Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network.

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.     

基因工程链激酶(r—SK)纯化及其单克隆抗体制备和鉴定

采用Rotofor等电聚焦和分子筛技术纯化大肠杆菌表达的重组链激酶(r—SK),其纯度为97%。比活性1×10⁶IU／mg,回收率41％。分析所纯化的r—SK　N端氨基酸顺序证实其1—15个氨基酸顺序与SK基因的核苷酸顺序所示3联密码子一致。以纯化r—SK为抗原,通过杂交瘤技术构建了分泌抗r—SK单克隆抗体(McAb)杂交瘤细胞(4D11),该McAb属IgG1亚型．特异地作用于r-SK和C组口溶血性链球菌分泌的SK。用底物显色法测定该McAb可抑制SK对纤溶酶原的激活。Western blot法证实该McAb能识别分子量为16 250Da的r—SK的CNBr裂解片段。推测SK蛋白中第71残基至第237残基间的氨基酸顺序参与SK与纤溶酶原的结合。     

抗人PAI—1单克隆抗体制备,鉴定和应用

以重组PAI-(rPAI-1)为抗原,通过杂交瘤技术获得6株阳性杂交瘤细胞(AP1、AP2、AP3、AP4、AP5和AP6),并用SPA亲和层析纯化了抗PAI-1单克隆抗体(McAb)。所有McAb均能识别rPAI和天然PAI-1,腹水滴度均为10~6以上。6种McAb对PAI-1亲和常数在3.45×10~7—1.05×10~9mol/L之间。AP2、AP3 McAb能完全抑制PAI-1活性,AP4、AP和AP6只能部分抑制PAI-1活性,AP1则不抑制PAI-1活性。6种McAb中只有AP1、AP4和AP5能识别PAI-1/t-PA复合物。利用AP1、AP3、和AP4 McAb制备免疫亲和柱一步纯化HepG_2细胞分泌的PAI-1,纯度大于98％,回收率92％,纯化倍数51倍。利用抗PAI-1 McAb建立了夹心法ELISA,测定了人血浆PAI-1水平,正常人血浆PAI-1平均含量(±D)为24.7±7.75ng/ml。     

湘北烟区烟青虫发生情况研究

烟青虫在湘北烟区1年发生5代,第一至四代危害烟草,第五代在其他作物上取食。自然条件下,世代重叠比较明显,幼虫发生与危害最大的是第一代,危害时期是5月下旬至6月下旬,同时,还得出了烟青虫各代的发生期、历期及其发育起点温度和有效积温。     

野生大豆种子蛋白含量差异的生理及结构基础的探讨

利用ＳＤＳ－聚丙烯酰胺凝胶电泳、电子显微镜、蛋白及酰脲含量测定等技术,对高蛋白含量（５０．７％ ）的５０３５９ 和低蛋白含量（４０．８％ ）的５０３０５ 两个野生大豆在种子发育过程中贮藏蛋白积累的速率、蛋白组分合成的起始时间、蛋白体发育的进程以及幼茎的酰脲含量进行了比较研究。结果表明：野生大豆５０３５９的高蛋白含量是与其种子发育过程中较高的植株酰脲含量、较早较快的贮藏蛋白合成及积累速率,液泡中高效的蛋白贮藏方式以及蛋白体在子叶细胞中占有较大体积相联系的     

油菜素内酯促进绿豆上胚轴伸长与蛋白质和核酸合成的关系

用饲喂蛋白质和核酸合成的放射性前体［３ Ｈ］－Ｐｈｅ、［３ Ｈ］－尿嘧啶和［３ Ｈ］－胸腺嘧啶证实了油菜素内酯（ＢＲ）能促进绿豆上胚轴的生长和蛋白质、ＲＮＡ 及ＤＮＡ 的合成。用蛋白质和核酸合成抑制剂（ＣＨ、Ａｃｔ．Ｄ、５－Ｆｕ）进一步探讨它们对上胚轴伸长的抑制作用与蛋白质、ＲＮＡ、ＤＮＡ 和ｍ ＲＮＡ 合成之间的关系。证明了上胚轴的伸长依赖于蛋白质和核酸的合成,尤其是依赖于ｍ ＲＮＡ 的合成。说明ＢＲ是在转录水平上调节基因的表达,进而促进上胚轴的伸长     

谷子显性雄性不育基因“Ms~（ch）”的细胞质转换

通过种间杂交和连续置换回交,选育出具有轮生狗尾草（Ｓ,ｖｅｒｔｉｃｉｌｌａｔａ（Ｌ．）Ｂｅａｕｖ．）细胞质的谷子（Ｓｅｔａｒｉａｉｔａｌｉｃａ（Ｌ．）Ｂｅａｕｖ．）核质杂种,以带有上位基因Ｒｆ的显性核不育植株为父本与此核质杂种杂交,已将显性核不育基因导入轮生狗尾草细胞质中,从其分离后代中选出了具有显性核不育基因的新核质杂种,为谷子的三系选育和细胞质研究创造了新工具,也为核互作杂优利用增加了核质互作优势新机制。     

β－内酰胺酶启动子C－17缺失对其强度的影响

通过酶学手段在β－内酰胺酶启动子间隔序列中造成Ｃ－１７的缺失突变,以氨苄青霉素为底物,检测突变前后β－内酰胺酶活力变化,并作了细菌的Ａｍｐ耐受性试验,实验结果表明,Ｃ－１７的缺失突变使启动子强度增加约６０％,含有突变启动子的细菌对Ａｍｐ的抗性明显增强,突变体生长的半抑制浓度为２８０μｇ／ｍｌ,而在此浓度下野生型菌体吸光度只有突变体的５０％左右,对启动子强度增加的原因进行了讨论。     

Zn对细胞保护作用机理的研究

应用扫描质子微探针和同步辐射ｘ荧光分析技术测定了细胞中元素的分布和组成,为确定Zn是细胞结构成分提供了直接的实验依据.用上述核技术结合有关生化指标,分析测定了正常和损伤细胞（脂质过氧化损伤）中Fe,Zn和丙二醛、SH基含量变化的相互关系.实验结果表明,当细胞发生脂质过氧化损伤时,Fe含量和丙二醛含量同步增高,而Zn含量和SH基量则降低.给细胞补充Zn后,提高了细胞质膜中的Zn含量,SH基量也随之增加,同时丙二醛量降低.提示Zn保护细胞完整性的作用机理之一是控制脂质过氧化作用.Zn可保护膜蛋白的SH基,减少和阻止被Fe所催化的过氧化反应.