Immune response to human influenza virus hemagglutinin expressed in Escherichia coli

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.     

Early events in calcium-induced liposome fusion

Calcium-induced interaction of liposomes composed of pure phosphatidylserine (PS) has been studied using a rapid-mixing, rapid-freeze device. Freeze-fracture electron microscopy of this material revealed that liposomes react very rapidly after addition of calcium ions. After only 10 ms (the resolution of the technique) vesicle fusion was apparent. At the same time, however, vesicles also collapsed, and appeared as aggregates of flattened membranes. This may explain controversies which have arisen over vesicle fusion studied with more indirect methods.     

用细胞融合的方法选育耐高酒精度的酿酒酵母

选择耐高渗透压、耐高酒精度和发酵终了产酒精量较高的黄酒酵母H—1和目前酒精工业生产用菌K号酒精酵母K—1,运用细胞融合技术选育发酵速度快、产酒精量高的工业用酒精酵母.双倍体黄酒酵母H—1和双倍酒精酵母K—1经过产孢前预培养和产孢培养后,蜗牛酶水解子囊孢子壁,离心收集单倍体子囊孢子,培养后得到单倍体黄酒酵母H—2和单倍体酒精酵母K—2.用硫酸二乙醇诱变处理单倍体细胞,得到单倍体黄酒酵母的维生素缺陷型H—3和单倍体酒精酵母的氨塞骏营养缺陷型K—3通过正交试验找出了H—3和K—3原生质体形成及再生的较优条件是:对数生长后期的细胞33℃、0.2%的β—巯基乙醇预处理15分钟,然后4%蜗牛酶作用2小时.用35%聚乙二醇和10mMCaC1_2诱导融合40分钟,于再生基本夹层培养基上培养获得营养互补融合子,并且考查了融合子的遗传稳定性.通过耐酒精度、一发酵速度和最终产酒精量的测定,筛选出融合子HK—6.HK—6与生产用K号酒精酵母相比,发酵速度相接近,而最终产酒精量提高了11%.可耐受16%的酒精度.     

Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm-egg fusion: inhibitor studies with human sperm and zona-free hamster eggs.

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy-L-phenylalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona-free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca(2+)-free medium and fusion in Ca(2+)-containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50-60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon-inhibitable MEP activity was released during the ionomycin-initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome-reacted, phosphoramidon-treated sperm resulted in a large reversal of the phosphoramidon-inhibitory effect on sperm-egg fusion. These results support the hypothesis that the acrosomal phosphoramidon-inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm-egg fusion.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Macromolecules Involved in the Amoeba-Bacteria Symbiosis1

ABSTRACT. In the Amoeba-bacteria symbiosis, rod-shaped Gram-negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host-symbiont interactions, although their respective roles are not yet fully known. The symbiont-derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29-kDa protein was most abundant in the host cytoplasm, while the 96-kDa protein and LPS were found mostly on the symbiosome membranes. The 67-kDa protein was a GroEL analog and stayed within the symbionts. The host-derived 43-kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225-kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29-kDa and 67-kDa proteins were cloned and sequenced. The 29-kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper-like motif, suggesting a possible DNA-binding function. The DNA sequence of the 67-kDa protein gene showed a 70% identity with heat-shock-protein genes of Escherichia coli and Coxiella burnetii.     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.