Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand

The genome of the human immunodeficiency virus (HIV) is known to contain eight open reading frames (ORFs) on the minus strand of the double-stranded DNA replicative intermediate. Data presented here indicate that the DNA plus strand of HIV contains a previously unidentified ORF in a region complementary to the envelope gene sequence. This ORF could encode a protein of approximately 190 amino acid residues with a relative molecular mass of 20 kilodaltons if translation began from the first initiation codon. The predicted protein is highly hydrophobic and thus could be membrane associated. It is possible, therefore, that the HIV genome encodes a protein on antisense messenger RNA.     

香蕉多酚氧化酶基因编码序列的电子克隆 和生物信息学分析

用电子克隆方法获得香蕉多酚氧化酶（polyphenol oxidase,PPO）基因编码序列。采用生物信息学方法,对此序列编码的蛋白质从氨基酸组成、理化性质尧疏水性/亲水性、亚细胞定位、高级结构及功能域等方面进行了预测和分析。结果表明：香蕉PPO 基因编码序列全长1602bp,包含1 602 bp开放阅读框（open reading frame, ORF）,含有PPO功能域和酪氨酸酶功能域,在序列组成、高级结构及活性位点等方面,与小麦等其他植物的PPO 基因具有高度的相似性。     

Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel

The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.     

IV型戊型肝炎病毒ORF3蛋白在人肝细胞中相互作用蛋白的筛选与鉴定

戊型肝炎病毒编码3个开放性阅读框:ORF1,ORF2和ORF3,其中ORF3蛋白的功能目前尚不明确。为了进一步了解其功能,研究构建了ORF3哺乳动物表达载体pcDNA3.1Flag-ORF3,利用免疫共沉淀和LC/MS的方法筛选并鉴定肝细胞中可以和ORF3蛋白相互作用的蛋白。结果显示,成功构建了Ⅳ型戊型肝炎ORF3的重组表达载体,并利用免疫印迹检测其可以在哺乳动物细胞中正常表达ORF3蛋白,在肝癌细胞株HepG2中筛选并鉴定到了7个可能和ORF3相互用的蛋白,并推测其中RPS18的可能性较大,进而推测HEV可能通过ORF3与核糖体直接相互作用,从而影响其所感染的细胞中蛋白的正常翻译过程。本研究为进一步认识戊型肝炎病毒ORF3的功能提供了线索,进而有利于揭示戊型肝炎病毒的致病机理。     

烟草丛顶病毒编码的沉默抑制子初步鉴定

 烟草丛顶病毒（Tobacco bushy top virus,TBTV）为幽影病毒属成员,能在烟草上造成严重危害。TBTV基因组编码4个ORF,其中ORF3和ORF4几乎完全重叠,可能在TBTV的致病性中发挥重要作用。分别将TBTV的ORF3和ORF4与载体pGR107连接,构建植物表达载体pGR107 ORF3,pGR107 ORF4。经农杆菌与含外源GFP基因的pGREEN208共浸润转GFP基因的本氏烟16c叶片,2～5d后（days post inoculation,dpi)在紫外灯下观察发现浸润区有明显绿色荧光,其中pGR107 ORF4在12dpi后浸润区绿色荧光区域扩大,推断ORF4编码产物可能具有局部抑制本氏烟16c对外源GFP基因进行沉默的功能;而pGR107 ORF3浸润区绿色荧光逐渐恢复红色,GFP基因发生沉默,说明ORF3编码产物不具有抑制基因沉默的功能。进一步利用real time PCR定量检测发现,pGR107 ORF4浸润15 dpi 的本氏烟叶片中GFP RNA的含量要比仅接种pGR107的对照高,比未接种的本氏烟16c稍低。推断是因为ORF4编码蛋白抑制了植物对外源GFP RNA产生的沉默,使GFP得以在本氏烟内表达。由此进一步证实ORF4编码产物是TBTV的基因沉默抑制子。     

Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel.

Modulation of the activity of potassium and other ion channels is an essential feature of nervous system function. The open probability of a large conductance Ca(2+)-activated K+ channel from rat brain, incorporated into planar lipid bilayers, is increased by the addition of adenosine triphosphate (ATP) to the cytoplasmic side of the channel. This modulation takes place without the addition of protein kinase, requires Mg2+, and is mimicked by an ATP analog that serves as a substrate for protein kinases but not by a nonhydrolyzable ATP analog. Addition of protein phosphatase 1 reverses the modulation by MgATP. Thus, there may be an endogenous protein kinase activity firmly associated with this K+ channel. Some ion channels may exist in a complex that contains regulatory protein kinases and phosphatases.     

1990: annus mirabilis of potassium channels

Voltage-gated potassium channels make up a large molecular family of integral membrane proteins that are fundamentally involved in the generation of bioelectric signals such as nerve impulses. These proteins span the cell membrane, forming potassium-selective pores that are rapidly switched open or closed by changes in membrane voltage. After the cloning of the first potassium channel over 3 years ago, recombinant DNA manipulation of potassium channel genes is now leading to a molecular understanding of potassium channel behavior. During the past year, functional domains responsible for channel gating and potassium selectivity have been identified, and detailed structural pictures underlying these functions are beginning to emerge.     

大麦黄矮病毒GPV株系运动蛋白基因原核表达载体的构建

大麦黄矮病毒GPV株系ORF4基因编码一种17 kDa的蛋白,利用一对特异性引物,通过RT-PCR对ORF4基因进行了扩增,序列测定表明其长度为456 bp,与已报道的序列一致,并构建了原核表达载体。     

甘蔗花叶病毒河南分离物HC-Pro基因的序列分析及其RNA沉默表达载体构建

采用RT-PCR方法从河南地区甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)感染的玉米叶片中,克隆了辅助成分-蛋白酶基因(helper component.proteinase,HC-Pro).该基因长1 380 bp,编码460个氨基酸.序列比对表明.HC-Pro编码的氨基酸序列与北京、山东地区SCMV分离物的相似性分别达到97%和98%.而与以前数据库登录的河南分离物的相似性仅98%,说明河南地区SCMV可能发生了变异.选取长582 bp的HC-Pro片段,构建了表达发夹RNA的基因沉默双元表达载体pRNAi-HC-Pro(±),成功转化了农杆菌LBA4404.     

Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels

Voltage-dependent ion channels are responsible for electrical signaling in neurons and other cells. The main classes of voltage-dependent channels (sodium-, calcium-, and potassium-selective channels) have closely related molecular structures. For one member of this superfamily, the transiently voltage-activated Shaker H4 potassium channel, specific amino acid residues have now been identified that affect channel blockade by the small ion tetraethylammonium, as well as the conduction of ions through the pore. Furthermore, variation at one of these amino acid positions among naturally occurring potassium channels may account for most of their differences in sensitivity to tetraethylammonium.     

A family of three mouse potassium channel genes with intronless coding regions

To understand the molecular mechanisms responsible for generating physiologically diverse potassium channels in mammalian cells, mouse genomic clones have been isolated with a potassium channel complementary DNA, MBK1, that is homologous to the Drosophila potassium channel gene, Shaker. A family of three closely related potassium channel genes (MK1, MK2, and MK3) that are encoded at distinct genomic loci has been isolated. Sequence analysis reveals that the coding region of each of these three genes exists as a single uninterrupted exon in the mouse genome. This organization precludes the generation of multiple forms of the protein by alternative RNA splicing, a mechanism known to characterize the Drosophila potassium channel genes Shaker and Shab. Thus, mammals may use a different strategy for generating diverse K+ channels by encoding related genes at multiple distinct genomic loci, each of which produces only a single protein.     

传染性法氏囊病病毒河南分离株（HN04）A片段cDNA的克隆及序列分析

利用RT-PCR技术,以传染性法氏囊病毒河南分离株(HN04)基因组RNA为模板,扩增并克隆IBDV HN04株基因组A片段cDNA。测序结果表明:克隆的A片段全长3260个核苷酸,包括2个部分重叠的开放阅读框(ORFl和ORF2)及两端的非编码区。ORFl和ORF2分别编码含有1012个氨基酸的结构蛋白(VP2-4-3)及含有145个氨基酸的VP5。IBDV HN04株基因组A片段核苷酸序列与来自GenBank IBDV血清Ⅰ型毒株核苷酸序列的同源性高达94.9%～99.4%,血清Ⅱ型毒株核苷酸序列同源性为83.8%。对IBDV HN04株的A片段核苷酸及其推导氨基酸进行序列分析,结果显示HN04株与国内外IBDV数株弱毒株的同源性在99.0%以上。     

The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide

Bovine papillomavirus (BPV-1) contains two independent transforming genes that have been mapped to the E5 and E6 open reading frames (ORF's). The E5 transforming protein was identified by means of an antiserum against a synthetic peptide corresponding to the 20 COOH-terminal amino acids of the E5 ORF. The E5 polypeptide is the smallest viral transforming protein yet characterized; it had an apparent size of 7 kilodaltons. The transforming polypeptide is encoded entirely within the second half of the E5 ORF and its predicted amino acid composition is very unusual; 68% of the amino acids are strongly hydrophobic and 34% are leucine. Cell fractionation studies localized this polypeptide predominantly to cellular membranes.     

油葵油酸去饱和酶基因的序列分析

该研究利用生物信息学软件对克隆得到的油葵基因fad2序列进行核苷酸结构和蛋白质结构分析,结果表明油葵中fad2基因编码382个氨基酸,含有完整的ORF框;其功能域为△-12脂肪酸去饱和酶类结构域,含有3个保守的组氨酸簇;蛋白质二级结构中包含14个α螺旋,7个β折叠,此外还含有17个具有二级结构趋势的无规则卷曲;蛋白质羧基末端以YXXKI结尾,符合Phi-X-X-K/R/D/E-Phi-COOH基序序列规则,是内质网滞留信号;从系统发育进化的角度可知油葵与橄榄的亲缘关系较近。     

Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR

Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.     

橡胶树乳管HbPMP1基因的克隆与表达分析

过氧化物酶体膜蛋白PMP是ABC转运蛋白ABCD亚家族蛋白之一．存在于过氧化物酶体膜上。研究表明．拟南芥中的ABCD亚家族ABC转运蛋白AtABCD1主要通过参与部分物质运输进入过氧化物酶体．其中包括茉莉酸合成的前体OPDA。本研究通过RACE技术克隆了一个ABCD亚家族ABC转运蛋白基因HbPMP1．并进行表达分析。结果表明：HbPMP1全长4011bp．编码1337个氨基酸残基,其表达产物与拟南芥AtABCDl有较高的相似性（氨基酸一致性为78％）：该基因在伤害及外源茉莉酸诱导下呈上调表达．推测其可能参与内源茉莉酸合成相关物质时的运输．     

木尔坦棉花曲叶病毒“C4 ORF”编码蛋白对病毒致病性的影响

【目的】通过分析发现木尔坦棉花曲叶病毒（cotton leaf curl Multan virus,CLCuMuV）C4开放阅读框（open reading frame,ORF）附近可编码3个不同大小的蛋白,而NCBI数据库登录的CLCuMuV各个分离物编码的C4蛋白大小也不尽相同,本研究试图通过分析该3个假定的“C4 ORF”编码蛋白对病毒致病性的影响,以明确CLCuMuV C4蛋白ORF的确切定位。【方法】根据双生病毒编码蛋白的保守性,在CLCuMuV C4 ORF附近查找到3个大小不等的ORF,分别标记为C4-L（567 nt）、C4-M（546 nt）和C4-S（303 nt）。利用同源重组的方法将该3个ORF分别构建到PVX异源表达载体,通过农杆菌介导的植物接种法,分析PVX介导的3个蛋白在本氏烟表达对本氏烟症状的影响。利用同源重组的方法构建CLCuMuV野生型及C4-L或C4-S缺失突变型的侵染性克隆,同样通过农杆菌介导的植物接种法,分别与其伴随DNA-β分子的侵染性克隆接种本氏烟,分析C4-L或C4-S缺失对CLCuMuV致病性的影响,并利用Southern blot和Western blot分别对接种烟草的病毒基因组的积累量和病毒C4蛋白的表达量进行分析。同时利用Gateway系统载体对C4-L和C4-S蛋白在本氏烟叶片表皮细胞中的亚细胞定位进行分析。【结果】PVX异源表达载体接种本氏烟,结果发现PVX-C4-L和PVX-C4-S接种的本氏烟叶片卷曲、叶柄伸长,并且PVX-C4-S症状更重,而PVX-C4-M接种的本氏烟症状较轻或基本无症状,Western blot试验也在PVX-C4-L和PVX-C4-S接种组检测到了C4蛋白的表达,说明C4-S ORF编码的蛋白对PVX异源病毒的致病性影响最大。侵染性克隆接种本氏烟,结果发现野生型（CLCuMuV）和C4-L突变型（CLCuMuV-ΔL）接种的本氏烟叶片皱缩增生、叶柄和茎秆扭曲,而C4-S突变型（CLCuMuV-ΔS）和对照组（Mock）接种的本氏烟未显示任何症状,并且前两者的植株高度明显矮于后两者,同时Southern blot试验在CLCuMuV和CLCuMuV-ΔL接种的本氏烟中均检测到大量病毒基因组的积累,而Western blot试验也在其中检测到C4蛋白的表达,说明C4-S ORF编码的蛋白在CLCuMuV诱导寄主产生症状过程中起关键作用。亚细胞定位发现YFP-C4-L主要定位在本氏烟叶片表皮细胞的叶绿体,YFP-C4-S则定位在细胞膜或细胞质周边,并有点状聚集体结构。【结论】C4-S ORF编码的蛋白对CLCuMuV的侵染必不可少,而C4-L和C4-M ORF编码的蛋白对CLCuMuV的侵染不是必需。     

枸杞LbMYB1基因的克隆与表达分析

在前期的枸杞花药蛋白质组学研究鉴定差异表达蛋白的基础上,采用RT-PCR技术,从宁夏枸杞"宁杞1号"花药中克隆了一个花药发育差异表达MYB类蛋白基因。生物信息学分析表明,该基因编码R2R3类MYB转录因子的典型结构域,与其他物种MYB蛋白的氨基酸的相似性达72%以上,属于R2R3类MYB蛋白基因家族。将该基因命名为Lb MYB1,Lb MYB1包含一个975 bp的开放阅读框,编码324个氨基酸残基,分子量79.2 kv,等电点为4.95。实时定量RT-PCR检测表明,Lb MYB1基因在花药四分体形成时期高丰度表达,在花中的表达量也较高,果实中表达量较低,在根、茎及叶中未检测到表达,推测Lb MYB1基因可能与花药发育中有关。本研究为后继进一步探讨Lb MYB1蛋白在枸杞花药发育过程中可能发挥的调控功能及其分子机理提供参考。     

Channelrhodopsin-1: a light-gated proton channel in green algae

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.     

K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse

The Drosophila Shaker gene on the X chromosome has three sister genes, Shal, Shab, and Shaw, which map to the second and third chromosomes. This extended gene family encodes voltage-gated potassium channels with widely varying kinetics (rate of macroscopic current activation and inactivation) and voltage sensitivity of steady-state inactivation. The differences in the currents of the various gene products are greater than the differences produced by alternative splicing of the Shaker gene. In Drosophila, the transient (A current) subtype of the potassium channel (Shaker and Shal) and the delayed-rectifier subtype (Shab and Shaw) are encoded by homologous genes, and there is more than one gene for each subtype of channel. Homologs of Shaker, Shal, Shab, and Shaw are present in mammals; each Drosophila potassium-channel gene may be represented as a multigene subfamily in mammals.