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91.
Lysozyme expression in Lactococcus lactis 总被引:1,自引:0,他引:1
Maarten van de Guchte Fimme Jan van der Wal Jan Kok Gerard Venema 《Applied microbiology and biotechnology》1992,37(2):216-224
Summary Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria. 相似文献
92.
A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome. 相似文献
93.
Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments 总被引:8,自引:0,他引:8
F Marcus S H Chamberlain C Chu F R Masiarz S Shin B C Yee B B Buchanan 《Archives of biochemistry and biophysics》1991,287(1):195-198
Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions. 相似文献
94.
T Yee 《BioTechniques》1991,10(6):786-789
A computer program for digital image processing is described which can be implemented using scanning densitometer hardware pre-existing in most biology departments plus computer video hardware which may either pre-exist in the biology department or would represent a moderate upgrade over an already planned computer purchase. The primary purpose of this computer program is to provide contrast enhancement of faint or low contrast autoradiograph images and to implement background subtraction and digital smoothing methods which permit visualization of blurry electrophoresis bands against noisy backgrounds. However, the program also has modest editing capabilities that allow its use in the routine preparation of images for publication. Finally, the program has facilities for deblurring, edge enhancement and multiple image averaging, which give it usefulness in other forms of photographic analysis. 相似文献
95.
A practical approach to routine immunostaining of paraffin sections in the microwave oven 总被引:1,自引:0,他引:1
M. E. Boon F. C. J. Hendrikse P. G. Kok P. Bolhuis L. P. Kok 《The Histochemical journal》1990,22(6-7):347-352
Summary The Streptavidin-Biotin Complex (SABC) Immunostaining method can be carried out by performing the rinsing and blocking steps and in addition the incubations with the primary and the secondary antibody sera in the microwave oven. Irradiation of the Streptavidin-Biotin Complex reduces stain activity due to destruction of horseradish peroxidase (HRP) (Boon & Kok, 1988). Therefore, we decided not to perform this step in the microwave oven. The microwave incubations can be performed using antisera dilutions of 1:1000 (instead of 1:50) in tissue fixed with Kryofix, allowing staining in staining racks. This keeps hands-on time low and simplifies the microwave steps. It is very difficult to obtain reproducible results in the microwave oven using droplet incubations due to problems with hot spots and antenna effects. These problems are avoided when cuvettes are used and air is blown through the solutions during microwave incubations. With effective temperature control the method is highly reproducible, and takes at least 100 min less than the conventional SABC method. It is, in particular, attractive when large series of slides must be routinely immunostained and when reproducible results are desired. 相似文献
96.
97.
Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype. 相似文献
98.
99.
Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein
Absorption changes accompanying the formation of light-induced P-700+ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700+ and A?2. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A2 were completely lost. These results provide evidence that the intermediate electron acceptor, A2, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A2 is different from that of P-430. 相似文献
100.
Enterobacteriaceae and Pseudomonas aeruginosa Recovered from Vegetable Salads 总被引:5,自引:4,他引:1 下载免费PDF全文
Klebsiella, Enterobacter, and Serratia were recovered frequently in high counts from vegetable salads. Pseudomonas aeruginosa, although isolated frequently, was in lower counts. 相似文献