中期染色体外鞘蛋白质的研究

从１２例硬皮病患者的抗染色体抗血清中发现４例是抗中期染色体鞘的,用它们和小鼠腹水癌细胞核及全细胞裂解液ＳＤＳ－ＰＡＧＥ的蛋白印迹相反应,结果显示它们和细胞核裂解液的１１条抗原蛋白相结合,而且和全细胞裂解液中除以上的１１条外的另８条相结合。     

谈谈植物的性别——由一道选择题引起的思考

此题的正确答案是A,许多学生错误选择了B。选错学生的理由是：人体细胞中有46条染色体,人类基因组计划测定的22条常染色体及X、Y染色体（共24条）的DNA碱基序列。那么,同理可以推出,此题中水稻基因组就是要测定11条常染色体及X、Y染色体（共13条）的DNA碱基序列。因此答案应该是B。     

CCCTC结合因子在HeLa细胞中调控X染色体连锁的凋亡抑制蛋白的表达

目的:确定HeLa细胞的CCCTC结合因子(CTCF)表达水平是否与细胞的抗凋亡能力相关,并研究其具体分子机制。方法:用顺铂和阿糖胞苷分别诱导HeLa细胞和CTCF敲降的HeLa-CTCF-II-11细胞凋亡,比较两者的凋亡率;用基因表达芯片检测CTCF敲降后HeLa细胞的表达谱变化,寻找并验证受CTCF调控的与凋亡相关的蛋白。结果:用顺铂和阿糖胞苷诱导后,HeLa-CTCF-II-11细胞的凋亡率显著高于HeLa细胞;HeLa细胞的CTCF敲降后,X染色体连锁的凋亡抑制蛋白(XIAP)的表达显著下降。结论:CTCF敲降使HeLa细胞的抗凋亡能力下降,CTCF对XIAP基因的表达调控在这个过程中起了重要作用。     

ScII类似蛋白是洋葱根端细胞核、染色体和染色体骨架的组成成分

采用机械破碎和蔗糖梯度离心方法从洋葱根端分生组织中分离出细胞核并制备出核骨架。细胞核SDS-PAGE谱带中135kD处有一多肽,免疫印迹实验结果表明,该多肽可被抗鸡ScⅡ抗体标记,核骨架中没有此多肽。经抗ScⅡ抗体和FITC偶联的二抗标记后,细胞核发出代表ScⅡ的特异性荧光,而核骨架中无荧光发出。经抗ScⅡ抗体和蛋白A胶体金处理后,金颗粒特异性地结合在核内染色质区域。说明ScⅡ类似蛋白是洋葱根端细胞核的组分,且位于核内染色质上,但该蛋白不是核骨架成分。免疫荧光和免疫电镜实验结果还说明ScⅡ类似蛋白是洋葱根端细胞染色体和染色体骨架的组成成分。     

利用相互染色体涂色技术分析两株人肺腺癌细胞系(A549和GLC-82)的核型特征

肺癌是全世界癌症死亡中的一个主要的原因。除吸烟外,一些肺癌患者的发病与氡气污染相关。该研究采用包括染色体分选、正向和反向染色体涂色技术,分析了两株肺腺癌细胞系A549和GLC-82的核型特征。A549和GLC-82细胞系都属于非小细胞肺癌细胞系,但诱因不同,后者来源于一个长期生活在氡气污染环境肺癌病人的癌组织。染色体涂色结果表明,这两株肺癌细胞系发生了复杂的染色体重排。在A549和 GLC-82细胞系中,除正常染色体拷贝数变化外,还分别存在13条和24条畸变染色体。约一半的畸变染色体是通过非相互易位形成的,其余的畸变染色体则是通过一些正常染色体的片段缺失或重复而产生的。尽管这两株肺癌细胞系都没有共同的畸变染色体, 但它们似共享两个染色体易位断裂点：HSA8q24和12q14。     

SMC蛋白的结构和功能

近年来新发现的一类蛋白－染色体结构维持蛋白（SMC蛋白,structural maintenance of chromosome proteins)与染色体结构细胞周期性的动态变化紧密相关,它们参与有丝分裂染色体的集缩和分离,性染色体的剂量补偿效应,姐妹染色单体的内聚作用（cohesion),遗传重组和DNA修复等过程,本从生化特性和生物学功能两方面叙述了对SMC蛋白的研究。     

人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

果蝇唾腺染色体实验的改进

果蝇唾腺巨型染色体是大约1000-4000多条同源染色线精确配对聚集而成的多线染色体,这是染色体多次复制,但细胞只生长、不分裂的结果.染色体长达0.5mm.粗细是一般体细胞染色体的100-200倍左右,其上有深浅间隔、宽窄不等的染色带,果蝇4对唾腺染色体上已确定了6000多条染色带,它们宽窄、疏密、顺序、数目恒定,有种的特异性,同种个体的是相同的,不同的种则不一样,因此果蝇多线染色体可以建立染色带及间带分布图,它们的表现和遗传学图大致平行,多数遗传学家认为这些横纹与基因有对应关系,     

ScⅡ类似蛋白是洋葱根端细胞核、染色体和染色体骨架的组成成分

采用机械破碎和蔗糖梯度离心方法从洋葱根端分生组织中分离出细胞核并制备出核 骨架。细胞核ＳＤＳ－ＰＡＧＥ谱带中１３５ｋＤ处有一多肽,免疫印迹实验结果表明,该多肽可被抗 鸡ＳｃⅡ抗体标记,核骨架中没有此多肽。经抗ＳｃⅡ抗体和ＦＩＴＣ偶联的二抗标记后,细胞核 发出代表ＳｃⅡ的特异性荧光,而核骨架中无荧光发出。经抗ＳｃⅡ抗体和蛋白Ａ胶体金处理 后,金颗粒特异性地结合在核内染色质区域。说明ＳｃⅡ类似蛋白是洋葱根端细胞核的组分, 且位于核内染色质上,但该蛋白不是核骨架成分。免疫荧光和免疫电镜实验结果还说明ＳｃⅡ 类似蛋白是洋葱根端细胞染色体和染色体骨架的组成成分。     

一个22p＋家系的临床及分子细胞遗传学研究

本文对一例男性性腺发育不全伴有22p 标记染色体的患者及其家庭成员进行了分子细胞及临床细胞遗传学研究,结果表明,该家系中共有6名成员有22p 染色体,它们来源于先证者的外祖母。标记染色体的p 部分几乎与22q等大,C－带呈深染,G－,R－带在p 中间分别可见一条较窄的浅,深带型,Ag-显带在p＋末端见到较大的银染区,部分p 出现双NOR,型分析未见其它,D,G组或Y染色体重排,rRNA基因的染色体原位杂交银颗粒沿整个p 分布,其数目是正常D,G组染色体短臂上平均数的3．9倍,家系研究表明,在6例22p 携带者中,2例女性具有多次自然流产史,4例男性中除一例年龄12岁末见明显性腺异常外,其余3人均有不同程度性腺或外生殖器异常,结合文献,我们认为此家系中22p 可能与上述异常表型存在着一定的关系。     

人类抗中心体自身抗体的研究

用间接免疫荧光法以小鼠腹水癌细胞为底物从硬皮病病人的自家抗血清中筛选出几个抗中心体的抗血清。因为中心体是化学构分复杂的细胞结构,而自家抗体又是多克隆的,故本文只用其中一个抗中心体抗血清作进一步研究。为了定位其抗原,同时也采用了L 929培养细胞为底物。发现这抗血清结合于微管,有丝分裂期的纺锤体,中心体以及其它一些核结构。同时也用此抗血清在细胞裂解液的免疫印迹膜上检出了分子量与微管蛋白相同的主要条带,此外还有几条较不明显的条带;后者肯定了间接免疫荧光的观察结果。     

蚕豆染色体鞘的免疫荧光显示和其抗原的分析

本文应用本实验室发现的着染HeLa细胞染色体鞘的一例红斑狼疮病人抗血清对分离出的蚕豆染色体及核和蚕豆根尖生长点压片进行了免疫荧光染色,从两种不同制片方法得到的染色体上均染出了清晰的染色体鞘免疫荧光。之后又用此血清对蚕豆三天龄幼苗中(去除子叶、种皮)提取出的总蛋白进行Western印迹分析,获得的带型显示分子量为41K,39K,18K,17K的蛋白质可与抗血清中的抗体特异性结合,另有分子量为87K,38K,35K,34K,32K的蛋白质也可能具有与抗体特异性结合的能力。     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor

A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Intradermal DNA Immunization: Antisera Specific for the Membrane Lectin MR60/ERGIC-53

The trafficking of intracellular membrane proteins in Golgi apparatus, endoplasmic reticulum or intermediate compartment has not yet been fully elucidated. The human MR60/ERGIC-53 and the rat p58 proteins are one such protein; and to study them in cell-free and in situ systems, high quality monospecific antisera are required. Highly specific antisera have been obtained after immunization of mice with plasmids containing a gene encoding either the full length or a truncated protein. The best results were obtained after intradermal injections of a plasmid encoding a truncated protein comprising both the luminal carbohydrate recognition domain and the stem down to a cysteine residue close to the C-terminal end, but neither the transmembrane nor the cytosolic domains. Such antisera have a very high titer and are very efficient tools to visualize the MR60 protein in situ or to selectively precipitate the MR60 proteins from a whole cell lysate.     

人γ心钠素在大肠杆菌中的高效表达

利用聚合酶链式反应（ＰＣＲ）技术扩增人γ心钠素（γ－ｈＡＮＰ）ｃＤＮＡ编码序列,在其５′和３′端分别引入ＥｃｏＲⅠ和ＢａｍＨⅠ限制性内切酶位点,定向克隆到表达质粒载体ｐＭＳ－３１ｂ,在大肠杆菌ｐｏｐ２１３６中高效表达出人γ心钠素融合蛋白,表达产物占菌体总蛋白的３０～４０％。双向免疫扩散法测定证明表达产物与人α心钠素（α－ｈＡＮＰ）抗血清呈阳性反应,纯化的表达产物经复性处理后能引起大鼠胸主动脉条舒张．     

Electrophoretic mobility and immunoblot analysis of the outer membrane proteins of Aeromonas hydrophila, A. sobria and A. caviae.

The outer membrane profiles of three species of the genus Aeromonas were examined by means of SDS-PAGE and immunoblotting to identify species-specific polypeptides and antigens which could presumably be applied to differentiate Aeromonas spp. at the species or subspecies level. Profiles on an 11% discontinuous SDS-PAGE showed common band sharing at the 52 kD position. Species-specific bands for the three strains could also be detected. Immunoblots using heterologous LPS-adsorbed polyclonal antisera revealed demarcated common and uncommon antigens within the three species. Outer membrane preparations were immunoblotted against whole cell polyclonal antisera. The previously documented host pathogenicity of A. sobria correlated well with the immunoblots which showed antigenicity, especially due to the LPS, when compared with the other two species.     

Antigens in chromatin associated with proliferating and nonproliferating cells

Xenoantisera were raised to total chromatin from the leukemia cell line K562, or materials released through limited deoxyribonuclease I digestion of nuclei or during the control incubation of nuclei without enzyme. The peroxidase-antiperoxidase method of antibody-antigen detection was employed to visualize individual antigens resolved on one-dimensional polyacrylamide gels following transfer to sheets of nitrocellulose (immunotransfers). Each antiserum contained multiple antigen specificities as evidenced by the diverse patterns of reactive bands displayed on the immunotransfers. The most striking difference in antigens recognized between the antisera was observed in the molecular weight region below 50,000, where two highly reactive bands were seen mainly with antiserum to nuclear materials released by deoxyribonuclease I digestion. The antigens detected with all of the antisera were present in chromatins prepared from proliferating cells, while the levels of antigens present in chromatin from non-proliferating peripheral blood lymphocytes were greatly reduced or not detected. Antigens in chromatin from proliferating cells that migrated with apparent molecular weights of 37,000 and 100,000 were not lost once the activities to antigens in lymphocyte chromatin were absorbed out. These two activities were absorbed from antisera with the same amount of chromatins from proliferating cells. Two antigens migrating at molecular weight 52,000 and 76,000 appeared more active in the chromatin from unstimulated lymphocytes than in chromatin from proliferating cells.     

通过“供体-受体”原生质体融合将裸燕麦部分核基因组转移给普通小麦(英文)

本文进行了小麦和裸燕麦悬浮细胞原生质体的电融合,并基于双亲失活(用IOA处理受体小麦原生质体,用γ-射线照射供体裸燕麦细胞系),获得可能的杂种愈伤组织。对7块愈伤组织进行了乙醇脱氢酶(Adh)同工酶筛选,发现5块表现出双亲特征酶带。对3个杂种细胞系进行5种同工酶分析,证实它们均为稳定的不对称体细胞核杂种细胞系;它们表现出小麦的完整谱带和裸燕麦的部分谱带。对2个杂种细胞系及亲本的核糖体DNA Southern分析结果表明只有一个杂种细胞系(HB 95)含有双亲的全部谱带。细胞学观察表明,2个杂种细胞系的染色体数目均显著高于双亲。从杂种细胞系HB 94中分化出叶原基等分化结构。Adh同工酶分析表明,这些分化结构具有和母体细胞系完全相同的杂种谱带。