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lanlan_zhu木蟲 (著名寫手)
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求翻譯文獻(xiàn) 生物類
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To isolate potential mutants in the general secretory pathway, we developed a two-step screening procedure. Initially, mutants were isolated which were unable to degrade starch on plates and, subsequently, these were screened for their ability to degrade gelatin. It was assumed that mutants unable to degrade either of these substrates were more likely to be affected in the general secretory pathway. As general secretory pathway mutants may lead to a lethal phenotype, screening was initially performed at 42 °C on colonies that had been grown for 3 d at 25 °C so that temperature-sensitive mutants could be selected. However, none of the mutants isolated from either the first starch plate screen or the subsequent gelatin plate screen displayed temperature sensitivity, i.e. the inability to degrade either substrate was displayed at both 25 and 42 °C. Of the 10 putative general secretory mutants isolated, 7 appeared to grow at the same rate as the parental strain but all showed reduced sporulation. None of these mutants when examined by fluorescence microscopy showed intracellular accumulation and all had fusion protein present in the walls and septa. These mutants were therefore disregarded. The lack of halo formation on starch and gelatin media may reflect a reduction in the overall ability of these strains to secrete proteins, resulting in delayed halo formation compared to the parent strain rather than a block in secretion per se. The remaining three putative general secretory pathway mutants all displayed poor growth at both 25 and 42 °C compared to the parental strain, a complete loss of sporulation and abnormal hyphal morphology. When examined under fluorescence microscopy, two mutants (gsp 26 and 29) displayed intracellular accumulation of GLA: : sGFP fusion protein with no detectable fluorescence in the walls or septa. The third mutant (gsp 31) showed no accumulation and fluorescence in the wall or septa and was disregarded. The pattern of accumulation of GLA: : sGFP fusion protein in gsp 26 and 29 differed significantly from each other. In gsp 26 (Fig. 9a), fluorescence was diffuse and evenly located throughout the hyphae, whereas in gsp 29 (Fig. 9b), accumulation in circular bodies ranging in size from approximately 0.5 to 2 μm diameter could clearly be seen. Thus, for both gsp 26 and 29, preliminary evidence strongly suggests the presence of defects in the general secretory pathway, leading to intracellular accumulation, and that these defects are likely to be affecting different points within the pathway. Further characterization of both gsp 26 and 29 is currently being undertaken to identify the sites at which accumulation occurs as well as examining the effects of inhibitors of secretion on the dynamics and organization of the secretory pathway. Thus the GLA: : sGFP constructs allow the direct visualization of protein secretion and localization in vivo in growing hyphae and have proved invaluable in the characterization of secretory mutants, allowing a quick and reliable method for confirming blocks in the secretory pathway and providing additional visual information on the sites of accumulation within the hyphae. We would like to thank Jen Sheen for sGFP(S65T), Roy Montijn for help in the analysis of transformants and Gerda Lamers for assistance with the microscopical techniques. The work carried out in the UK was funded by a BBSRC studentship to C.G. and a scholarship from the Pasteur Institute of Iran to V.K. [ Last edited by lanlan_zhu on 2010-5-31 at 20:19 ] |
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freshblack
金蟲 (正式寫手)
- 翻譯EPI: 15
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★ ★
wypward(金幣+2):歡迎新蟲，辛苦啦！ 2010-06-01 09:57:57
lanlan_zhu(金幣+50, 翻譯EPI+1): 2010-06-01 10:19:52
wypward(金幣+2):歡迎新蟲，辛苦啦！ 2010-06-01 09:57:57
lanlan_zhu(金幣+50, 翻譯EPI+1): 2010-06-01 10:19:52
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為了分離在一般分泌途徑的潛在突變，我們制定了一個(gè)兩步驟的篩選程序。首先（第一步），篩選出無法降解平板中的淀粉的突變體，然后（第二步），再篩選了它們是否能降解明膠。假設(shè)突變體無法降解這些營(yíng)養(yǎng)物質(zhì)更可能是由于本身處于一般分泌通路的影響。一般的分泌途徑突變可能導(dǎo)致致命的表型。在42攝氏度條件下篩選25攝氏度培養(yǎng)了3天的克隆子，篩選出溫度敏感突變體。但是，兩次分離出的突變體都沒有顯示出對(duì)溫度的敏感性，比如在一般分泌通路中，假設(shè)分離出10株在25和42攝氏度無法降解淀粉和明膠的突變株，7株顯示出與親代相同的生長(zhǎng)速率，但其產(chǎn)生孢子的數(shù)量均有所減少。熒光顯微鏡檢查顯示這些突變株均無細(xì)胞內(nèi)的積累，細(xì)胞膜和細(xì)胞壁上也均無融合蛋白。因此，這些突變體是沒有研究意義的。在淀粉和明膠的培養(yǎng)基上沒有形成水解圈，可能反映了這些菌株的缺少分泌蛋白的能力，導(dǎo)致與親代相比它們水解圈形成的延遲性。其余3株突變體與其親代相比， 在25和42℃增長(zhǎng)緩慢，完全不產(chǎn)孢子并且菌絲形態(tài)異常。使用熒光顯微鏡觀察兩個(gè)突變體（gsp26和29）顯示，細(xì)胞膜和細(xì)胞壁上無熒光，但胞內(nèi)積累有GLA：：sGFP融合蛋白。剩余的一株突變（gsp31）顯示胞內(nèi)無積累且細(xì)胞膜細(xì)胞壁上無熒光，因此沒有研究?jī)r(jià)值。Gsp26和gsp29對(duì)GLA：：sGFP融合蛋白胞內(nèi)積累的方式又全然不同。Gsp26（圖9A），熒光均勻地分散于整個(gè)菌絲，而在gsp29（圖9B）中，可以看到熒光形成小球狀，小球直徑從約0.5至2微米。因此，無論gsp26和29，初步證據(jù)強(qiáng)力證明在一般分泌途徑中缺陷的存在，這些缺陷導(dǎo)致細(xì)胞內(nèi)的積累，并很可能會(huì)影響在代謝通路中不同的阻斷點(diǎn)。對(duì)gsp26和29特點(diǎn)的進(jìn)一步研究目前正在開展，以確定在通路中的哪些點(diǎn)發(fā)生積累，以及動(dòng)態(tài)和組織的分泌途徑中抑制劑的作用。因此，GLA：：sGFP的結(jié)構(gòu)使分泌的蛋白質(zhì)可視化，并且能在活菌體體內(nèi)定位，證明了突變體的分泌特性非常具有研究?jī)r(jià)值，確定了在一個(gè)特定的分泌途徑中快速、可靠檢測(cè)分泌阻斷的方法，提供了菌絲體內(nèi)胞內(nèi)積累物的額外的可視化信息。 我們要感謝Jen Sheen提供sGFP（S65T），ROY Montijin在轉(zhuǎn)化工作中的幫助和Gerda Lamers在顯微分析技術(shù)中的幫助。這項(xiàng)工作在英國(guó)完成的部分是由BBSRC對(duì)CG的助學(xué)金和伊朗巴斯德研究所獎(jiǎng)學(xué)金支持。 |
2樓2010-06-01 09:55:08
freshblack
金蟲 (正式寫手)
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LZ看看可否，請(qǐng)指教~3樓2010-06-01 09:57:30
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