTA) for 24 h.

實驗流程 - 準備 Strain, culture conditions and RNA extraction 15 To evaluate the effects of iron, 10 6 cells/mL of yeast were transferred to 50 mL of LIM or LIM-Fe. Cells were grown for two distinct periods of time (3 h or 12 h) in LIM or LIM-Fe at 37 °C. Cells were harvested by centrifugation and immediately frozen in liquid nitrogen before RNA extraction. Total RNA was isolated using the RNeasy mini kit, and cDNA was synthesized using the SMART PCR synthesis kit. First-strand cDNA synthesis was performed with reverse transcriptase from 500 ng of total RNA. An aliquot of 5 μL of first-strand cDNA was used as the template for second-strand synthesis.

實驗流程 - 步驟 Representational difference analysis (RDA) 16 cDNA from C. gattii grown for 3 h and 12 h in a low-level iron medium (LIM) as the “tester” and cDNA from C. gattii grown for 3 h and 12 h in iron-replete medium (LIM+Fe) as the “driver”. All cultures were grown at 37 °C with shaking. A ds-cDNA sample from each experimental condition was digested with Sau3AI and resulting products were purified using the illustra GFX PCR DNA and gel band purification kit. The RBam24/12 adapters were ligated to the digested cDNA in order to be used as a tester.

實驗流程 - 步驟 Representational difference analysis (RDA) 17 The first differential product (Dp1) was obtained by hybridization (20 h at 67 °C) of the driver and cDNA mixed at a 10:1 ratio, followed by PCR amplification with an RBam24 primer. In order to generate the second (Dp2) and third (Dp3) differential products, NBam and JBam adapters were ligated to the tester in each round of subtractive hybridization and the driver/tester ratio was increased to 100:1 and 1000:1, respectively.

實驗流程 - 步驟 Cloning and bioinformatics analysis of the RDA products 18

結論 19

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參考資料 21

22 2 代表性差異分析 (representational difference analysis ， RDA) RDA ［ 4-8 ］是 Lisitsyn 等於 l993 年在差減雜交的技術基礎上 發展的分析基因組 DNA 差異的方法， Hubank 等於 l994 年對其 作改良后用於 cDNA 的分析. 该技術是將差減雜交与 PCR 有機 結合形成的一種方法［ 9 ］， 主要步骤包括 : ①將對照組（ driver) 和待測組 (tester)mRNA 逆轉錄成 cDNA 片段群，接上接頭進行第 1 次 PCR 擴增 ②將 tester cDNA 和 driver cDNA 用限制性內切酶酶切，形成 平均長度 256bp 的 cDNA ③將接頭消化，在 tester cDNA 末端接上新的接頭， tester cDNA 和 driver cDNA 按 1 ∶ 100 比例混合. 過量的 per cDNA 可與 tester cDNA 中互補的部分形成雙鏈 ④復性，以新接頭為引物進行第 2 輪 PCR. 三種雜合體中只有自 身退火形成的 tester/tester 雜合體才能和引物配對，並以指數 形式擴增； tester/driver 雜合體只能是線性遞增；而 driver/driver 雜合體則無法擴增. 該方法的主要优點就是能夠 使差異表達的基因高度富集，重復性較好，假陽性少. 利用 RDA 人們已經發現了多個新基因. 如白血病雕亡相关基因 TFAR19 ，人 乳腺癌相關基因 TSPS0 等. Arava 等［ 10 ］利用 RDA 分析了胰腺 β 細胞和 α 細胞，發現 26 個基因在 β 細胞中高表達，其中 l4 個为已知 基因，如 PKA 调節亞基、 STAT6 等，其余為未知基因，闡明這些 未知基因的作用對於了解 β 細胞的功能及 1 型糖尿病的發病機制具 有重要意義. 免費論文下載中心 /8/3

23 生物資訊生物資訊學中， BLAST （英語： Basic Local Alignment Search Tool ）它是一個用來英語 比對生物序列的一級結構（如不同蛋白質的胺基酸序列或不同基因的 DNA 序列）的算法。生物序列一級結構蛋白質胺基酸基因 DNA 已轉錄序列 - 蛋白 BLAST （ blastx) 已知一段已經轉錄的序列，藉由這個程式對這段序列的 6 個 ORF 對上用戶所選擇的蛋白質 資料庫， 比對最相似的序列。其功用可以找出在基因體 DNA （ genomic DNA ）上轉譯出 蛋白質的序列。 ORF轉譯 蛋白質 維基百科 %E8%A8%8A%E5%AD%B8% /07/07 %E8%A8%8A%E5%AD%B8%29

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