A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.

Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.     

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes.

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.     

番茄种子经激光和咖啡因处理后其当代生物效应的研究

咖啡因具有抑制一些酶的活性,可减弱或消除生物体自身对激光引起的生物效应的修复作用。试验证明咖啡因对番茄种子萌发有强烈的抑制作用,咖啡因和激光复合处理生物效应显著,可降低番茄种子的发芽,并促进番茄果实增大和生长前期速率加快。     

柳牡蛎蚧生命表的研究

1 引言 柳牡蛎蚧(Lepidosaphes salicina Bersch)(同翅目:盾蚧科)是一种以为害杨树为主的刺吸式害虫之一,广泛分布于“三北”防护林区,该蚧一年发生一代,以卵越冬。通过刺吸式口器刺入树皮内,吸取树木养分和水分,并使树木表皮栓化。同时,由于枝干被蚧壳所覆盖,对呼吸及光合作用也有影响,特别是幼树被害后,一般     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

An improved Eschehchia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7·9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10⁵ cfu μg^-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus , it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.     

黄腐酸增强小麦抗旱能力的生理生化机制初探

叶面喷施黄腐酸可显著提高小麦幼苗的保水能力,表现为降低叶面蒸腾强度,增加气孔扩散阻力,提高幼苗的生物量,在干旱条件下尤为明显。喷施黄腐酸可使干旱条件下叶片内脯氨酸含量提高近一倍,并在水分充足时,也能使叶片脯氨酸含量增加78%。     

应用地理信息系统模拟森林景观动态的研究

根据地理信息系统的结构,通过数据文件的转换,把它与森林动态模型有机地结合起来,可实现对森林景观动态的模拟和预测。这种模拟方法的数据输入灵活,运行速度快,尤其是它可以获得一些以各种图表的形式输出的模拟结果。本文用文字和框图详细描述了地理信息系统的组成与结构,及森林动态模型的选择与运行方式,并以长白山森林景观为例,叙述了这种模拟方法的整个过程。     

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.