Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.     

半滑舌鳎GAPDH基因开放读码框的克隆及序列分析

甘油醛-3-磷酸脱氢酶(GAPDH)广泛存在于真核生物中,对许多生理活动具有重要作用.参考金头鲷和斑马鱼的甘油醛-3-磷酸脱氢酶(GAPDH)基因序列设计一对简并引物,提取半滑舌鳎肌肉组织总RNA,采用RT-PCR技术对GAPDH基因进行克隆测序及序列分析.结果表明,从半滑舌鳎肌肉组织中成功克隆了GAPDH基因的开放读码框(ORF),序列全长1002 bp,编码333个氨基酸,分子量为36.0235 ku,等电点PI为7.75.序列同源性分析显示,该序列与其他鱼类的GAPDH氨基酸序列具有较高的同源性.     

Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs

Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.     

Reversal of the TCR stop signal by CTLA-4

The coreceptor cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is pivotal in regulating the threshold of signals during T cell activation, although the underlying mechanism is still not fully understood. Using in vitro migration assays and in vivo two-photon laser scanning microscopy, we showed that CTLA-4 increases T cell motility and overrides the T cell receptor (TCR)-induced stop signal required for stable conjugate formation between T cells and antigen-presenting cells. This event led to reduced contact periods between T cells and antigen-presenting cells that in turn decreased cytokine production and proliferation. These results suggest a fundamentally different model of reverse stop signaling, by which CTLA-4 modulates the threshold for T cell activation and protects against autoimmunity.     

利用mRNA差异显示技术克隆恶疫霉侵染诱导表达的草莓基因

为了解草莓与恶疫霉互作的分子机制,以接种后3、5和7 d的感病草莓品种‘FDP821’的冠部为材料,利用mRNA差异显示技术研究‘FDP821’与恶疫霉亲和互作前后基因表达的差异,获得78条差异表达的cDNA片段,通过分析从中分离出恶疫霉侵染诱导表达的10个草莓基因cDNA片段。BLASTX在线分析表明,这些草莓基因的功能涉及呼吸作用、衰老和核苷运输与代谢等。为阐明草莓与恶疫霉互作的分子机制提供了重要的线索。     

URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.     

外源褪黑素诱导下Hippo信号通路调控山羊绒生长mRNA和lncRNA的生物信息学分析

为分析绒山羊皮肤中受Hippo信号通路调控的绒生长候选基因集,本研究选取6只罕山白绒山羊随机分为对照组和褪黑素处理组,每组设3个重复。以自然年为试验周期,每月采集皮肤样本进行RNA测序,使用Bowtie、TopHat、Cufflinks、Cuffmerge、Cuffdiff、Cuffcompare、CPAT和CPC软件分析DE-mRNA和DE-lncRNA,联合PCA分析它们对次级毛囊生长周期的应答,利用WGCNA挖掘调控山羊绒生长的基因模块,GSEA分析候选基因集的生物学功能。结果表明：1)筛选得到组间DE-mRNA 2 024个和DE-lncRNA 329个,它们应答了绒山羊皮肤次级毛囊的生长周期。2)获得有生物学意义的对照组7个和试验组9个基因模块。3)深入分析富集到平衡哺乳动物皮肤生长分化、毛囊形态发生的Hippo信号通路基因模块,揭示了外源褪黑素诱导下调控山羊绒生长候选基因集191个mRNA和49个lncRNA的共表达调控网络关系。4)发现候选基因集与皮肤发育生物学过程(|NES|>1且FDR<25%)、Hippo信号通路(|NES|>1且FDR<25...     

Initial function determination for the open reading frame (ORF) region of Pib gene via rice transformation

Three plant binary expression vectors—pNAR501, pNAR502 and pNAR503—were constructed, carrying fragments of exon2-exon3, 5′partial deletion exon1 and 5′partial deletion exon1-exon2-exon3 of Pib gene driven by 35S. These three vectors were transformed into the japonica rice variety Nipponbare through agrobacterium-mediated transformation. More than 30 transgenic rice plants were obtained and confirmed by polymerase chain reaction (PCR), Southern hybridization and the hygromycin resistance test in seed germination of their progeny. A rice blast resistance test for in vitro leaves of T_o transgenic plants in the tillering stage showed higher resistance to the races of E1, F1 and G1 of rice blast than that of the control Nipponbare. However, results of rice blast resistance test for seedlings of T₁ transgenic plants in the 3-to 4-leaf stage were different. All T₁ transgenic seedlings had a lower level of resistance to E1, F1 and G1 races than that of the control Nipponbare. Translated from Journal of Nanjing Agricultural University, 2006, 29(3): 1–5 [译自: 南京农业大学学报]     

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.     

Memory disruption by electrical stimulation of substantia nigra, pars compacta

Electrical stimulation of the substantia nigra, pars compacta, of albino rats while they were learning a simple foot shock task of withdrawal and response suppression disrupted retention of that task 24 hours after original learning. Stimulation in the reticular zone of the substantia nigra was without effect on retention performance. Stimulation through electrodes in the medial lemniscus, red nucleus, or brainstem regions surrounding the substantia nigra, pars compacta, was also ineffective. Original learning performance, measured as time to criterion, was unimpaired by the stimulation. Posttrial stimulation in the substantia nigra, pars compacta, but not in adjacent structures, also disrupted retention performance.     

牛至香酚对LPS所致的免疫应激小鼠脾脏细胞因子及核转录因子mRNA表达的影响

将80只小鼠随机分成5组(空白对照组、模型组及牛至香酚低、中、高剂量组),牛至香酚低、中、高剂量组小鼠分别按25、50、100 mg·kg~(-1)的剂量灌胃牛至香酚,空白对照组、模型组灌胃相同体积的橄榄油,每日1次,连续14 d.第15天时,牛至香酚试验组、模型组小鼠腹腔注射8 mg·kg~(-1)脂多糖(LPS),6 h后采集脾脏组织,用qRT-PCR检测小鼠脾脏细胞因子(TNF-α、IFN-γ、IL-6、IL-10、IL-1β)、NF-κB及核转录因子(T-bet、GATA-3)mRNA的表达量,研究牛至香酚对LPS所致的免疫应激小鼠脾脏细胞因子及核转录因子mRNA表达的影响.结果表明:与空白对照组相比,模型组小鼠TNF-α、IFN-γ、IL-6、IL-10、IL-1β、NF-κB、T-bet mRNA的表达量提高(P<0.01),GATA-3 mRNA的表达量降低(P<0.01);与模型组相比,牛至香酚低、高剂量组IL-1β mRNA的表达量降低(P<0.01),低、中、高剂量组IL-6、TNF-α mRNA的表达量降低(P<0.01、P<0.05),低、中、高剂量组IFN-γ mRNA的表达量极显著或显著降低(P<0.01或P<0.05),高剂量组NF-κB mRNA的表达量降低(P<0.05),低、中、高剂量组T-bet mRNA的表达量降低(P<0.05),中剂量组GATA-3 mRNA的表达量提高(P<0.05).由本试验结果可知,牛至香酚通过提高或降低小鼠脾脏细胞因子、核转录因子的表达,调节免疫应激小鼠的免疫功能.     

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.     

利用转录组测序技术分析ADC和NOS基因mRNA在鹅等级前卵泡中的表达

为研究精氨酸代谢的关键酶精氨酸脱羧酶(Arginine Decarboxylase,ADC)和一氧化氮合酶(Nitric-oxide Synthase,NOS)mRNA在鹅等级前卵泡中的表达情况。采用高通量测序技术对鹅等级前卵泡进行转录组测序分析,并通过荧光定量PCR对结果进行验证。通过FPKM值计算分析,这2个基因在鹅等级前卵泡中均有表达,且随着卵泡的发育,NOS mRNA的表达呈现先降低后增加再降低的趋势,在初级卵泡中的表达量达到最高值,在中白卵泡中的表达量最低。ADC mRNA的表达则呈现先增加后降低再增加的趋势,在初级卵泡中的表达量最低,在小白卵泡中的表达量最高。荧光定量PCR结果与转录组测序结果基本相同。     

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.     

Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges

The recently identified plant photoreceptor UVR8 (UV RESISTANCE LOCUS 8) triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light through an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan "pyramid" responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine retunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.     

露地瓠瓜无公害搭架栽培技术

从地块选择、科学育苗、移栽、田间管理、病虫害防治、适时收获、留种管理等方面总结露地瓠瓜无公害搭架栽培技术。     

以图书馆为主导的全民阅读合作模式研究

开展全民阅读活动,已逐渐成为中国的一项基本公共文化政策,然而,这是一个复杂的社会系统工程,只有社会各界鼎力合作才能达到良好的效果,图书馆作为传承文化、传递知识的公共文化服务平台,有责任发挥自身优势,创新服务模式,与社会各界积极合作,共同推动全民阅读,为全民阅读的良好风气在全国推行做出自己的努力。