实验十一 ＤＮＡ的酶切
实验十一 ＤＮＡ的酶切
Restriction Endonucleases: An Overview Restriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this "restriction" of viral growth stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these "restriction enzymes" to be purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific sites, generating discrete, gene-size fragments that could be re-joined in the laboratory. Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression. As the use of restriction enzymes spread among molecular biologists in the late 1970’s, companies such as New England Biolabs began to search for more. Except for certain viruses, restriction enzymes were found only within prokaryotes. Many thousands of bacteria and archae have now been screened for their presence. Analysis of sequenced prokaryotic genomes indicates that they are common-all freeliving bacteria and archaea appear to code for them. Restriction enzymes are exceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to the giant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have been purified and characterized, more than 250 different sequence-specificities have been discovered. Of these, over 30% were discovered and characterized at New England Biolabs
Restriction Endonucleases: An Overview Restriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this "restriction" of viral growth stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these "restriction enzymes" to be purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific sites, generating discrete, gene-size fragments that could be re-joined in the laboratory. Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression. As the use of restriction enzymes spread among molecular biologists in the late 1970’s, companies such as New England Biolabs began to search for more. Except for certain viruses, restriction enzymes were found only within prokaryotes. Many thousands of bacteria and archae have now been screened for their presence. Analysis of sequenced prokaryotic genomes indicates that they are common-all freeliving bacteria and archaea appear to code for them. Restriction enzymes are exceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to the giant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have been purified and characterized, more than 250 different sequence-specificities have been discovered. Of these, over 30% were discovered and characterized at New England Biolabs
The search for new specificities continues, both biochemically, by the analysis of cell-extracts, and computationally, by the analysis of sequenced genomes. Although most activities encountered today turn out to be duplicates-isoschizomers-of existing specificities, restriction enzymes with new specificities are found with regularity. Beginning in the early 1980’s, New England Biolabs embarked on a program to clone and overexpress the genes for restriction enzymes. Cloning improves enzyme purity by separating enzymes from contaminating activities present in the same cells. It also improves enzyme yields and greatly simplifies purification, and it provides the genes for sequencing and analysis, and the proteins for x-ray crystallography. Restriction enzymes protect bacteria from infections by viruses, and it is generally accepted that this is their role in nature. They function as microbial immune systems. When a strain of E.coli lacking a restriction enzyme is infected with a virus, most virus particles can initiate a successful infection. When the same strain contains a restriction enzyme, however, the probability of successful infection plummets. The presence of additional enzymes has a multiplicative effect; a cell with four or five independent restriction enzymes could be virtually impregnable
The search for new specificities continues, both biochemically, by the analysis of cell-extracts, and computationally, by the analysis of sequenced genomes. Although most activities encountered today turn out to be duplicates-isoschizomers-of existing specificities, restriction enzymes with new specificities are found with regularity. Beginning in the early 1980’s, New England Biolabs embarked on a program to clone and overexpress the genes for restriction enzymes. Cloning improves enzyme purity by separating enzymes from contaminating activities present in the same cells. It also improves enzyme yields and greatly simplifies purification, and it provides the genes for sequencing and analysis, and the proteins for x-ray crystallography. Restriction enzymes protect bacteria from infections by viruses, and it is generally accepted that this is their role in nature. They function as microbial immune systems. When a strain of E.coli lacking a restriction enzyme is infected with a virus, most virus particles can initiate a successful infection. When the same strain contains a restriction enzyme, however, the probability of successful infection plummets. The presence of additional enzymes has a multiplicative effect; a cell with four or five independent restriction enzymes could be virtually impregnable
Restriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cell’s own DNA from cleavage by the restriction enzyme. Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it. Together, a restriction enzyme and its "cognate" modification enzyme(s) form a restriction-modification (R-M) system. In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification enzyme
Restriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cell’s own DNA from cleavage by the restriction enzyme. Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it. Together, a restriction enzyme and its "cognate" modification enzyme(s) form a restriction-modification (R-M) system. In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification enzyme
Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level there are many more than three different kinds. Type I enzymes are complex, multisubunit, combination restriction-andmodification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns
Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level there are many more than three different kinds. Type I enzymes are complex, multisubunit, combination restriction-andmodification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns