Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein

Genome-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin fragment or of alpha-synuclein. Of 4850 haploid mutants containing deletions of nonessential genes, 52 were identified that were sensitive to a mutant huntingtin fragment, 86 that were sensitive to alpha-synuclein, and only one mutant that was sensitive to both. Genes that enhanced toxicity of the mutant huntingtin fragment clustered in the functionally related cellular processes of response to stress, protein folding, and ubiquitin-dependent protein catabolism, whereas genes that modified alpha-synuclein toxicity clustered in the processes of lipid metabolism and vesicle-mediated transport. Genes with human orthologs were overrepresented in our screens, suggesting that we may have discovered conserved and nonoverlapping sets of cell-autonomous genes and pathways that are relevant to Huntington's disease and Parkinson's disease.     

转录因子Sp1对成肌细胞中猪SKIP的表达调控作用

利用基因组PCR步移方法获得猪SKIP转录起始位点上游2 075 bp启动子序列。生物信息学分析发现该序列中存在4个Sp1结合位点和1个CpG岛。将启动子序列构建到pGL3-basic的双荧光素酶报告基因载体上,再利用RNA干扰技术分析转录因子Sp1对SKIP转录的影响。荧光素酶活性分析发现Sp1的表达抑制使成肌细胞中SKIP启动子活性显著下降,表明转录因子Sp1可能通过顺式作用元件GC-Box对成肌细胞分化过程中SKIP基因的转录激活起正向调控作用。     

Impairment of the ubiquitin-proteasome system by protein aggregation

Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.     

奶牛乳脂性状候选基因EEF1D突变位点功能分析

提高乳脂含量、提升奶质已成为当前奶牛业研究的重点方向之一。前期经GWAS方法研究发现,EEF1D基因5′非翻译区(5′-UTR)的G/A突变与乳脂率极显著相关,为了进一步对候选基因EEF1D的突变位点进行功能分析,首先通过人工合成获得了EEF1D基因突变型与野生型片段,并在细胞水平上对其开展了功能验证实验。研究发现,EEF1D基因5′-UTR的G/A突变引起Sp3转录起始位点后移33 bp,并且突变型的活性大约是野生型的3倍。研究结果表明,该突变可改变转录因子的转录起始位点,从而使EEF1D的表达上调。     

水稻空间诱变突变系苗期养分效率性状的差异

利用水培方法对农艺性状明显变化的几个水稻空间诱变突变系Sp211、Sp151和Sp8的苗期氮、磷、钾的吸收和利用效率进行评价。结果表明：3个突变系的植株生物量、根系、植株养分吸收量在养分正常供应和养分胁迫条件下均低于野生型;除Sp8在养分正常供应条件下氮浓度高于野生型外,其他系的植株体内养分浓度在两种条件下与野生型无显著差异;3个突变系均是根系性状的突变导致其养分吸收能力的变化。因此,可利用空间诱变创造的突变体开展植物养分效率的遗传机制研究。     

ACC合成酶和ACC氧化酶基因家族在番茄乙烯过表达突变体Epinastics果实中的差别表达(英文)

利用 Ｎｏｒｔｈｅｒｎ 杂交和核酸保护分析（ＲＰＡ）检测了番茄ＡＣＣ 合成酶（ＡＣＳ）和 ＡＣＣ氧化酶（ＡＣＯ）基因家族在番茄突变体Ｅｐｉｎａｓｔｉｃｓ （Ｅｐｉ）果实中的表达特性,同时测定了Ｅｐｉ果实自动催化乙烯合成系统的特性⒚ＥＰＩ等位基因的突变诱导了ＬＥＡＣＳ２ 和ＬＥＡＣＯ１ 两个基因的过表达,这是Ｅｐｉ果实的乙烯过表达的主要成因⒚ＥＰＩ突变同时显著抑制了ＬＥＡＣＳ４ 和 ＬＥＡＣＯ３ 的表达强度⒚这些结果初步鉴别了这两个基因家族在番茄果实上表达的各个成员的转录调节途径⒚同时表明,番茄单基因乙烯反应突变体是研究ＡＣＳ和ＡＣＯ基因家族各成员转录调节特性的一种有用工具⒚     

γ-聚谷氨酸高产菌株的鉴定及诱变选育

从豆豉中分离筛选出一株产γ-聚谷氨酸（γ-PGA）的菌株,经过形态、生理生化鉴定及16SrRNA基因序列分析,确定为枯草芽孢杆菌。其中Bacillus subtilis HD11γ-PGA产率最高,达6.8g/L。以HD11作为出发菌株,采用紫外线、硫酸二乙酯和亚硝基胍对其进行复合诱变,分离筛选得到一株稳定的高产突变株Bacillus subtilisHD-F9,经摇瓶发酵γ-聚谷氨酸的含量达到10.72g/L,较出发菌株提高56.3%。     

Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice

The most common inherited [correct] form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motor neurons, is caused by dominant mutations in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1). In chimeric mice that are mixtures of normal and SOD1 mutant-expressing cells, toxicity to motor neurons is shown to require damage from mutant SOD1 acting within nonneuronal cells. Normal motor neurons in SOD1 mutant chimeras develop aspects of ALS pathology. Most important, nonneuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant-expressing motor neurons.     

Coupling of the TCR to integrin activation by Slap-130/Fyb

SLAP-130/Fyb (SLP-76-associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb-deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.     

一个水稻T-DNA插入类病斑突变体的初步研究

从T-DNA插入突变体中筛选到一个类病斑突变体AZT91,主要表现为生长缓慢、植株矮小、叶片出现条状褐斑,最后死亡。对突变体及其后代分离群体进行潮霉素抗性检测,证明该突变体是由T-DNA插入突变引起的,突变性状与T-DNA共分离。PCR和TAIL-PCR分析进一步证明了上述的观点。利用TAIL-PCR扩增了左边界侧翼序列,通过分析,初步推测该突变体可能是由于T-DNA插入后激活了单加氧酶基因的过量表达,破坏正常代谢途径,导致突变体死亡。该材料可用于水稻代谢调控机理的研究。     

Exercise and genetic rescue of SCA1 via the transcriptional repressor Capicua

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by up-regulation of epidermal growth factor and consequential down-regulation of Capicua, which is an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of all disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reduction of Capicua levels--either genetically or by exercise--mitigated the disease phenotypes by dampening the toxic gain of function. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases.     

小鼠胞内氯离子通道5基因启动子核心功能区域鉴定及转录调控分析

为研究胞内氯离子通道5基因(Chloride intracellular channel 5,CLIC5)广泛参与调节细胞内的各项生理活动与生化反应,并探讨该基因自身的表达调控机制,以小鼠基因组序列为模板,利用PCR技术扩增小鼠CLIC5基因5′上游调控序列,将其插入荧光素酶报告基因表达载体(pGL3-Basic)中,同时采用5′侧翼区缺失的方法构建了7个缺失不同DNA片段的荧光素酶表达载体。重组质粒与海肾荧光素酶载体(phRL-TK)共同瞬时转染HEK-293细胞,经双荧光素酶报告基因活性分析后,确定CLIC5基因的核心启动子区。利用生物信息学方法预测其中转录因子结合位点及启动子区甲基化状况。结果表明,CLIC5基因启动子缺乏TATA盒,但含有典型的GC盒及其他潜在转录因子结合位点;双荧光素酶报告基因活性分析表明,CLIC5基因-329～+1、-624～+1、-917～+1和-2 230～+1区域的启动子活性较高,其中-624～+1区域的启动子活性最强。进一步分析表明,启动子区-624～-329存在负性调控元件,预测存在转录因子结合位点RXR heterodimer binding sites与GC-Box factorsSp1/GC,-420～-283范围内存在CpG岛位点。