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ABSTRACT: BACKGROUND: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. RESULTS: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30degreesC to 42degreesC. However, using Escherichia coli DH5alpha as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30degreesC, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42degreesC. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5alpha[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42degreesC. CONCLUSIONS: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production. 相似文献
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Abstract Results are presented on vascular species richness in three representative alpine plant communities at 1040–1410 m on Mt Burns in the perhumid Fiordland region, a hotspot of alpine plant diversity, in south‐western South Island, New Zealand. Overall species richness was not dissimilar between the three communities in any of the eight plot sizes (mean values of 20.8–24.4 species in the largest plots of 100 m2), even though coefficients of floristic similarity were small (17.9; 23.5) between both low‐alpine communities (snow tussock‐shrubland and snow tussock grassland) and the high‐alpine cushion fellfield. Vascular species richness was generally similar to that in the few other oceanic New Zealand alpine communities for which data are available. The decline in richness from the low‐alpine to high‐alpine zones, revealed in more comprehensive records from two other regions with generally similar oceanic environments, was not recorded, indeed was reversed, on Mt Burns. Whether the recognized biodiversity hotspot of Fiordland has a generally richer high‐alpine flora than other regions in New Zealand needs further examination. The general pattern of alpine floristic richness in relation to elevation, in New Zealand, also prevails in most alpine regions abroad, usually under much more extreme continental environments. This pattern is usually ascribed to the associated decrease in temperature. Both the small size of the land mass and/or associated environmental conditions may be implicated but clarification awaits further data, preferably collected with standardized procedures. 相似文献
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J. PEARSON D. M. WELLS K. J. SELLER A. BENNETT A. SOARES J. WOODALL & M. J. INGROUILLE 《The New phytologist》2000,147(2):317-326
Mosses have been used as biomonitors of atmospheric pollution for some years, but few studies have been carried out on the effect of NOx emissions from traffic on moss tissue N. Eight species of moss (102 samples) growing on walls or roofs next to roads exposed to different traffic densities were collected from urban and rural sites in the UK. The shoots were sampled for total N, their stable isotope 15 N/14 N content (δ15 N) and heavy metal content (Pb, Zn). There was a lack of correlation between tissue total N and traffic exposure, but a very good correlation between traffic exposure and tissue δ15 N. Plants collected near motorways or busy urban roads had δ15 N values ranging between +6 and −1‰, while in rural areas with hardly any traffic these ranged from −2 to −12‰. In a separate survey of mosses, the average δ15 N of shoots from busy roadsides in London was +3.66‰, whereas from samples collected from farm buildings near poultry or cattle pens it was −7.8‰. This indicates that the two main atmospheric N sources, NOx and NHx , have different δ15 N signatures, the former tending to be positive and the latter negative. Tissue concentrations of both Pb and Zn show a strong positive correlation with traffic exposure, with Zn in particular being greater than Pb. The results are discussed with regard to the use of moss tissue Zn as a means for monitoring or mapping pollution from vehicles, and of δ15 N as an aid to distinguish between urban (NOx ) and rural (NHx ) forms of N pollution. 相似文献
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Synthesis of Cell Coat in Normal and Transformed Cells 总被引:6,自引:0,他引:6
THE surface of transformed cells has been a focus of considerable attention recently because some of the properties which distinguish these cells from their precursors, such as decreased cell adhesiveness, altered cell orientation and loss of contact and density dependent inhibition1–3, may relate to changes on their surface. A common feature of vertebrate cells is the cell coat, a glycoprotein structure surrounding the plasma membrane4. Electron microscopy has revealed that transformed cells have a thicker coat than normal cells5 and we have now found that coat synthesis in cells transformed by an oncogenic DNA virus and in cells transformed by a chemical carcinogen occurs faster than in normal controls whereas only in the virus-transformed cells is the coat significantly thicker. 相似文献
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A Emami-Khoyi DA Hartley RH Cruickshank LJ Boren JG Ross 《New Zealand journal of zoology.》2016,43(4):322-335
New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic. 相似文献
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