In the final step, they transplanted the synthetic M. In this Spotlight, you'll find a broad range of resources to help you gain a deeper understanding of how restriction enzymes affected the field of molecular biology and our ability to manipulate DNA, as well as how they continue to serve as an invaluable tool for research scientists.
Watch scientists answer questions about the fundamentals of these fascinating enzymes. Read about the discovery of REs and how scientists use them. Read about how REs operate at the molecular level and how they interact with DNA at the structural level.
Learn how REs are used for hereditary disease diagnosis, paternity testing, and forensics. Watch a video about how REs helped sequence the human genome. Learn how REs play an important role in creating genetically modified organisms. Read about how REs helped build a synthetic bacterial cell.
Since , this database has organized information about REs, methylases, and the bacteria they originated from. Watch Hamilton Smith, Nobel laureate for his seminal RE research, discuss the future of synthetic genomes with a student.
New England Biolabs. Sponsorship Close. Restriction Enzymes. Email your Friend. Submit Cancel. What are Restriction Enzymes? Hear from Scientists. This page has been archived and is no longer updated. Swiss microbiologist Werner Arber was one of the recipients of the Nobel Prize in Physiology or Medicine, an award he earned for his discovery with Stuart Linn of restriction enzymes, otherwise known by his daughter Sylvia as "servants with scissors.
Bacteriophages are viral particles that invade bacteria and replicate their own DNA independently of the bacterial chromosomal DNA. Those phages that grew poorly were said to be "restricted" by their host. Arber wanted to know why. Arber proposed that bacterial cells in this case, E. Specifically, he theorized that only those bacteriophages that had previously been in contact with the same bacterial strain could successfully infect new host cells, and that the previous exposure somehow modified the phage DNA in a way that protected it from restriction.
Phages with unmodified DNA, on the other hand, were immediately broken down by enzymes. This occurred because the host cell enzymes recognized these phages as foreign, cleaving their DNA and restricting their growth. Arber further proposed that there were specific sites in the genome at which restriction activities occurred.
Arber and Linn referred to the enzyme responsible for this "endonucleolytic scission" as endonuclease R, a name later changed to EcoB. It didn't take long for other scientists to identify a second restriction enzyme in E. Soon after the discovery of EcoB and EcoK, microbiologists Hamilton Smith and Kent Wilcox isolated and characterized the first restriction enzyme from a second bacterial species , Haemophilus influenzae.
The first three letters of a restriction enzyme's name are abbreviations of the bacterial species from which the enzyme has been isolated e. Roman numerals are also used as part of the name when more than one restriction enzyme has been isolated from the same bacterial strain. Today, scientists recognize three categories of restriction enzymes: type I, which recognize specific DNA sequences but make their cut at seemingly random sites that can be as far as 1, base pairs away from the recognition site; type II, which recognize and cut directly within the recognition site; and type III, which recognize specific sequences but make their cut at a different specific location that is usually within about 25 base pairs of the recognition site.
As originally postulated by Arber, all restriction enzymes serve the purpose of defense against invading viruses. Bacteria protect their DNA by modifying their own recognition sequences, usually by adding methyl CH 3 molecules to nucleotides in the recognition sequences and then relying on the restriction enzymes' capacity to recognize and cleave only unmethylated recognition sequences.
Also, as Arber suspected, bacteriophages that have previously replicated in a particular host bacterial strain and survived are similarly modified with methyl-labeled nucleotides and thereby protected from cleavage within that same strain.
Within just a few years of the initial discoveries of EcoB, EcoK, and HindII, scientists were already testing ways to use restriction enzymes. The first major application was as a tool for cutting DNA into fragments in ways that would make it easier to study and, in particular, identify and characterize genes. A second major use was as a device for recombining, or joining, DNA molecules from different genomes, usually with the goal of identifying and characterizing a gene or studying gene expression and regulation Heinrichs, Nathans and Danna then used the enzyme to cut, or digest, the DNA of the eukaryotic virus SV40 into 11 unique linear fragments.
Found in both monkeys and humans, SV40 has the capacity to cause tumors and was being intensively studied at the time for its cancer-causing potential. Finally, they separated the fragments using gel electrophoresis , a technique developed in the s and still commonly used as a way to sort nucleic acid molecules of different sizes Figure 1. Clearly, he must have had a vision at the very beginning of this that just the simple idea of being able to separate the fragments of viral DNA into specific pieces would have enormous applications" Brownlee, Today, scientists still use restriction enzyme digestion, followed by electrophoresis , as a way to separate DNA fragments.
Many scientists also use what is known as a probe , or a DNA or RNA molecule with a base sequence that is complementary to a DNA sequence of interest, to identify where in the genome i. This basic procedure is outlined in Figure 2. After separating the DNA fragments through electrophoresis, the fragments are transferred from the gel to a solid medium, or membrane. When DNA fragments are separated and transferred in this manner, the process is known as Southern blotting , named after the scientist who developed the technique, Edwin Southern Southern, After transfer, the membrane is immersed in a solution of either radioactive or chemically labeled probes.
The probes bind to their complementary sequences on the membrane, if any are present. The membrane is then washed, leaving only bound probes that can be detected using autoradiography , if the probes are radioactive, or other means. At the time, scientists had identified the specific site and sequence of cleavage for only one restriction enzyme, HindII.
With HindII, cleavage occurred in the middle of a six-base-pair recognition site, yielding what are known as blunt-end fragments see Figure 3, in which PvuII similarly produces blunt-end fragments. Mertz and Davis discovered that another restriction enzyme, EcoR1, by contrast, cleaves its recognition site in a staggered way that generates fragments with single-stranded overhanging ends known as cohesive, or sticky, ends.
After two fragments with complementary sticky ends are joined, the DNA backbone may be covalently sealed using another enzyme called DNA ligase. This gives molecular biologists powerful tools to create nearly limitless combinations of recombinant DNA. Far fewer Type I, III and IV restriction enzymes have been characterised, but studies of these enzymes are providing rich information on DNA—protein interactions and catalysis, protein family relationships, control of restriction activity and plasticity of protein domains.
In Salvador Luria and his graduate student Mary Human based at the University of Illinois noted a strange phenomenon when conducting experiments to study the breakup of DNA in phage-infected bacteria. They had started their work using Escherichia coli and then switched to the use of Shigella bacteria following an accidental breaking of their test tube full of phage-sensitive Escherichia coli culture.
To their surprise they observed that exposure to Shigella prevented the growth of the phages viruses in Escherichia coli but not in other bacteria species. In a series of further experiments with other phages and a range of Escherichia coli and Shigella hosts they noted that some phages disappeared and then reappeared when cultured with different bacteria and that the phages could once again grow in the original host one generation later.
Whatever had caused the modification in the viruses, they concluded, had not been a genetic mutation because it was not a permanent change. Rather it seemed to be caused by the host bacteria. They labelled the process a 'host induced' genetic variation, later called restriction-modification phenomenon. In Werner Arber and his doctoral student, Daisy Dussoix, based on experiments they had conducted with with lambda phage, proposed the phenomenon could be explained by restriction and modification enzymes produced by bacteria to defend themselves against invading viruses.
When elaborating on this, Arber hypothesised that bacteria produced a digestive enzyme which cut viral DNA into smaller pieces at specific sites and an enzyme to catalyse methylation, or modification, of its own DNA to protect it from the restrictive enzyme. In Arber argued that should it be possible to isolate and characterise restriction enzymes, they could be used as a laboratory tool to cleave DNA. Three years later Werber and his postdoctoral student Stuart Linn identified the first restriction enzyme EcoB in Escherichia coli and demonstrated its action.
Soon after, in July , Hamilton Smith and Kent Wilcox announced that they had isolated and characterised a restriction enzyme HindII in a second bacterial species, Haemophilus influenza, and demonstrated that it degraded the DNA of a foreign phage.
This confirmed Arber's hypothesis that restrictive enzymes are highly selective in where they make their cuts. Within a short time the basic action of restriction enzymes had been understood and in Smith's colleague, Daniel Nathans and his postgraduate student Kathleen Danna demonstrated HindII cleaved DNA of the SV40 virus into 11 well defined fragments and how o piece these fragments together to construct the complete genetic map of SV40 DNA. This laid the foundation for the adoption of restriction enzymes for DNA research.
Restriction enzymes are used for many different purposes in biotechnology. They are named after the genus and species of the organism they were isolated from and are given a number to indicate the order in which they were found. DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix.
Sma I is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end. Other restriction enzymes, like Eco RI , cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.
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