DNA plasmid

FIGURE 5.3.14 The plasmid is a packet of supplemental DNA material found outside the bacterial chromosome. Plasmids often contain DNA molecules that serve specific purposes supplemental to chromosomal DNA. Genes for specific antibiotic resistance are found in plasmids, and genes conferring virulence are also found there. Exchange of plasmid DNA among bacteria is relatively easy. (From Amibile-Cuevas, C.E, Am. ScL, 91, 138, 2003. With permission.) 

Bacteria, far from being opportunistic loners, are highly social creatures that incessantly chatter among themselves, with the hosts they infect, and even with other species of bacteria. 

Autism is a mental condition where people, mostly children, crave routine, have trouble communicating, and don t understand intuitive social rules. Some autistics barely speak, while others have some very large vocabularies. Some are riotously overstimulated, while others are isolated and withdrawn. 

It is thought that the causes for autistic behavior are a combination of genetic makeup and environmental factors. Although the right genes must be present, not all children with these genes develop autism. Speculate on possible environmental causes. 

There has been found a genetic variation present in 47% of the U.S. population that codes for a protein active in the brain, gastrointestinal tract, and immune system. This gene is associated with autism. It is likely that there is a genetic predisposition to the condition that is triggered by unknown environmental events. 

Immune responses can be elicited in rodents by direct transfer of DNA, e.g. by injecting an expres.sion plasmid containing the specific gene directly into muscle or other tissue. Some of the muscle cells rvill express the specific gene and the recombinant protein mtiy activate the host immune system. Alternatively, naked DNA or gold particles coated with low concentrations of the p)a.smid DNA can be transferred into the tissue using a pneumatic gun or by particle bombardment. These procedures circumvent the need to express the proteins in vi tro and details of their application are given in refs 12 and 13. 

Mix the peptide and protein carrier (KLH. OVA. or BSA) in a 1 1 ratio, e.g. pipette 250 (J.1 of each into a 5 ml glass beaker on a magnetic stirrer. Small fleas can be made from pieces of paper clip sealed in polythene tubing by heating. Add 5 p.1 of 25% glutaraldehyde and stir for 15 min at room temperature. 

Block excess glutaraldehyde by adding 100 pil of 1 M glycine and stirring for a ftirther 15 min. 

Transfer of antibiotic resistance genes among bacteria in nature has created serious problems in the treatment of many infectious diseases such as tuberculosis, gonorrhea, pneumonia, staphylococcus, and others. The widespread use of antibiotics in agriculture and in hospitals has resulted in the selection and evolution of pathogenic microorganisms that are resistant to many, sometimes all, of the antibiotics normally used to treat infections by these microorganisms. 

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Figure Bl.19.9. Plasmid DNA (pUClS) on mica imaged by STM at high resolution. The inset is a cut-out of a zoomed-in image taken inunediately after the overview. (Taken from [42], figure 2.)...

Hansma H G, Vesenka J, Siegerist C, Kelderman G, Morrett H, Sinsheimer R L, Bustamante C, Elings V and Hansma P K 1992 Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope Science 256 1180... 

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

The synthetic and plasmid DNAs are mixed and join their sticky ends spontaneously. They are covalently bound together by DNA ligases, when the resulting hybrid plasmid is inserted into bacterial cells. Dilute calcium chloride solutions render the bacterial membranes permeable and allow the passage of ONA into the cells. 

Plasmid DNAs. Plasmids are nucleic acid molecules capable of intracellular extrachromosomal repHcation. Usually plasmids are circular DNA species, but linear and RNA plasmids are known. In nature, plasmids can assume a variety of lifestyles. Plasmids can recombine into the host chromosome, be packaged into vims particles, and repHcate at high or low copy number relative to the host chromosome. Additionally, their information can affect the host phenotype. Whereas no single plasmid is usually capable of all these behaviors, the properties of various plasmids have been used to constmct vectors for a variety of purposes. 

Most plasmids are topologically closed circles of DNA. They can be separated from the bulk of the chromosomal DNA by virtue of their resistance to alkaline solution. The double-stranded stmcture of DNA is denatured at high pH, but because the two strands of the plasmid are topologically joined they are more readily renatured. This property is exploited in rapid procedures for the isolation of plasmid DNA from recombinant microorganisms (5,6). 

Plasmid Vectors for Facile Introduction of Passenger DNA and Selection of Recombinants. The map of a commonly used plasmid vector, pUC19 (7), is shown in Figure 2. Three parts of the vector are key to its utility. The origin sequence, oh, allows the repHcation of plasmid DNA in high copy number relative to the chromosome. A gene, amp, encoding the enzyme beta-lactamase, which hydrolyzes penicillin compounds, allows... 

Toyopearl HW-75 resin, with pores larger than 1000 A, have been used in place of ultracentrifugation steps for the purification of plasmid DNA. Ultracentrifugation is a time-consuming process and requires expensive chemicals, such as cesium chloride. Toyopearl HW-75 resin provides superior separation performance for plasmid DNA and also provides high yields (54). 

FIGURE 13.2 Foreign DNA sequences can be inserted into plasmid vectors by opening the circnlar plasmid with a restriction endonnclease. The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. 

O Suspend 20 ng plasmid DNA + lo E.eoli cells in CaCh.. solndon. 

Continuous. Long fermentation run times, with many generations, increase the chance of mutation or loss of plasmid DNA. 

Addition of antibiotics to the fermentation broth may be used to avoid problems associated with growth revertants (eg auxotrophic back mutation) ensure that genetic material (eg plasmid DNA) is maintained within the process micro-organism. 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

TLRs are the basis for the fact that plasmid DNA of prokaryotic origin, as is used in all DNA vaccination... 

Liu MA, Ulmer JB (2005) Human clinical trials of plasmid DNA vaccines. Adv Genet 55 25-40... 

Presently, 1,260 gene-therapy trials are underway worldwide (Tables 1 and 2). For details see http // www.wiley.co.uk/genetherapy/clinical/. Almost three fourths of all trials are based on viral vectors. The vast majority of nonviral gene-therapy trials use naked/ plasmid DNA (18% of all gene-therapy trials). 

Genetic material can be transferred horizontally between bacterial cells either as free DNA by transformation or as plasmid DNA via conjugational... 

Class C Serine p-lactamases AmpC enzymes of coti, Shigella spp., Enterobacterspp., C. freundii, M. morganii, Providencia spp. and Serratia spp. cephalos-porinases with wide spectrum of activity CMY, LAT, BIL, MOX, ACC, FOX and DHA types. All genes are ampC genes that have been mobilized by transfer to plasmid DNA. 

Griffin DE III, Hill WE. 1978. In vitro breakage of plasmid DNA by mutagens and pesticides. Mutat Res 52 161-169. 

S., Hamer, )., Escobedo, J., Cohen, F., Radhakrishnan, R., Dwarki, V, and ZuCKERMANN, R.N. Lipitoids — novel cationic lipids for cellular delivery of plasmid DNA in vitro. Chem. Biol. 1998, 5, 345-354. 

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