Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase. In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.     

Novel tumor-associated antigen of hepatocellular carcinoma defined by monoclonal antibody E4-65

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.     

聚苹果酸生产菌出芽短梗霉CCTCC M2012223的全基因组测序及序列分析

【目的】解析出芽短梗霉CCTCC M2012223的基因组序列信息,分析其代谢产物聚苹果酸、黑色素、普鲁兰多糖合成相关基因,为深入研究遗传多样性和代谢工程改造提供序列背景信息。【方法】使用Illumina Hi Seq高通量测序平台对出芽短梗霉CCTCC M2012223菌株进行全基因组测序,并对测序数据进行序列拼接,基因预测与功能注释,COG/GO聚类分析,比较基因组学分析等。下载其他5株出芽短梗霉基因组序列,比较分析6株菌的种内同源基因、全基因组进化以及代谢产物合成相关基因。【结果】出芽短梗霉CCTCC M2012223基因组序列全长30756831 bp,GC含量47.49%,编码9452个基因。比较基因组分析表明出芽短梗霉CCTCC M2012223的基因组组装长度最长,6株菌的同源基因数达到7092个,普鲁兰多糖和聚苹果酸合成相关基因的蛋白序列有很高的保守性。出芽短梗霉CCTCC M2012223和Aureobasidium pullulans var.melanogenum亲缘关系最近,而这2株菌的黑色素合成相关基因的蛋白序列有一些插入和突变。【结论】本研究解析了出芽短梗霉CCTCC M2012223的基因组序列信息,获得黑色素、普鲁兰多糖和聚苹果酸合成相关基因,为后续的代谢机制解析和改造提供相关依据。     

人类免疫缺陷病毒Ⅰ型核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

在大肠杆菌中,利用新构建的含T7g-10L RBS以及λ-PR启动子的新型原核表达载体,通过表达gag-pol基因片段,获得了具有天然序列的人类免疫缺陷病毒1型(HIV-1)核心蛋白p24的高效表达。克隆的gag-pol基因片段在其阅读框架移位区域插入了4bp碱基,其表达的病毒蛋白酶在阅读框架上与gag一致,从而实现了对gag-pol融合蛋白的有效加工,产生成熟的核心蛋白p24及其它产物。重组p24以可溶形式存在,可以被抗p24的单克隆抗体特异识别。测定的N端8个氨基酸序列与从病毒纯化的p24完全一致。在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到95％以上的纯度。结果表明,纯化的p24可以作为特异性很强的试剂而用于HIV感染的诊断及病情的预后,并可用于p24的生化及结构分析。     

Prediction of protein structure class by coupling improved genetic algorithm and support vector machine

Structural class characterizes the overall folding type of a protein or its domain. Most of the existing methods for determining the structural class of a protein are based on a group of features that only possesses a kind of discriminative information for the prediction of protein structure class. However, different types of discriminative information associated with primary sequence have been completely missed, which undoubtedly has reduced the success rate of prediction. We present a novel method for the prediction of protein structure class by coupling the improved genetic algorithm (GA) with the support vector machine (SVM). This improved GA was applied to the selection of an optimized feature subset and the optimization of SVM parameters. Jackknife tests on the working datasets indicated that the prediction accuracies for the different classes were in the range of 97.8–100% with an overall accuracy of 99.5%. The results indicate that the approach has a high potential to become a useful tool in bioinformatics.     

‘Green tides’ are overwhelming the coastline of our blue planet: taking the world’s largest example

A broad spectrum of events that come under the category of green tide are recognized world-wide as a response to elevated levels of seawater nutrients in coastal areas. Green tides involve a wide diversity of sites, macroalgal species, consequences, and possible causes. Here we review the effect of natural and man-induced environmental fluctuations on the frequency and apparent spread of green tides. This article highlights the need for interdisciplinary research aimed at shedding light on the basic mechanisms governing the occurrence and succession of green algae in coastal seas. This will result in more effective management and mitigation of the effects of green tides, thus safeguarding the intrinsic and commercial value of coastal marine ecosystems.     

黄芪对镉致大鼠睾丸支持细胞超微结构及波形蛋白、E-钙粘蛋白表达改变的保护作用

目的 探讨黄芪对镉致大鼠睾丸支持细胞损伤的保护作用.方法 21只成年雄性SD大鼠随机分成镉组(0.1％氯化镉腹腔内注射,1mg/Kg体重/天,5天/周,处理后1、2、3、4周取材)、镉加黄芪组(注射氯化镉的同时注射黄芪,10g/Kg体重/天,5天/周,处理后2、4周取材)和对照组(腹腔内注射等量生理盐水).睾丸取材作光镜、免疫组织化学染色和图像分析及超微结构观察.结果 光镜H.E染色对照组支持细胞核不规则,染色浅,核仁明显,镉处理后胞浆内有空泡形成,镉加黄芪组支持细胞未见明显改变.对照组波形蛋白阳性产物在支持细胞靠近基室腔的胞浆中表达,E-钙粘蛋白阳性产物则主要定位于生精上皮近腔室的支持细胞和部分生精细胞胞浆中.镉处理后支持细胞胞浆中波形蛋白和E-钙粘蛋白阳性产物表达的平均光密度值均明显降低(P＜0.05),镉加黄芪组阳性产物表达虽较对照组减弱但明显高于相应镉组(P＜0.05).镉处理组支持细胞胞质特化区和紧密连接破坏,镉加黄芪组支持细胞超微病变较相应镉组为轻.结论 镉降低大鼠睾丸支持细胞波形蛋白和E-钙粘蛋白的表达并造成支持细胞的超微结构损伤,黄芪具有较好的保护效果.     

微生物菌种改良的新方法新策略

自然分离得到的原始菌种远不能达到工业生产要求,通过菌种改良获得高产菌种是有效的手段。传统方法虽然无需了解过多遗传背景就能取得成效,但往往耗时费力。随着DNA重组技术、原生质体融合、组学研究的应用日益广泛,微生物菌种改良的新方法和新策略诸如代谢工程、基因组改组和系统生物技术、核糖体工程、表观遗传修饰等逐步发展起来。以下综述了近年来菌种改良相关领域方法和策略特别是首次报道的表观遗传修饰的最新进展。