结合基因表达数据和ChIP-chip数据的酵母转录调控模块的识别

活细胞依赖其众多的转录调控模块来实现复杂的生物功能,识别转录调控模块对深入理解细胞的功能及其转录机制有着重要的意义。本文结合酵母基因表达数据和ChIP-chip数据,提出了一种转录调控模块识别算法。该算法通过采用不同的P值阈值分别得到了核心集和粗糙集,然后对核心集和粗糙集进行判别,最后对基因进行扩展之后得到基因转录调控模块。将该算法运用到两个酵母基因表达数据中,得到了一些具有显著生物学意义的基因转录调控模块。与其它算法相比,该算法不仅可以识别含有较多基因的转录调控模块,而且可以识别一些其它算法不能识别的基因转录调控模块。识别得到的基因转录调控模块有着不同的生物学功能,并且有助于进一步理解酵母的转录调控机制。     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

Identification of Prognostic Biomarkers by Combined mRNA and miRNA Expression Microarray Analysis in Pancreatic Cancer

Pancreatic cancer is the fourth leading cause for cancer-related death, and early diagnosis is one key to improve the survival rate of this disease. Molecular biomarkers are an important method for diagnostic use in pancreatic cancer. We used data from three mRNA microarray datasets and a microRNA dataset (GSE16515, GSE15471, GSE28735, and GSE41372) to identify potential key genes. Differentially expressed genes (DEGs) and microRNAs (DEMs) were identified. Functional, pathway enrichment, and protein-protein interaction analyses were performed on common DEGs across all datasets. The target genes of the DEMs were identified. DEMs targets that were also DEGs were further scrutinized using overall survival analysis. A total of 236 DEGs and 21 DEMs were identified. There were a total of four DEGs (ECT2, NR5A2, NRP2, and TGFBI), which were also predicted target genes of DEMs. Overall survival analysis showed that high expression levels of three of these genes (ECT2, NRP2, and TGFBI) were associated with poor overall survival for pancreatic cancer patients. The basic expression of DEGs in pancreas stood lower level in various organ tissues. The expression of ECT2 and NRP2 was higher in different pancreatic cancer cell lines than normal pancreas cell line. Knockout of ECT2 by Crispr Cas9 gene editing system decreased proliferation and migration ability in pancreatic cancer cell line MiaPaCa2. In conclusion, we think that data mining method can do well in biomarker screening, and ECT2 and NRP2 can play as potential biomarker or therapy target by Crispr Cas9 in pancreatic cancer.     

基因表达与mRNA结构的关系

基因的转录调控和转录后水平的调控在基因表达过程中起着重要作用。mRNA的结构与基因表达调控的关系非常密切。目前对于mRNA结构对表达的影响因素,主要集中于起始密码子和S-D序列的结构和间隔长度、基因和基因间的间隔区序列和长度,5’末端与3’末端非翻译区、多聚（A）尾、内含子序列对翻译起始效率、发夹结构对mRNA的稳定性的影响和mRNA翻译起始区等对基因表达影响。     

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

Mining of Candidate Maize Genes for Nitrogen Use Efficiency by Integrating Gene Expression and QTL Data

Breeding maize varieties for high nitrogen (N) use efficiency (NUE) by marker-assisted selection using NUE quantitative trait locus (QTL) or by genetic transfer of NUE-associated genes is a viable approach for increasing grain yield in N-limited production areas. In this investigation, we evaluated a set of introgression line populations under N supply and N deficiency conditions. From 42 QTLs for grain yield and yield components, 23 were identified under N supply conditions and 33 from N limited conditions. Meta-analysis of published maize NUE QTLs revealed 37 “consensus” QTLs, of which, 18 was detected under low N conditions. In addition, 258 unique ESTs associated with low N stress response, N uptake, transport, and assimilation were aligned on the maize genome by in silico mapping. Integrating the EST map with the QTL map has resulted in the identification of candidate NUE-associated genes of the following functional categories: N uptake, transport, and assimilation; carbon (C) metabolism and assimilation; and cascades of stress response and signal transduction genes. Nine candidates that have been introgressed into Ye478 significantly altered grain yield/yield components. It is suggested that the dynamics of interactions between C and N metabolism are important for maize yield. A high NUE variety should have a highly efficient C assimilation per unit N and actively express CO₂ assimilation-related genes under N-limited conditions.     

麻疯树脂酶全长基因克隆、表达及其蛋白质结构预测

脂酶(Lipase,EC3.1.1.3)是普遍应用于皮革、饲料及生物柴油工业的工业酶制剂,具有广泛的应用价值。目前对植物来源的脂酶研究较少。本研究用在生物柴油中具有应用前景的油料植物——麻疯树(Jatrophacurcas)作为研究对象,克隆了该物种的脂酶基因(JcLIP)。通过多序列比对并结合物种的亲缘关系设计了具有较高特异性的简并引物,通过使用RT-PCR和RACE技术,最终获得了麻疯树脂酶基因的全长序列并成功地在大肠杆菌中表达,酶活测定结果表明,麻疯树脂酶在大肠杆菌中表达在包涵体中,但是能产生具有活力的蛋白质,酶活约为0.8U.mL-1。结构预测和比较表明,JcLIP蛋白质具有脂酶的结构核心和催化活性中心,而在非核心区具有较毛霉脂酶更多的插入和随机卷曲,这可能是决定二者之间酶活差异的重要原因。     

Identification of Novel miRNAs and miRNA Expression Profiling in Wheat Hybrid Necrosis

MicroRNAs (miRNAs) play essential roles in a vast array of biological processes, including growth and development, defense against viral infection, and responses to environmental changes in plant. Wheat hybrid necrosis is an interesting genetic phenomenon observed frequency and it is lethal or semi lethal, resulting in gradual death or loss of productivity. However, the molecular basis and mechanisms associated with hybrid necrosis in wheat are still not well understood. Here, we report the population and expression profiles of miRNAs in wheat hybrid necrosis. We identified a total of 57 conserved miRNA families as well as 182 putative novel miRNAs. Expression profiling revealed that expression of 49 known miRNAs and 165 novel miRNAs was changed in hybrid necrosis. And the expression levels of some miRNAs and their predicated targets have been confirmed by qRT-PCR. These results indicate that these miRNAs, especially miR159, miR166, miR167 and miR5072 could be involved in the extensive regulation of gene expression in response to hybrid necrosis.     

Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.     

Gene Structure, Chromosomal Location, and Expression Pattern of Maleylacetoacetate Isomerase

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

中国株HIV-1核心蛋白真核表达载体的构建与表达

运用限制性内切酶XbaⅠ、SalⅠ对pKSGAG进行双酶切,获得HIV-1 gag基因,并与真核表达载体pCI-neo连接,构建含有中国流行株HIV-1 核心蛋白真核表达载体pCI-neoGAG.经XbaⅠ/SalⅠ双酶切及测序鉴定证实,成功地构建了HIV-1 核心蛋白真核表达载体pCI-neoGAG.通过脂质体将pCI-neoGAG转染入p815细胞,G418筛选4周后,使用间接免疫荧光方法检测表达产物.结果表明所构建的HIV-1 核心蛋白真核表达载体能在p815细胞中高效表达,为下一步进行HIV-1 DNA疫苗研究奠定了基础.