Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.     

猪源冠状病毒聚合酶基因的克隆及特性分析

参照GenBank中Purdue株序列对TGEVTS株聚合酶基因(ORF1)设计特异性引物,经RT-PCR扩增获得了20054bp的片段,与预期大小相符。TS株与Pur46-MAD株的ORF1核苷酸和氨基酸序列同源性分别为98.8%和99.0%,与FIPV、PEDV、HCV299E及SARS氨基酸同源性分别为88%、57%、57%和45%。不同冠状病毒比较结果显示RdRp有很高的保守性而且有重要的作用,序列分析标明TS株在ORF1a和ORF1b重叠区有核糖体剪切位点UUUAAAC,且相应区域RNA二级结构形成3个茎环结构。     

猪源冠状病毒聚合酶基因的克隆及特性分析

参照GenBank中Purdue株序列对TGEV TS株聚合酶基因(ORF1)设计特异性引物,经RT-PCR扩增获得了20054bp的片段,与预期大小相符.TS株与Pur46-MAD株的ORF1核苷酸和氨基酸序列同源性分别为98.8%和99.0%,与FIPV、PEDV、HCV299E及SARS氨基酸同源性分别为88%、57%、57%和45%.不同冠状病毒比较结果显示RdRp有很高的保守性而且有重要的作用,序列分析标明TS株在ORF1a和ORF1b重叠区有核糖体剪切位点UUUAAAC,且相应区域RNA二级结构形成3个茎环结构.     

油桐尺蠖核型多角体病毒DNA聚合酶基因的克隆和序列分析

用PCR方法从油桐尺蠖核型多角体病毒 (BusuNPV)中扩增出DNA聚合酶基因片段 ,经pGEM T载体克隆到大肠杆菌DH5α菌株中。经自动序列分析仪测出DNA聚合酶基因 2 379bp长的核苷酸序列 ,推导出 793的氨基酸序列。氨基酸同源性比较显示 ,BusuNPV与HzSNPV的同源性最高 ,达 57% ;与OpMNPV的同源性最低 ,为 39.6 %。     

桑泛素延伸蛋白基因片段的克隆及序列分析

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。     

毛尖紫萼藓泛素延伸蛋白基因克隆及序列分析

目的:该文首次从毛尖紫萼藓中克隆到泛素延伸蛋白基因的全长,为深入研究其功能打下基础。方法:提取植物的总RNA,经反转录得到cDNA,根据实验室得到的毛尖紫萼藓泛素延伸蛋白基因的EST序列设计引物,基于3’RACE(Rapid Ampli-fication of cDNA End)技术克隆得到全长序列,使用分子生物学软件进行序列分析。结果:获得了cDNA全序列,GenBank登录号为JQ659260,序列全长为673bp,开放阅读框为471 bp,编码156个氨基酸残基。结论:通过Blast P对其编码的氨基酸序列进行比对结果显示:毛尖紫萼藓泛素延伸蛋白与小立碗藓、北美云杉、葡萄、蓖麻、毛果杨、紫茎泽兰的泛素延伸蛋白的同源性均在96%以上。     

牡丹泛素延伸蛋白基因片段的克隆及序列分析

目的：利用巢式PCR技术克隆牡丹泛素延伸蛋白基因,为泛素蛋白降解系统研究奠定基础,也为牡丹基因表达水平研究提供内参基因。方法：从牡丹（Paeonia suffruticosa）叶片、花瓣、花萼中提取总RNA,反转录得到cDNA,根据已报道的泛素延伸蛋白基因设计巢式引物进行PCR扩增。结果：得到一条389bp的牡丹泛素延伸蛋白基因片段,该片段编码129个氨基酸残基。结论：通过Blastn比对分析表明：牡丹泛素延伸蛋白基因与番茄、水稻、拟南芥等植物的泛素延伸蛋白基因一致性达到100%,编码的氨基酸序列同源性达94%以上。确认该片段即牡丹泛素延伸蛋白基因。     

中国棉铃虫核型多角体病毒DNA聚合酶基因的克隆和序列分析

应用谷实夜蛾核型多角体病毒（ＨｚＳＮＰＶ）ＤＮＡ聚合酶基因ＨｉｎｄⅢ／ＰｓｔⅠ３５９６ｂｐ片段作探针,经Ｓｏｕｒｔｈｅｒｎｂｌｏｔ杂交,克隆了中国棉铃虫核型多角体病毒（ＨａＳＮＰＶ）完整的ＤＮＡ聚合酶基因,大小约为３．４ｋｂ。限制性内切酶分析表明,ＨａＳＮＰＶＤＮＡ聚合酶基因限制性内切酶图谱与ＨｚＳＮＰＶ相似。用双脱氧链终止法测定该基因部分核苷酸序列（８０５ｂｐ）,推导出编码区２０６ａ。序列同源性比较显示,ＨａＳＮＰＶＤＮＡ聚合酶与ＨｚＳＮＰＶ之间具有高度的同源性;与ＬｄＭＮＰＶ、ＡｃＭＮＰＶ、ＢｍＳＮＰＶ、ＣｆＭＮＰＶ和ＯｐＭＮＰＶ也具有一定的同源性     

应用聚合酶链反应突变和克隆HIV-lgag基因片段

作者设计并合成了一对突变引物PGO1和PGO2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、BamHI和ATG及TAA序列,以便于HlV-1gag基因序列的定向克隆和表达。用PCO1和PGO2作引物,采用PCR方法从HIV-1基因组DNA中扩增出一个长504bp的DNA片段,用EcoRl和BamHI双酶切位点将此片段定向克隆入pUC19质粒。将克隆基因插入M13mp18进行DNA序列分析,结果表明,该基因序列及读框完全正确,且在其5′末端突变出EcoRI位点和ATG起始码,3′末端突变出TAA终业码和BamHI位点,从而为该基因的表达研究奠定了基础。     

利用套叠PCR和高保真DNA聚合酶进行基因多位点突变的研究

利用套叠PCR和高保真DNA聚合酶对人工合成的牛口蹄疫病毒VPI基因(FMDV-VPI,Foot-Mouth Disease Vims-VPI)的5个突变位点进行修复,修复的成功率为100％。     

Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from Thermococcus kodakaraensis KOD1

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 ‘KOD DNA polymerase’. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.     

Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene

The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms “pre-P” by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.     

栽培大豆染色体改构复合体基因SNP5的克隆及序列分析

根据大豆耐盐基因表达芯片上提供的表达量下调的EST序列信息,借助了电子克隆方法,根据获得的假定序列设计引物,通过RT-PCR,从栽培大豆克隆了染色体改构复合体SNP5类同源基因。其片段长度为812bp,编码240个氨基酸的多肽,分子重量为27.4kD,等电点pI为5.37。NCBI保守结构域分析表明,其属于SNF5超家族,因此我们将其命名为Gm-SNP5(GenBank登录号:HM068595)。Southern杂交表明,GmSNF5基因在栽培大豆基因中至少有一个拷贝。氨基酸序列及系统进化树分析表明,GmSNF5与其同科的豌豆PsSNF5有较高的同源性,氨基酸序列一致性高达92%。由于其部分EST序列在大豆耐盐芯片中表达量下调,因此推测此基因在功能上与栽培大豆的耐盐性相关。     

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD''s overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism.     

Gene Expression Phenotypes of Oncogenic Signaling Pathways

No abstract available.     

Schizophrenia Gene Networks and Pathways and Their Applications for Novel Candidate Gene Selection

Background

Schizophrenia (SZ) is a heritable, complex mental disorder. We have seen limited success in finding causal genes for schizophrenia from numerous conventional studies. Protein interaction network and pathway-based analysis may provide us an alternative and effective approach to investigating the molecular mechanisms of schizophrenia.

Methodology/Principal Findings

We selected a list of schizophrenia candidate genes (SZGenes) using a multi-dimensional evidence-based approach. The global network properties of proteins encoded by these SZGenes were explored in the context of the human protein interactome while local network properties were investigated by comparing SZ-specific and cancer-specific networks that were extracted from the human interactome. Relative to cancer genes, we observed that SZGenes tend to have an intermediate degree and an intermediate efficiency on a perturbation spreading throughout the human interactome. This suggested that schizophrenia might have different pathological mechanisms from cancer even though both are complex diseases. We conducted pathway analysis using Ingenuity System and constructed the first schizophrenia molecular network (SMN) based on protein interaction networks, pathways and literature survey. We identified 24 pathways overrepresented in SZGenes and examined their interactions and crosstalk. We observed that these pathways were related to neurodevelopment, immune system, and retinoic X receptor (RXR). Our examination of SMN revealed that schizophrenia is a dynamic process caused by dysregulation of the multiple pathways. Finally, we applied the network/pathway approach to identify novel candidate genes, some of which could be verified by experiments.

Conclusions/Significance

This study provides the first comprehensive review of the network and pathway characteristics of schizophrenia candidate genes. Our preliminary results suggest that this systems biology approach might prove promising for selection of candidate genes for complex diseases. Our findings have important implications for the molecular mechanisms for schizophrenia and, potentially, other psychiatric disorders.