Plasmids ColEl

Fig. 3. Example of cniciform formation from an inverted-repeat sequence of DNA which occurs in the plasmid ColEl. The bolded bases are paired with...

ColEl Regulation by RNA Hairpins. Rephcation of the E. coli plasmid ColEl is regulated by two short RNA molecules and a protein in a system that provides an example of the unique stmcmral elements accessible to RNA molecules. Multidimensional heteronuclear nmr spectroscopy has been used to characterize the complex formed between the two RNAs (25). Each of the RNA molecules fold back on the other to form a pair of hairpin... 

In addition to their chromosomal DNA, bacteria often carry extra small pieces of DNA as permanent parts of their genome. These plasmids (sometimes called episomes), which are about the size of the DNA of viruses, replicate independently of the host chromosomes. Each bacterial cell usually contains more than a single copy of the plasmid. For example, the "colici-nogenic" plasmid ColEl, that infects E. coli is a circular piece of DNA of molecular mass 4.2 x 106 Da. Over 20 copies are normally found per cell but in the presence of a suitable concentration of the drug chloramphenicol the number may rise to 1000-2000. 

Plasmids carry a variety of genes which are often useful to bacteria. Some proteins encoded by plasmid genes confer drug resistance to a bacterium. Some are antibiotics. For example, a protein encoded by a gene in plasmid ColEl is toxic to other strains of E. coli. Some plasmids carry genes for enzymes needed for the oxidation of hydrocarbons. Some plasmids contain... 

Plasmids pET28a ColEl Pt7j.c Kan Novagen, Madison, USA... 

Plasma amine oxidase 886 Plasma antithrombin III 177 Plasma membrane 12, 379 Plasmalemma. See Plasma membrane Plasmalogens 383s, 384 Plasmids 5, 248249 ColEl 248 drug resistance 248 Plasmin 634 Plasminogen 634 Plasmodesmata 10... 

Figure 28-8 Simplified scheme for control of replication of the ColEl type plasmid by antisense RNA. The primer for DNA synthesis is RNA II whose initial transcript extends past the replication ori. It is cut by RNase H at ori and then primes replication of the upper strand as shown in the figure. The antisense RNA is RNA I. It hinds to protein Rop whose gene location is also indicated in the figure. Rop assists RNA I and RNA II in undergoing a complementary interaction. However, both RNAs apparently maintain a folded tertiary structure, and only some segments interact. The interaction with the Rop protein evidently in some way prevents initiation of replication until the Rop concentration falls because of replication of the host cell.167,168...

Extrachromosomal DNA molecules called plasmids are harbored in some strains of E. coli. The normal copy number of the plasmids is small, between 2 and 10 however, if these strains of E. coli are grown in the presence of chloramphenicol, up to 3000 copies may be replicated per cell. Plasmid DNA has been demonstrated to be a useful vehicle in molecular cloning. This experiment describes a method for the growth of E. coli and amplification of the ColEl plasmids. The plasmids will be isolated from E. coli cells by one of two methods, a large-scale boiling method or a microscale alkaline lysis method. The DNA plasmids will be measured for molecular size by agarose electrophoresis. 

The presence of ColEl and other plasmids in a bacterial cell may be confirmed by genetic screening of antibiotic resistance. However, it is sometimes necessary to isolate plasmid DNA for further characterization and manipulation. Isolation and purification of plasmids are usually carried out for one of the following reasons ... 

Several methods for isolating plasmid DNA have been developed some lead to a more highly purified product than others. All isolation methods have the same objective, separation of plasmid DNA from chromosomal DNA. Plasmid DNA has two major structural differences from chromosomal DNA (1) plasmid DNA is almost always extracted in a covalendy closed circular configuration, whereas isolated chromosomal DNA usually consists of sheared linear fragments, and (2) ColEl plasmids and other potential vehicles are much smaller than chromosomal DNA. 

Escherichia coli slant, slabs, plate or liquid culture. Recommended strains are RR1, HB101, GM48, 294, SK1591, JC411Thy /ColEl, or CR34/ColEl. The strain must contain a plasmid such as ColEl, pBR322, pUR290, or the pUC series of plasmids. 

Students may use isolated and purified plasmids from Experiment 14. Alternatively, several purified plasmids, including ColEl, pBR322, and pUBllO, are available from commercial sources. The plasmids may be cleaved with various restriction enzymes and the products analyzed by agarose gel electrophoresis. 

Restriction enzymes, 1 unit/pY. Keep stored in freezer until ready to use. Recommended enzymes are BamYtt. and coRI for A. phage DNA,. fooRI and Taq for plasmids (pBR322 or ColEl), and Hpal for Ad 2 DNA. 

As an example, we use RNAI, an antisense repressor of the replication of some Escherichia coli plasmids with a ColEl origin of replication. 

Tomizawa, J.I. (1986) Control of ColEl plasmid replication binding of RNA I to RNA II and inhibition of primer... 

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