Mobile genetics elements (viruses and plasmids)

Plasmids:
-Extrachromosomal molecules of cirucular, double stranded DNA
-Carry from a few to a few dozen genes
-FOund in many prokaryotes, some yeast, ...
-PLasmids usually confer some useful property to host
*Can carry genes that enable bacteria to live in inhospitable environments
-Contaminated soil with toxic metals
-Industrial chemicals, petroleum by-products
-COmpetition with other bacteria
*Can carry resistance factors that destroy/modify antibiotics
*Can carry genes for transferring plasmid via conjugation
-Have their own origin of replication
*REplication is not synchronized with replication of host chromosome, but wil
l not happen in absense of host machinery
-Can be present in multiple copies in cell
*"Copy number" varies
*Naturally occuring plasmids: copy number is 1, 2, or 10
-Can be moved between or among species of bacteria
*Major consequence: antibiotic resistant bacteria
-PLasmids also have beneficial uses in genetic engineering
*Modified plasmids (and ohages( can be used as transport molecules to move DN
A between cells
-Modified carriers are called vectors
-We can use endonucleases to "open" the circular DNA, insert new fragmen
t of double strnaded DNA, and then use
ligase to "close" the cirlce back up
*Endonucleases can cut DNA strands anywhere inside the strand
-THe nucleases we use are found naturally in bacteria
-Called restriction enzymes too
Restriction enzymes (endonucleases)
-Found in most bacteria
-None in eukaryotes
-Made by the bacteria to degrade foreign DNA
*Given names based on organism which produced it
-Ex. EcoRI from E. coli
-Cut double stranded DNA in vvery specific areas
*Called recognition sequences or sites
*Usually 4 or 6 base pairs, but can be 5, 8, or 12, etc
-Even uf one bp is changed, enzyme will not cut that site
-QUESTION: If restriction enzymes recognize specific sequences in double strande
d DNA, and bacteria have genomes of double
stranded DNA, why don't the restriction enzymes degrade host DNA?
*Bacteria modifies its own DNA to mark it
-Is methylated in host
-What's notable about restriction sequences?
*Are palindromes for both strands of DNA
-REads the same in the 5'-3' direction on both strands
-Type II restrction enzymes are used
-EX. 5' - GGATCC - 3'
3' - CCTAGG - 5'
-When they cut double strnaded DNA, two types of ends are possible
*Sticky or staggered
-CAn be brought together and religated
*Blunt
-Recogniztion sequences occur randomly throughout the genome
*IF you know how roughly how often they cut, andyo know roughly how many base
pairs there are in a given
DNA molecule, you can estimate the size and number of frangments that will r

esult from complete digestion

witha given enzyme
-FInd probability of finding that sequence
*EX. AATT -> 1/4 x 1/4 x 1/4 x 1/4 = 1/265
-Multiply with number of base pairs
*SO you can use restriction enymes to cut large DNA molecules into smaller s
ized fragments
THe process of replicating rexombinant DNA in a high copy number vector is calle
d cloning
-Amplifying copies of a DNA copies