Plasmids are similar to viruses

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..........plasmids as dna Vectors.........

Plasmids are similar to viruses, but lack a protein coat and cannot move from cell to cell in the same fashion as a virus.

Plasmid vectors are small circular molecules of double stranded DNA derived from natural plasmids that occur in bacterial cells. A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for the same restriction endonuclease.

The plasmid and the foreign DNA are cut by this restriction endonuclease (EcoRI in this example) producing intermediates with sticky and complementary ends. Those two intermediates recombine by base-pairing and are linked by the action of DNA ligase. A new plasmid containing the foreign DNA as an insert is obtained. A few mismatches occur, producing an undesirable recombinant.

The new plasmid can be introduced into bacterial cells that can produce many copies of the inserted DNA . This technique is called DNA cloning.

the plasmids can replicate independently of the genome, and are found in numbers ranging from one per cell to hundreds per cell (this is called "copy number"). Plasmids frequently carry genes for antibiotic resistance. While antibiotic resistance is becoming an increasingly important problem medically, it is a useful marker in Recombinant DNA technology. Such markers, along with the small size and potentially high copy number, make plasmids indispensible tools in Molecular Biology. The following figure is an example of a typical plasmid, called pACYC184, which is 4,240 base pairs (bp, or 4.24 kilobases, kb).
This plasmid map has 3 marked regions on it. Two of them are antibiotic resistance genes, one for Tetracycline resistance (Tc) and the other for Chloramphenicol resistance (Cm). The arrows denote the direction the genes are transcribed. Remember that transcription can only occur in the 5' --> 3' direction, b

ut plasmids are double-stranded, so the direction needs to be explicitly stated so it is known which strand is used as template for transcription. The other marked region (ori) is the origin of replication, the region where the DNA replication machinery assembles

Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.

Genes on plasmids with high numbers of copies are usually expressed at high levels. In nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one or more antibiotics.

Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes.

An Example

(courtesy of David Miklos and Greg Freyer of the Cold Spring Harbor Laboratory, who used these plasmids as the basis of a laboratory introduction to recombinant DNA technology that every serious biology student — high school or college — should experience!)

pAMP

4539 base pairs
a single replication origin
a gene (amp^r)conferring resistance to the antibiotic ampicillin (a relative of penicillin)
a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5'
that, as we saw above, is cut by the restriction enzyme BamHI
a single occurrence of the sequence
5' AAGCTT 3'
3' TTCGAA 5'
that is cut by the restriction enzyme HindIII

Treatment of pAMP with a mixture of BamHI and HindIII produces:

a fragment of 3755 base pairs carrying both the amp^r gene and the replication origin
a fragment of 784 base pairs
both fragments have sticky ends

pKAN

4207 base pairs
a single replication origin
a gene (kan^r) conferring resistance to the antibiotic kanamycin.
a single site cut by BamHI
a single site cut by HindIII

Treatment of pKAN with a mixture of BamHI and HindIII produces:

a fragment of 2332 base pairs
a fragment of 1875 base pairs with the kan^r gene (but no origin of replication)
both fragments have sticky ends

These fragments can be visualized by subjecting the digestion mixtures to electrophoresis in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates toward the positive electrode (anode) when a direct current is applied. The smaller the fragment, the farther it migrates in the gel.

the images are selected from many sites in the web.