中国番茄黄化曲叶病毒编码的RNA沉默抑制子AC4蛋白的核酸结合特性

农杆菌共浸润试验结果表明:中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)Y10分离物编码AC4蛋白是RNA沉默抑制子.为了研究RNA沉默抑制子AC4蛋白的作用机制,将TYLCCNV-Y10的AC4基因插入到原核表达载体pET-32a的多克隆位点上,构建重组原核表达载体pET32a-YIOACA.将重组载体导入BL21(DE3)pLysS进行ACA蛋白表达,并在自然条件下纯化蛋白.利用原核表达的ACA蛋白进行电泳迁移率变动试验(electrophoretic mobility shift assay,EMSA),分析ACA蛋白分别与单、双链siRNA和单、双链长RNA的结合情况.试验结果表明:TYLCCNV-Y10编码的ACA蛋白具有结合单链siRNA和单链长RNA特性,而不与双链siRNA和双链长RNA结合.     

甘蔗黄叶病毒P0蛋白分子特性及其抑制RNA沉默活性

【目的】甘蔗黄叶病（yellow leaf,YL）是一种主要的甘蔗病毒病害,在全球多数甘蔗种植国家或地区普遍发生,已对甘蔗生产造成严重威胁。其病原为甘蔗黄叶病毒（Sugarcane yellow leaf virus,SCYLV）,属于黄症病毒科马铃薯卷叶病毒属成员。研究SCYLV编码的沉默抑制子P0蛋白的分子特征、保守结构域及其在自然寄主甘蔗上抑制RNA沉默的功能,为下一步从RNA沉默水平解析SCYLV致病分子机理奠定基础。【方法】利用RT-PCR克隆技术获得SCYLV中国分离物CHN-FJ4编码的RNA沉默抑制子基因P0及其两个缺失突变体P0^Δ2-15（N端缺失15个氨基酸）和P0^Δ155-256（C端缺失102个氨基酸）,然后定向克隆到单子叶植物表达载体pUbi-nos（空载体）上,获得重组质粒。利用甘蔗嫩叶组织瞬时表达系统鉴定病毒RNA沉默抑制子技术,结合ImageJ软件定量分析P0及其两个缺失突变体抑制外源基因EYFP瞬时表达活性的变化。番茄丛矮病毒（Tomato bushy stunt virus,TBSV）编码的P19作为沉默抑制子阳性对照。【结果】P0核苷酸序列的遗传进化分析结果显示,所获得的SCYLV病毒分离物CHN-FJ4为BRA基因型,与其他BRA基因型分离物氨基酸序列一致性为96.1%-98.4%。利用MEME在线软件预测P0蛋白的保守结构域,结果表明P0蛋白含有3个显著的保守区。分别位于第1-60、76-125和161-210氨基酸残基。通过Datamonkey在线服务器（http://www.datamonkey.org）提供的5种运算方法分析P0蛋白氨基酸位点的选择压力,结果显示P0蛋白具有8个氨基酸正向选择位点。将含有P0及其缺失突变体的重组质粒连同含EYFP报告基因的pTEM12质粒采用基因枪轰击法分别导入甘蔗嫩叶组织细胞,轰击后48-120 h,P0和P19逐渐地提高EYFP荧光表达点数和荧光表达水平;在120 h,与pUbi-nos空载体（不含沉默抑制子）对照组相比,P0和P19均显著地提高甘蔗嫩叶组织EYFP荧光表达点数和荧光表达水平,分别达1.6倍和4.0倍以上。P0与P19抑制RNA沉默活性水平没有显著差异（P>0.05）。P0蛋白N端缺失15个氨基酸（P0^Δ2-15）和C端缺失102个氨基酸（P0^Δ155-256）均丧失了沉默抑制子的功能,荧光表达与不含沉默抑制子对照组相当。【结论】在甘蔗嫩叶组织EYFP瞬时表达体系上,P0能够显著地提高外源基因EYFP表达水平。P0蛋白N端15个氨基酸和C端102个氨基酸含有显著的保守区域,是P0发挥沉默抑制子功能所必需的。     

南方水稻黑条矮缩病毒S6编码一个沉默抑制子

 【目的】分析南方水稻黑条矮缩病毒（SRBSDV）基因组S6编码的SP6蛋白的抑制子活性,明确SRBSDV是否编码RNA沉默抑制子来干扰植物的沉默。【方法】将分别含有SP6与GFP质粒的农杆菌共浸润转GFP基因的16c本氏烟纯合系,观察SP6对局部沉默和系统沉默的抑制作用;将含有SP6,GFP和dsGFP质粒的农杆菌三者共浸润,观察SP6对由dsRNA引起的沉默的抑制作用;在同一植株不同部位接种GFP和SP6,观察SP6对RNA沉默信号传导的影响;通过马铃薯X病毒（Potato virus X,PVX）在本氏烟上表达SP6,观察SP6是否能增强PVX的致病性。【结果】SP6能抑制由GFP正义RNA介导的沉默,但其抑制作用较弱,仅能延缓局部沉默和系统沉默的产生。SP6能灭活RNA沉默信号,阻止沉默信号的长距离传导,回复GFP的沉默,但不能抑制由dsRNA引起的沉默。利用PVX在本氏烟上表达SP6能增强PVX的致病性。【结论】SP6是病毒编码的RNA沉默抑制子,在RNA沉默的起始和信号传导阶段起作用。     

Reading frame selection and transfer RNA anticodon loop stacking

Messenger RNA's are translated in successive three-nucleotide steps (a reading frame), therefore decoding must proceed in only one of three possible frames. A molecular model for correct propagation of the frame is presented based on (i) the measured translational properties of transfer RNA's (tRNA's) that contain an extra nucleotide in the anticodon loop and (ii) a straightforward concept about anticodon loop structure. The model explains the high accuracy of reading frame maintenance by normal tRNA's, as well as activities of all characterized frameshift suppressor tRNA's that have altered anticodon loops.     

Collagen synthesis in Xenopus oocytes after injection of nuclear RNA of frog embryos

A messenger RNA fraction from polysomes of frog larvae or RNA preparations from isolated nuclei of developing frog embryos were injected into growing Xenopus laevis oocytes that were incubated with labeled proline. Column chromatography of protein hydrolyzates revealed labeled hydroxyproline after injection of the messenger RNA fraction and neurula nuclear RNA, indicating that the injected material had promoted collagen synthesis.     

53BP1, a mediator of the DNA damage checkpoint

53BP1 binds to the tumor suppressor protein p53 and has a potential role in DNA damage responses. We used small interfering RNA (siRNA) directed against 53BP1 in mammalian cells to demonstrate that 53BP1 is a key transducer of the DNA damage checkpoint signal. 53BP1 was required for p53 accumulation, G2-M checkpoint arrest, and the intra-S-phase checkpoint in response to ionizing radiation. 53BP1 played a partially redundant role in phosphorylation of the downstream checkpoint effector proteins Brca1 and Chk2 but was required for the formation of Brca1 foci in a hierarchical branched pathway for the recruitment of repair and signaling proteins to sites of DNA damage.     

Mutational analysis of the tyrosine phosphatome in colorectal cancers

Tyrosine phosphorylation, regulated by protein tyrosine phosphatases (PTPs) and kinases (PTKs), is important in signaling pathways underlying tumorigenesis. A mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs (PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, PTPN14), affecting 26% of colorectal cancers and a smaller fraction of lung, breast, and gastric cancers. Fifteen mutations were nonsense, frameshift, or splice-site alterations predicted to result in truncated proteins lacking phosphatase activity. Five missense mutations in the most commonly altered PTP (PTPRT) were biochemically examined and found to reduce phosphatase activity. Expression of wild-type but not a mutant PTPRT in human cancer cells inhibited cell growth. These observations suggest that the mutated tyrosine phosphatases are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.     

甘蓝脯氨酸脱氢酶基因克隆与RNAi表达载体构建

通过RT-PCR、同源克隆和RACE等方法由甘蓝总RNA扩增得到了甘蓝脯氨酸脱氢酶基因cDNA全长(1 719 bp),其中包含了一个1 497 bp的完整开放阅读框,编码498个氨基酸,与已发表的十字花科植物ProDH基因均具有85%以上的同源性。在此基础上设计并克隆干扰片段,利用酶切连接的方法将该基因干扰片段正反向插入到载体pFGC-1008的GUS内含子两侧,经限制性内切酶酶切和测序鉴定,证明植物表达载体pFGC-gPDH已构建成功,为进一步研究该基因的功能创造了条件。     

植物病毒编码RNA沉默抑制子的研究进展

RNA沉默是真核生物用来抵抗病毒入侵的一种普遍而又古老的防御机制，而病毒在进化过程中也相应地产生了一些与之抗衡的功能，其中编码沉默抑制子就是对抗RNA沉默的有效策略。它们分别能够在不同阶段，以不同的方式干扰来自于寄主的RNA沉默，从而有效地建立侵染。本文主要针对目前已经发现的由植物病毒编码RNA沉默抑制子的作用特征、鉴定方法以及作用副效应进行综述，并讨论了RNA沉默抑制子的研究及应用前景。     

Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling

The ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) plays a central role in the cellular response to changes in oxygen availability. pVHL binds to HIF only when a conserved proline in HIF is hydroxylated, a modification that is oxygen-dependent. The 1.85 angstrom structure of a 20-residue HIF-1alpha peptide-pVHL-ElonginB-ElonginC complex shows that HIF-1alpha binds to pVHL in an extended beta strand-like conformation. The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. Although the beta sheet-like interactions contribute to the stability of the complex, the hydroxyproline contacts are central to the strict specificity characteristic of signaling.     

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

Construction and recovery of viable retroviral genomes carrying a bacterial suppressor transfer RNA gene

The integration of retroviral genomes into cellular DNA can induce mutations by altering the expression of nearby cellular genes and can serve to identify the gene affected. The construction of a retrovirus that stably carries a suppressor transfer RNA gene from Escherichia coli has allowed facile recovery of the viral genome in vectors marked with amber mutations. This virus can be used for rapid isolation of cellular sequences at the site of proviral insertion.     

Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping

Most mutations in the dystrophin gene create a frameshift or a stop in the mRNA and are associated with severe Duchenne muscular dystrophy. Exon skipping that naturally occurs at low frequency sometimes eliminates the mutation and leads to the production of a rescued protein. We have achieved persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA of the mdx mouse, by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. We report the sustained production of functional dystrophin at physiological levels in entire groups of muscles and the correction of the muscular dystrophy.     

Measurement of suppressor transfer RNA activity

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.     

几种水稻病毒非结构蛋白研究进展

对水稻病毒编码的病毒基质、管状蛋白、逆转录酶、基因沉默抑制子、核酸结合活性蛋白、转录调节功能蛋白6种非结构蛋白及其功能进行了介绍,为发现水稻病毒的潜在药物靶标以及研究病毒与寄主互作关系提供了参考。     

Suppressor T cell action inhibits the expression of an excluded immunoglobulin gene

Cells of the murine plasmacytoid line MOPC-315 synthesize two distinct immunoglobulin light chains: a normal lambda II protein, which is incorporated into secretory and surface-bound immunoglobulin, and a truncated, nonfunctional lambda I protein found only in the cytoplasm. Idiotype-specific suppressor T lymphocytes selectively inhibit the expression of both lambda II- and lambda I-specific messenger RNA by MOPC-315 cells. This finding demonstrates that phenotypically excluded light chain genes can be subject to immunoregulatory control and suggests that the expression of divergent lambda isotypes may be coordinately regulated in immunoglobulin-secreting cells.     

盐胁迫对甘薯叶片氮代谢的影响

以胜利百号、栗子香、徐薯１８等３个甘薯品种为材料,对盐胁迫下甘薯氮代谢进行了研究．结果显示,盐胁迫下甘薯叶片总氮含量下降,游离氨基酸迅速累积,叶绿素含量下降,ＲＮＡ及ＤＮＡ降解、ＲＮＡ／ＤＮＡ值下降,游离脯氨酸迅速累积,硝酸还原酶活力下降且伴随着其底物（ＮＯ３－）累积和产物（ＮＯ２－）下降．说明盐胁迫下甘薯氮代谢系统失调是造成其植株失绿变黄,并最终导致其减产的主要原因之一．     

Azure mutants: a type of host-dependent mutant of the bacteriophage f2

A new type of host-dependent mutant, azure mutant, of bacteriophage f2 has been isolated. Growth of these mutants was restricted specifically by amber suppressor genes in the host bacteria. Restriction of the formation of infective centers by different bacterial suppressor genes was 98 percent, 90 percent, and 70 percent with Su-3, Su-1, and Su-2 genes, respectively. Restriction, like suppression, was the dominant phenotype. The block in growth of the mutants occurred in an early stage of the infection cycle. Once infection was established, however, an infected cell produced approximately the same number of progeny phage as a cell without the suppressor genes did. It is proposed that the azure codon is the same as the amber codon (uracil, adenine, guanine) and that restriction results from improper termination of protein chains of the phage RNA polymerase. Similar mutants may exist in other systems.