Plasmid molecules

Once in the cytoplasm, a proportion of plasmid molecules are likely degraded by cytoplasmic nucleases, effectively further reducing transfection efficiencies. There are two potential routes by which plasmid DNA could reach the nucleus ... 

As well as chromosomal DNA many bacteria contain closed circular double-stranded DNA structures in their cytoplasm called plasmids. These plasmids are able to replicate independently of the chromosome and usually carry one or more genes that are responsible for a useful characteristic displayed by the host bacteria, e.g. antibiotic resistance. The number of plasmid molecules in each bacterium is known as the copy number and varies between different species of bacteria and types of plasmid. 

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

Figure 7.6. Schematic representation of in vitro synthesis of RNA. Shown is a plasmid molecule containing a cloned DNA that is flanked by T3 and T7 promoter sequences. The recombinant plasmid DNA is linearized in such a way that the transcription from one of the promoter elements generates RNA molecules corresponding to the cloned insert DNA and not the plasmid vector DNA. At the end of the reaction plasmid DNA is removed after enzymatic digestion with DNase I, and the pure RNA species is ethanol precipitated.

The reason is speed. At only days after the availability of an expression vector, milligrams to hundreds of milligrams of a recombinant protein can be delivered into the hands of the researcher. Vectors for transient expression do not require a selection marker - the goal is to deliver DNA to a maximal number of cells in the population. Several vectors can be transfected simultaneously into cells and will be expressed simultaneously. With calcium phosphate as a vehicle, it was shown that approximately 20000 plasmid molecules per cell can be delivered [132]. After a few days, the copy number of plasmid molecules will decline in the nucleus and the production ofmRNA ceases. Depending on the protein at the time point of the highest accumulated yield, the product is harvested and cells are discarded. A new production can be re-started at any time when sufficient fresh cells can be provided and a new DNA-vehicle preparation is ready for transfection. 

Folger, K. R., Wong, E.A., Wahl, G., and Ca-pecchi, M.R. (1982) Patterns of integration of DNA microinjected into cultured mammalian cells evidence for homologous recombination between injected plasmid molecules. Mol. 

We hypothesize that at the endosomal pH of 5.0, the amino acid groups in POE 2 would protonate and the positively charged groups would bind electrostatically with the negatively charged DNA plasmid molecules. Thus, release of DNA plasmid would be delayed until sufficient POE 2 hydrolysis took place to liberate the DNA plasmid. 

A second effect of platination was noted (18) on the electrophoretic mobility of relaxed circular DNA. The mobility of this form increased with increasing platinum binding, as opposed to the initial decrease and subsequent increase in mobility of supercoiled DNA. This behavior was attributed to a shortening of the DNA due to platiniim induced crosslinks. Further evidence was obtained from electron micrographs of the platinated DNA samples (18,24)f which showed a dramatic decrease in contour length of relaxed plasmid molecules upon platination (see Figure 6 below). 

First determine how many plasmid molecules are present in the bacterial cell culture 10 cells/ml = 10 cells/L, and 10 celhL x 100 plasmids = 10 plasmid molecules in the culture. Then use the molecular weight of a base pair (-660 g/mol bp) in the plasmid, the length of the plasmid in base pairs (4.4 x 10 bp), and Avogadro s number to determine the mass of 10 plasmid molecules. 

In most organisms, DNA exists in a supercoiled form. Supercoiled DNA can exist in a wide variety of topological conformations with variations in twisting and writhing. A simple plasmid molecule purified from bacterial cells will exist as a naturally occurring... 

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