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Central Dogma
Proof that DNA Restriction Modification |
Restriction Modification SystemPhage (or viruses) invade all types of
cells. Bacteria are one favorite target. Defense mechanisms have been
developed by bacteria to defend themselves
from these invasions. The system they possess for this defense is the
restriction-modificiation system. This
system is composed of a restriction endonuclease enzyme and a methylase
Restriction enzyme - an enzyme that cuts DNA at internal phosphodiester bonds; different types exist and the most useful ones for molecular biology (Type II) are those which cleave at a specific DNA sequence Methylase - an enzyme that adds a methyl group to a molecule; in restriction-modification systems of bacteria a methyl group is added to DNA at a specific site to protect the site from restriction endonuclease cleavage Several different types of restriction enzymes have been found but the most useful ones for molecular biology and genetic engineering are the Type II restriction enzymes. These enzymes cut DNA at specific nucleotide sequences. For example, the enzyme EcoRI recognizes the sequence:
5' - G A A* T T C - 3' *The site of methylation protection from restriction enzyme cleavage is the 3' adenine. This enzyme always cuts between the 5'
G and A residues. But if we look at the sequence we can see that both
5' - G A A T T C - 3' Not all Type II restriction enzymes generate
staggered ends at the target site. Some cut and leave blunt ends. For
5' - T G G C* C A - 3' is cut at the point of symmetry to produce:
5' - T G G C C A - 3' (Note: * The site of methylation protection from restriction enzyme cleavage; 5' cytosine) We began this discussion by stating that the restriction-modification system is used to protect bacteria from invasion by viral DNA molecules that may subvert the gene expression system of the bacteria to its own use. But how does this system actually work? The bacterial cell uses the restriction enzyme to cut the invading DNA of the virus at the specific recognition site of the enzyme. This prevents the virus from taking over the cellular metabolism for its own replication. But bacterial DNA will also contain sites that could be cleaved by the restriction enzyme. How is the bacterial cell protected? This
protection is offered by the action of the methylase. The methylase
Methylation of Plant DNA and Its Effect on Restriction DigestionThe following discussion is based on the
experiments described by Grenbaum et al. in the paper "Methylation of
It was noted that 5-methyl cytosine (5mC)
is found to be a component of plant DNA much more frequently than
What is the experimental explanation for such a high level of 5mC? 1. 5mC occurs at 70-80% of the 5'-CG-3' dinucleotides. What are the occurrences of the dinucleotide in the two kingdoms?
2. 5mC also occurs at the 5'-CXG-3' sequence in plants but does not occur in animals. How often are these sequences methylated in plants?
What ramifications does this have for performing restriction digests of plant? Restriction digestion and subsequent hybridizations are important for genomic and RFLP analysis of plants. For this analysis to be informative, the DNA must be digested to completion. Thus it is important to chose the correct enzymes for analysis. Because plant DNA is highly C-methylated, it is important to chose an enzyme for digestion experiments that are not affected by C- methylation. Do Not Use These Enzymes to Analyze Plant DNA 1. Restriction site has 5'-CXG-3'
(Base preceeding the * is methylated.) 2. C at or near end of site;if next base in DNA is G, it will be methylated
(Base preceeding the * is methylated.) Use These Enzymes Instead to Cut Plant DNA
(Base preceeding the * is methylated.)
Copyright © 1998. Phillip McClean |
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