马铃薯Y病毒pipo基因的分子变异及结构特征分析

为揭示马铃薯Y病毒(Potato virus Y, PVY) pipo基因的分子变异和结构特征, 文章根据文献报道的马铃薯Y病毒属(Potyvirus) pipo 基因保守区序列设计一对简并引物, 从感染PVY的马铃薯病叶中克隆获得pipo基因的cDNA全长序列, 分析其核苷酸序列和氨基酸序列的特征, 并基于氨基酸序列使用贝叶斯法重建了Potyvirus的系统发育树。结果显示：20个PVY分离物成功扩增出预期大小(约235 bp)的特异性片段, 其核苷酸序列与已报道的其它PVY 株系的pipo基因核苷酸序列一致性均在92%以上; 5′端均含有典型的G1-2A6-7 基序(motif), 无碱基插入/缺失, 所有的核苷酸变异都是碱基置换, 共发现13个多态性位点, 其中4个简约信息位点, 9个单一变异位点, 表明该基因高度保守, 但不同分离物也存在一定的分子变异; PIPO蛋白理论等电点11.26~11.62, 无信号肽和跨膜区, 是可溶的亲水性蛋白; 整个蛋白含有3个保守区, 其中位于10~59aa的基序最为保守。该蛋白主要定位于线粒体中, 可能是线粒体导肽。系统发育分析结果显示, 源于PVY不同株系优先相聚成簇, 而向日葵褪绿斑驳病毒(Sunflower chlorotic mottle virus, SuCMoV)与辣椒重花叶病毒(Pepper severe mosaic virus, PepSMV)的亲缘关系较PVY相比更近, 与前人的结果相一致, 表明PIPO蛋白可以作为研究Potyvirus系统发育关系的新的分子标记。     

植原体tuf基因与其上游部分基因结构和相关基因启动子保守区域特征及活性分析

植原体寄主种类多, 危害范围广, 开展其遗传多样性、关键基因调控等方面研究有助于提高该病害综合防治水平。通过长片段PCR引物扩增我国PaWB-sdyz、PaWB-fjfz和LY-fjya1植原体株系tuf基因及其上游6个基因的片段, 进行植原体基因启动子保守区域序列特征和多位点序列分析。利用启动子探针载体pSUPV4检测植原体tuf基因上游序列的启动子活性。扩增获得PaWB-sdyz、PaWB-fjfz、LY-fjya1株系tuf基因上游12,745-12,748 bp序列, 比较分析发现PaWB-sdyz、PaWB-fjfz、LY-fjya1、OY-M、AYWB、PAa、SLY、AT植原体株系tuf与其上游6个基因的结构顺序皆为5’-rplL-rpoB-rpoC-rps12-rps7-fusA-tuf-3’。推测出可能的植原体启动子保守区域模式序列: T₉₀T₁₀₀G₉₂T₇₅G₆₇A₈₅ (-35区); T₉₀A₉₆T₉₂A₉₈T₇₃T₉₀ (-10区)。基于8个植原体株系的rplL-tuf核苷酸序列编码基因、非编码序列、氨基酸序列的多位点序列分析可将不同植原体株系以较高的支持率清晰地区分, 不同植原体株系rplL-tuf核苷酸非编码区变异水平更高。16SrI组植原体tuf基因上游序列存在3种变异类型, 其代表株系PaWB-fjfz、LY-fjya1 tuf基因上游130 bp片段和CWB-hnsy1 tuf基因上游129 bp片段皆具有启动子活性。     

马铃薯Y病毒多基因系统发育分析及其在株系鉴定中的应用

为实现马铃薯Y病毒(Potato virus Y, PVY)常见株系的快速鉴定,本文以PVY的P1、HC-pro、VPg和CP 4个基因为研究对象建立了快速准确的多基因联合体系。根据基因的不同组合建立5个不同数据集,分别进行系统发育分析,并通过贝叶斯标签关联显著性(Bayesian tip-association significance, BaTS)分析各数据集中代表分离物与株系的关联性,以确定实现PVY快速鉴定的最佳组合。不同数据集的系统发育及BaTS分析结果显示,除了联合P1、VPg和CP 3个基因数据集外,其他4个数据集均无法实现PVY常见株系的准确鉴定。采用不同建树方法对联合P1、VPg和CP 3个基因数据集比较分析显示,基于ML法和NJ法的系统发育树在拓扑结构上基本一致,均优于基于贝叶斯算法的最大分支置信(maximum clade credibility, MCC)树。同时,以HLJ26分离物为研究对象,对建立的多基因联合体系进行实际应用,结果显示该分离物与PVY^NTN-NW株系的3个分离物SYR-Ⅱ-2-8、SYR-Ⅱ-Be1和SYR-Ⅱ-DrH以高置信值聚为一亚簇,表明该分离物可能属于PVY^NTN-NW株系(SYR-Ⅱ型)。重组分析显示,HLJ26基因组存在4个潜在的重组信号,分别位于P1、HC-pro/P3、VPg和CP的5°-末端,与PVY^NTN-NW株系(SYR-Ⅱ型)的重组位点相一致,表明其属于PVY^NTN-NW株系(SYR-Ⅱ型)。同时,应用多重RT-PCR成功扩增出约为1000 bp和400 bp的2个特异性片段,与PVY^NTN-NW株系(SYR-Ⅱ型)的特异条带大小相一致。这些结果进一步支持了多基因联合体系的鉴定结果。联合P1、VPg和CP 3个基因数据集系统发育分析,可以实现PVY常见株系的准确鉴定。     

不同寄主来源的马铃薯Y病毒群体遗传结构的比较分析

文章以马铃薯Y病毒(Potato virus Y, PVY) P3和pipo基因为分子标记,比较分析烟草和马铃薯两个寄主的PVY遗传多样性和群体分化,并评估突变、选择、基因流等遗传力所起的作用。通过P3和pipo基因计算获得的烟草和马铃薯群体分化指数F_ST分别为0.116和0.120,且统计检验差异显著,表明烟草和马铃薯寄主的PVY之间中度分化。变异分析结果显示,烟草分离物P3和pipo基因的核苷酸序列一致性分别为85.2%~100%和76.5%~100%,而马铃薯分离物的P3和pipo基因的核苷酸序列一致性分别为95.7%~100%和93.0%~100%,表明烟草PVY变异程度高于马铃薯。同时,P3基因内检测到大量的净化选择位点,表明该基因大部分位点的变异为有害突变,在进化过程中被剔除。此外,P3基因内还检测到两个显著正向选择位点,表明这两个位点的变异为有益突变,有利于病毒的生存竞争。在pipo基因中未检测到显著的选择位点,表明该基因上的变异基本不受自然选择影响。通过P3和pipo基因计算烟草和马铃薯群体间的基因流Nm值分别为1.91和1.83,表明这两个群体间存在较强的基因交流。以上结果表明,来源于烟草和马铃薯寄主的PVY遗传差异显著,突变、自然选择以及基因流都影响两者的遗传多样性及遗传分化程度。     

全基因组序列的系统发育与重组分析鉴定我国马铃薯Y病毒株系

为明确我国马铃薯Y病毒(Potato virus Y,PVY)株系组成及其在不同寄主上的分布特征,文章对31条PVY中国分离物的基因组编码区进行了系统发育及重组分析。系统发育分析结果显示,最大似然法(maximum likelihood,ML)和邻接法(neighbor-joining,NJ)重建的系统发育拓扑结构基本一致,GF_YL20、FZ10、DF、SD1、1116和9703-3等6个分离物均未与已知株系相聚,其他株系来源相同的分离物以较高的自展值相聚成簇,显示出较强的株系特异性。重组分析显示,31个PVY分离物中,共有28个PVY分离物均检测到具有显著的重组信号,这些分离物主要包括PVY~(N-Wi)、PVY~(NTN)和PVY~(NTN-NW)三种株系。进一步分析显示,分离物1104、DF、SD1、1116和9703-3为未报道过的PVY新重组分离物。株系统计结果显示PVY重组株系已上升为优势株系,生产上需要密切注意这些株系,尤其新重组分离物的发展动态。以上结果表明,综合应用PVY全基因组序列的系统发育和重组分析,可以实现PVY株系的准确鉴定。     

呼吸道合胞病毒B亚型分离株的G蛋白基因分析

对一株长春地区B亚型分离株(CC169)的G蛋白基因进行了 序列分析,结果表明：我国呼吸道合胞病毒(RSV)分离株CC169同RSV B亚型原型株CH18537的 核苷酸同源性为94%,核苷酸的有义突变率达65%。由核苷酸推导出氨基酸序列的同源性为894%,氨基酸的变异全部发生在胞外区,并主要集中在一个高度保守区的两端,胞内区和跨 膜区保守不变。氨基酸的变异导致了分离株既有糖基化位点的改变,又有蛋白长度的变异。 此外还初步探讨了我国RSV B 亚型分离株CC169的G蛋白基因同原型株之间的变异与疫苗研制 中的意义。     

中国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30基质蛋白基因和融合蛋白基因的分子特征分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行基质蛋白(M)和融合蛋白(F)基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出M和F基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。China/Tib/Gej/07-30的M基因由1483个核苷酸组成,编码335个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为92.4%～97.7%和97.0%～98.2%。F基因由2411个核苷酸组成,编码546个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为85.5%～96.1%和94.3%～98.2%。China/Tib/Gej/07-30的F蛋白含有信号肽序列和跨膜结构域,序列高度变异。F蛋白第104～108位和第109～133位氨基酸位点分别是高度保守的裂解位点和融合肽结构域。F蛋白还含有序列高度保守的三个七肽重复区。China/Tib/Gej/07-30的M基因3′端的非编码区(UTR)长度为443个核苷酸,GC含量高达68.4%,与其他PPRV毒株的同源性为82.4%～93.5%。China/Tib/Gej/07-30的F基因5′UTR区长度为634个核苷酸,GC含量高达70.0%,与其他PPRV毒株序列相似性为76.2%～91.7%。     

马铃薯Y病毒pl基因的克隆与序列分析

利用依据马铃薯Y病毒(PVY)pl基因序列设计合成的一对引物YP1,YP2,以带毒烟草总RNA为模板,通过RT-PCR方法扩增得到了0.83kb的目的条带,测序结果表明为PVY pl基因。通过对PVY P1蛋白氨基酸序列分析发现PVY不同分离物间P1蛋白氨基酸序列存在明显差异,氨基酸序列同源性在64％～94％间。依据P1蛋白氨基酸序列建立了PVY系统关系树并对PVY进行了类型分析。     

小西葫芦黄化花叶病毒分离物的3′末端序列多态性研究

研究了来自中国大陆9个小西葫芦黄化花叶病毒（ZYMV）分离物的基因组3′末端核苷酸序列及所推导的外壳蛋白（CP）氨基酸序列以及3′末端非编码区（UTR）序列,并与其它地区所报道的16个ZMYV分离物进行了同源性比较。ZYMV CP基因核苷酸序列具有一定的寄主相关性和地域相关性,但总体上其关联程度不明显;同时,CP氨基酸序列的寄主适应性程度明显高于地域相关性。25个ZYMV分离物的CP氨基酸序列根据其变异程度分为2个区： N端约41个氨基酸为高度变异区,CP核心区和C端氨基酸序列为保守区。研究结果初步揭示了ZYMV作为单链RNA病毒通过与寄主相互作用而表现寄主适应性变异的趋势。     

鹅新城疫病毒P基因的RNA编辑及其相关蛋白的分子特征

应用RT-PCR方法对鹅新城疫病毒安徽分离株WF00GP基因进行了RNA编辑分析,发现w‰GP基因在保守的编辑位点上插入一个G,使得P基因增加编码V蛋白,而且它的最大ORF的长度为1188bp,编码395个氨基酸的P蛋白。wkG株和参考毒株的P基因的同源性和系统发育分析表明,WF00G株与水禽源毒株NA．1、ZJ1、FPI／02、PX2／03、Duck／1／05和SF02等亲缘关系较近,与传统疫苗株或弱毒株HB92、LaSota和Clone30以及F48E9等的亲缘关系相对较远。进一步对其V蛋白羧基端序列分析发现,虽然编辑位点氨基酸序列（132KKG134）和羧基端的5个Cys残基位点是高度保守的,但是V蛋白c末端氨基酸存在较大变异,APMV-1毒株V蛋白羧基端氨基酸的突变呈现“基因型一致性”。     

13C and 1H solid state NMR investigation of hydration effects on gluten dynamics

The effect of hydration on the molecular dynamics of soft wheat gluten was investigated by solid state NMR. For this purpose, we recorded static and MAS ¹H spectra and SPE, CP, and other selective ¹³C spectra under MAS and dipolar decoupling conditions on samples of dry and H₂O and D₂O hydrated gluten. Measurements of carbon-proton CP times and several relaxation times (proton T₁, T_1ρ and T₂, and carbon T₁) were also performed. The combination of these techniques allowed both site-specific and domain-averaged motional information to be obtained in different characteristic frequency ranges. Domains with different structural and dynamic behaviour were identified and the changes induced by hydration on the dynamics of different domains could be monitored. The proton spin diffusion process was exploited to get information on the degree of mixing among different gluten domains. The results are consistent with the “loop and train” model proposed for hydrated gluten.     

Systematic synthesis, structural characterization, and reactivity studies of vanadium(V)–citrate anions [VO2(C6H6O7)]2, isolated from aqueous solutions in the presence of different cations

The aqueous chemistry of vanadium with physiologically relevant ligands constitutes a subject of burgeoning research, extending from bacterial metalloenzymic functions to human-health physiology. Vanadium, in the form of VCl₃ and V₂O_5, reacted expediently with citric acid, in a 1:2 molar ratio in water at pH4, and, in the presence of various cations, afforded crystalline materials bearing the general formula (Cat)₂[V₂O₄(C₆H₆O₇)₂]·nH₂O (A) (Cat⁺=Na⁺, NH₄ ⁺, n=2; Me₄N⁺, K⁺, n=4). Exploration of the reactivity of A toward H₂O₂ yielded the peroxo-containing complexes (Cat)₂[V₂O₂(O₂)₂(C₆H₆O₇)₂]·2H₂O (B) (Cat⁺=K⁺, NH₄ ⁺). Both classes of compounds were characterized analytically and spectroscopically. The X-ray structures of complexes A and B emphasize the exceptional stability of the dimeric rhombic unit V^V ₂O₂, which is retained upon H₂O₂ reaction, and the preserved mode of coordination of the citrate ligand as a doubly deprotonated moiety. In these complexes, typical six and eight coordination numbers were observed for the Na⁺ and K⁺ counter-ions, respectively. The variety of synthetic approaches leading to A, along with the stepwise and direct assembly and isolation of peroxo-compounds (B), denotes the significance of reaction pathways and intermediates in vanadium(III–V)–citrate synthetic chemistry. Hence, a systematic investigation of reactivity modes in aqueous vanadium–citrate systems emerges as a crucial tool for the establishment of chemical interconnectivity among low MW complex species, potentially participating in the intricate biodistribution of that metal ion in biological fluids.     

Inhibitory effect of heavy water on mutability of chemically injured Escherichia coli cells

After E. coli cells (WP2 and WP2uvrA) were treated with chemical mutagens (methyl methanesulfonate, MMS; N-methyl-N-nitrosourea, MNU; 4-nitroquinoline 1-oxide, 4NQO) in 1/15 M phosphate buffer, the mutability of the treated cells plated on a D₂O-agar plate was compared with that plated on an ordinary H₂O-agar plate. The mutation frequency decreased more or less on the D₂O-agar plate. The D₂O-substitution effects, as termed by the relative mutation frequencies (MF_D2O/MF_H2O), are 0.92 for MMS, 0.29 for MNU, and 0.42 for 4NQO in WP2, and 0.68 for MMS, 0.49 for MNU, and 0.16 for 4NQO in WP2uvrA. The D₂O effect seemed to be partly related to the function of the uvrA gene-associated products. The pH dependence of mutability was discussed in connection with the D₂O-substitution effect.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mito-chondrial F, ATPase, which we have been recently characterized (FEBS Letters 1996,379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H₂O₂, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H₂O₂ thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F₁ATPase in a molar ratio 1:1 rapidly reacts with H₂O₂ mediating a 30% enzyme inactivation. These results support the presence in mitochon-drial and thermophilic F₁ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F₁ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F, ATPase undergoes a higher inactivation (75%) upon exposure to H₂O₂. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dis-mutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H₂O₂.     

Interpretation of the low-angle meridional neutron diffraction patterns from collagen fibres in terms of the amino acid sequence

Measurement of fully corrected, low angle meridional neutron diffraction intensities from native collagen fibres, in a full range of H₂O/D₂O contrasts, is described. The observed contrast dependence of the intensities of the first 12 orders of 670 A (D) axial periodicity is fitted to a general quadratic theory of contrast variation. The observed first order contrast dependence is compared to predictions based on the amino acid sequence, assuming different extents of chemical H/D exchange, and found to be consistent with complete non-carbon linked H/D exchange except for 1 to 1.6 hydrogen atoms per gly-X-Ytriplet involved in H-bonding. Both X-ray and neutron diffraction data in a variety of contrasts are consistent with a unique model for the axially projected structure of native collagen fibrils based on the amino acid sequence. This model if characterized by average axial residua translations in the NH₂ and COOH terminal, non-triple helical telopeptides, expressed as multiples of the triple helical residue translation, of 0.85 ± 0.05 and 0.7 ± 01 respectively, with D = 235 ± 1 residues.     

Leaf alkanes in Persea and related taxa

The normal-alkane range in leaf cuticular waxes from 22 Persea species and cultivars and from the closely related genus Beilschmiedia was C₂₃ H₄₈ to C₃₅, H₇₂; alkanes with an odd number of carbon atoms predominated, C₃₃ H₆₈ usually constituting about half of the total wax. The alkane profiles gave good agreement with established taxonomy. Beilschmiedia showed an alkane distribution quite different from that of the Persea taxa. Amongst Persea species, the geographical and phylogenetic distinctiveness of P. indica and P. donnell-smithii were reflected in the distinctiveness of their alkanes. Within the subgenus Persea. the morphologically most distinct entity. P. schiedeana, also had a distinct alkane profile. Cultivars of hybrid origin indicated in their alkane proportions pronounced gene interaction.