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lanlan_zhu

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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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wypward(金币+2):欢迎新虫，辛苦啦！ 2010-06-01 09:57:57
lanlan_zhu(金币+50, 翻译EPI+1): 2010-06-01 10:19:52

为了分离在一般分泌途径的潜在突变，我们制定了一个两步骤的筛选程序。首先（第一步），筛选出无法降解平板中的淀粉的突变体，然后（第二步），再筛选了它们是否能降解明胶。假设突变体无法降解这些营养物质更可能是由于本身处于一般分泌通路的影响。一般的分泌途径突变可能导致致命的表型。在42摄氏度条件下筛选25摄氏度培养了3天的克隆子，筛选出温度敏感突变体。但是，两次分离出的突变体都没有显示出对温度的敏感性，比如在一般分泌通路中，假设分离出10株在25和42摄氏度无法降解淀粉和明胶的突变株，7株显示出与亲代相同的生长速率，但其产生孢子的数量均有所减少。荧光显微镜检查显示这些突变株均无细胞内的积累，细胞膜和细胞壁上也均无融合蛋白。因此，这些突变体是没有研究意义的。在淀粉和明胶的培养基上没有形成水解圈，可能反映了这些菌株的缺少分泌蛋白的能力，导致与亲代相比它们水解圈形成的延迟性。其余3株突变体与其亲代相比，在25和42℃增长缓慢，完全不产孢子并且菌丝形态异常。使用荧光显微镜观察两个突变体（gsp26和29）显示，细胞膜和细胞壁上无荧光，但胞内积累有GLA：：sGFP融合蛋白。剩余的一株突变（gsp31）显示胞内无积累且细胞膜细胞壁上无荧光，因此没有研究价值。Gsp26和gsp29对GLA：：sGFP融合蛋白胞内积累的方式又全然不同。Gsp26（图9A），荧光均匀地分散于整个菌丝，而在gsp29（图9B）中，可以看到荧光形成小球状，小球直径从约0.5至2微米。因此，无论gsp26和29，初步证据强力证明在一般分泌途径中缺陷的存在，这些缺陷导致细胞内的积累，并很可能会影响在代谢通路中不同的阻断点。对gsp26和29特点的进一步研究目前正在开展，以确定在通路中的哪些点发生积累，以及动态和组织的分泌途径中抑制剂的作用。因此，GLA：：sGFP的结构使分泌的蛋白质可视化，并且能在活菌体体内定位，证明了突变体的分泌特性非常具有研究价值，确定了在一个特定的分泌途径中快速、可靠检测分泌阻断的方法，提供了菌丝体内胞内积累物的额外的可视化信息。
我们要感谢Jen Sheen提供sGFP（S65T），ROY Montijin在转化工作中的帮助和Gerda Lamers在显微分析技术中的帮助。这项工作在英国完成的部分是由BBSRC对CG的助学金和伊朗巴斯德研究所奖学金支持。