双拷贝人α型心钠素基因的组建及大肠杆菌分泌型克隆与表达

将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。     

Expression of Characteristics of Ammonium Nutrition as Affected by pH of the Root Medium

To study the effect of root-zone pH on characteristic responsesof -fed plants, soybeans (Glycine max {L.}Merr. cv. Ransom) were grown in flowing solution culture for21 d on four sources of N (1.0 mol m^–3 , 0.67 mol m^–3 plus 0.33 mol m^–3, 0.33 mol m^–3 plus 0.67 mol m^–3 , and 1.0 mol m^–3) with nutrient solutions maintained at pH 6.0, 5.5, 5.0, and 4.5. Amino acid concentration increased inplants grown with as the sole source of N at all pH levels. Total amino acid concentration in the rootsof -fed plants was 8 to 10 times higher than in -fed plants, with asparagine accounting for more than 70% of the total in the roots of these plants.The concentration of soluble carbohydrates in the leaves of-fed plants was greater than that of -fed plants, but was lower in roots of -fed plants, regardless of pH. Starch concentration was only slightlyaffected by N source or root-zone pH. At all levels of pH tested,organic acid concentration in leaves was much lower when was the sole N source than when all or part of theN was supplied as . Plants grown with mixed plus N sources were generally intermediate between - and -fed plants. Thus, changes in tissue compositioncharacteristic of nutrition when root-zone pH was maintained at 4.5 and growth was reduced, still occurredwhen pH was maintained at 5.0 or above, where growth was notaffected. The changes were slightly greater at pH 4.5 than athigher pH levels. Key words: Ammonium, nitrogen nutrition, root-zone pH, soybean, tissue composition     

1,4—二取代酰氨基硫脲类化合物(DATCDs)的植物生理效应与应用研究——Ⅴ.DATCD-A对离体黄瓜子叶扩张及核酸代谢的影响

本实验以离体黄瓜子叶为材料,研究了 DATCD—A 对子叶扩张及核酸代谢的影响。结果表明,DATCD—A 可显著地促进离体黄化子叶扩张,使其鲜重明显增加;并且在处理后期逐渐提高子叶干重。在离体子叶扩张变绿的过程中,DATCD—A 促进离体黄化子叶 RNA、DNA 含量和 DNA/RNA 比率明显上升,且 RNA 的增加发生在 DNA 合成增加之前。凝胶电泳证明,RNA 的增加主要是25s 和18s 的 rRNA。     

基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

The RCE: a Riparian, Channel, and Environmental Inventory for small streams in the agricultural landscape

1. The Riparian, Channel and Environmental (RCE) Inventory has been developed to assess the physical and biological condition of small streams in the lowland, agricultural landscape. It consists of sixteen characteristics which define the structure of the riparian zone, stream channel morphology, and the biological condition in both habitats. 2. The inventory is based on the view that in landscapes where non-point source pollution and agriculture dominate, the environmental condition of small streams can be assessed by an appraisal of the physical condition of the riparian zone and stream channel. It is assumed that disturbance of this physical structure is a major cause for reduction of stream biological structure and function. This assumption is supported by a case study using fifteen Italian stream locations in which the RCE was found to be positively correlated to the benthic macroinvertebrate community as measured by the Extended Biotic Index (r = 0.80, P < 0.001) and the Shannon Diversity Index (r = 0.73; P < 0.001). 3. The inventory is designed for quick use to cover a large number of streams in a short period of time. When used it generates a numerical score which can be used to compare the physical and biological condition between different streams within a region. The numerical score is divided into five, colour-coded classes to facilitate use in streammonitoring programmes and to allow comparison with biological indices.     

Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans.

Chinese hamster ovary lines with two mutations, one causing accumulation of Man5GlcNAc2-P-P-dolichol and a second resulting in defective N-acetylglucosaminyltransferase I activity, synthesize asparagine-linked glycans with the structure Man3GlcNAc2. As a result, the asparagine-linked glycans produced by these lines are smaller and less heterogeneous than those produced by other currently available animal cell lines.