Given the vast genetic diversity among bacteria, it follows that different bacterial strains express different restriction enzymes, allowing them to balance their own genes against those of invading bacteriophages.
The known variety of restriction enzymes is staggering: To date, more than 4, different restriction enzymes that collectively recognize more than different recognition sequences have been isolated from a wide variety of bacterial strains. Based on DNA sequence analysis, scientists know that there are many more restriction enzymes out there waiting to be characterized.
The recognition sequences of these enzymes are typically four to six base pairs in length, and they are usually palindromic, which means that their recognition sequence reads the same in the 5' to 3' direction on both DNA strands. There are four different categories of restriction enzymes.
Type I restriction enzymes cut DNA at random locations far from their recognition sequence, type II cut within or close to their recognition sequence, type III cut outside of their recognition sequence, and type IV typically recognize a modified recognition sequence.
Type II restriction enzymes, which cut within their recognition sequence, are the most useful for laboratory experiments. When they act on a DNA molecule, restriction enzymes produce "blunt" ends when they cut in the middle of the recognition sequence, and they yield "sticky" ends when they cut at the recognition sequence in a staggered manner, leaving a 5' or 3' single-stranded DNA overhang. Any two blunt ends can be joined together, but only sticky ends with complementary overhangs can be connected to each other.
Restriction enzyme digestion continues to be one of the most common techniques used by researchers who carry out DNA cloning experiments. Today, researchers rely on restriction enzymes to perform virtually any process that involves manipulating, analyzing, and creating new combinations of DNA sequences.
Among the many new combinations are DNA cloning, hereditary disease diagnosis, paternity testing, forensics, genomics e. Indeed, without the discovery of restriction enzymes, the fields of recombinant DNA technology, biotechnology, and genomics as we know them today would not exist.
In , forty years after he purified the first restriction enzyme, Smith was part of the research team that used these very enzymes to build the first synthetic bacterial cell. Led by Craig Venter, this team of scientists used machines to chemically synthesize the one million base-pair Mycoplasma mycoides M. Along the way, Venter and his colleagues used restriction enzymes to help clone and analyze the synthetic genome.
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. 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. Today, scientists are mixing and matching DNA fragments from different species in ways that continue not only to demonstrate the power of this method, but also to raise serious ethical and social questions.
Arber, W. DNA modification and restriction. Annual Review of Biochemistry 38 , — Brownlee, C. It was the first bacterial genome to be deciphered. Later on he helped in the genomic sequencing efforts for the fruit fly and humans at Celera Genomics. Arber, 'Host-controlled modification of bacteriophage', Annual Review Microbiology, 19 , They found that bacteria protect themselves against invading viruses by producing two types of enzymes.
One cut up the DNA of the virus and the other restricted its growth. Arber believed these two enzymes could provide an important tool for cutting and pasting DNA, the method now used in genetic engineering. Restriction enzymes are now workhorses of molecular biology. They are essential in the development of recombinant DNA and were pivotal to the foundation of the biotechnology industry. Arber was the first to discover the enzymes; Smitth demonstrated their capacity to cut DNA at specific sites and Nathans showed how they could be used to construct genetic maps.
With their ability to cut DNA into defined fragments restriction enzymes paved the way to the development of genetic engineering. They are produced as part of an effort to generate restriction-modification enzymes with longer recognition sites without having to screen bacteria and microorganisms. Kim, Cha, Chandrasegaran Johns Hopkins University Nathans was the first scientist to demonstrate how restriction enzymes could be used to cleave DNA and how to piece together its fragments to construct a complete map of DNA.
Werner Arber was born in Granichen, Switzerland. First observation of the modification of viruses by bacteria. Idea of restriction and modification enzymes born. Werner Arber predicted restriction enzymes could be used as a labortory tool to cleave DNA. First restriction enzyme isolated and characterised.
The cuts are always made at specific nucleotide sequences. Different restriction enzymes recognise and cut different DNA sequences. Restriction enzymes are found in bacteria. Bacteria use restriction enzymes to kill viruses — the enzymes attack the viral DNA and break it into useless fragments. Like all enzymes, a restriction enzyme works by shape-to-shape matching. When it comes into contact with a DNA sequence with a shape that matches a part of the enzyme , called the recognition site , it wraps around the DNA and causes a break in both strands of the DNA molecule.
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