Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.     

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

黄皮种子发育过程中脱水敏感性与细胞膜透性的关系

黄皮（Ｃｌａｕｓｅｎａ ｌａｎｓｉｕｍ （Ｌｏｕｒ．） Ｓｋｅｅｌｓ）胚轴与完整种子的发育模式以及发育中电解质渗漏率变化有些不同． 种子生理成熟前、后的胚轴对脱水的反应也不同,前者经轻微脱水可提高萌发率和活力指数,后者不耐任何程度的脱水．活力指数的急剧下降伴随着电解质渗漏率的迅速上升．实验表明,黄皮种子在发育过程中没有形成耐脱水性． 细胞膜透性变化可反映脱水对种子的伤害程度     

川百合花粉管的生殖细胞分裂过程中微管骨架的分布变化

应用透射电镜辅以免疫荧光定位技术研究了川百合 (Lilium davidii Duch.)花粉管中生殖细胞分裂过程中染色体动态和微管分布的关系。在生殖细胞分裂前和有丝分裂前期 ,电镜观察一直未见微管结构 ,但免疫荧光图象显示生殖细胞中有微管蛋白存在。直到分裂的前中期—中期 ,染色体出现 ,它们沿花粉管的长轴前后排列 ,横向的着丝点对相应地一对对地纵向排列。这时 ,生殖细胞中才出现大量微管 ,它们分布于细胞周质区和染色体之间 ,并跨越染色体的整个长度。前中期—中期开始时 ,只有 1～ 2对着丝点从横向转为纵向 ,微管垂直插入着丝点形成着丝点微管 ,而非前人用免疫荧光方法观察到的微管与着丝点侧向联接的图象。随着横向的着丝点对逐渐转变成纵向的过程 ,着丝点微管数量逐渐增多 ,但不形成典型的纺锤体。分裂后期 ,染色体交错分离 ,微管的分布与前中期—中期的基本相同。晚后期 ,染色体呈明显的两群 ,除极区和细胞中央区有微管残余外 ,大部分微管消失。通过染色体长度的测量 ,间接证明了分裂后期 B的存在。分裂末期的晚期 ,核膜形成后 ,在两精核之间的区域 ,微管数量开始增多。此区可能代表用免疫荧光所观察到的微管重叠区。细胞板出现后 ,微管消失     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…     

可育的抗除草剂溴苯腈转基因小麦

报道了采用微粒轰击（Ｍｉｃｒｏｐｒｏｊｅｃｔｉｌｅ ｂｏｍ ｂａｒｄｍ ｅｎｔ） 幼胚将除草剂抗性基因导入小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ Ｌ．）的转化研究。实验共使用了１３ 个小麦品种, 从开花后１４～１８ ｄ 的籽粒中剥取幼胚, 植物表达质粒含有ＣａＭＶ ３５Ｓ启动子控制的除草剂溴苯腈抗性基因ｂｘｎ 以及筛选标记基因ＮＴＰⅡ。采用高压放电基因枪,用质粒ＤＮＡ 包被的钨粒轰击预培养３ ｄ 的幼胚。在含有卡那霉素类似物ｇｅｎｅｔｉｃｉｎ Ｇ４１８ｓｕｌｐｈａｔｅ 的ＭＳ培养基上, 经过多步骤筛选和分化, 从８００ 多个幼胚中获得了１６ 株转化苗。除草剂抗性鉴定和Ｓｏｕｔｈｅｒｎ 杂交分析证明, 其中４ 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为６ 个月, 因此, 该方法是一项快速有效的基因导入技术     

芸苔属脱外壁花粉的分离与人工萌发

芸苔属青菜（Ｂｒａｓｓｉｃａ ｃｈｉｎｅｎｓｉｓ）与紫菜苔（Ｂ． ｃａｍ ｐｅｓｔｒｉｓｖａｒ． ｐｕｒｐｕｒｅａ）的花粉经低温水合、热激、渗激三步程序,分离出大量具萌发能力的脱外壁花粉,脱外壁率可高达６０％ 以上。在含有碳源与氮源及Ｒｏｂｅｒｔｓ培养基盐成分的碱性ＰＥＧ 培养基中,首次使芸苔属脱外壁花粉萌发,萌发率可达３３％ ～４１％ 。在扫描电镜下观察了花粉脱外壁与萌发的过程。讨论了不同植物花粉脱外壁的方法与花粉壁生物学特点的对应关系,以及外壁对花粉萌发的可能作用