Progress in any scientific field purely depends on the techniques and methods available for the conduction of experiments. The one such progress which has revolutionised the scientific world is through the emergence of genetic engineering. Genetic engineering is the biochemical manipulation of genes, that is poised to usher in the era of both animal and plant breeding. There are several terms coined to genetic engineering. Some of them include gene manipulation, gene cloning, genetic modification and recombinant DNA technology. It is one of the fastest emerging areas because it is now routine practice to isolate a gene to determine its biological function and its base sequence. It finds numerous applications in forensic science, genome sequencing and biotech industries. This discovery has put the life sciences in a highly enviable position. There is a growing awareness about the vast potential that genetic engineering has for humanity.
The basic principles of gene manipulation are very simple despite involving diverse and complex techniques. Basically, the technique involves four steps: generation of DNA fragments of interest, joining the fragment to a molecular vehicle (vector), introduction of the recombinant/chimaeric DNA into a host cell for amplification and selection of the gene of interest. Techniques for manipulating prokaryotic, as well as eukaryotic DNA, witnessed a remarkable progress during the later part of the 20th century. Manipulation essentially involves the breakage and rejoining of DNA fragments. The resulting end product produced is the recombinant DNA, bringing about the recombination, which forms the core of genetic engineering. Using this technique, genetic changes can be induced in the cells, which alter the characteristics of the organisms at the cellular and molecular level and to design forms of life that possess desired traits.
Before we consider the actual techniques involved in genetic engineering, it would be worthwhile to consider the important properties of the molecule to be manipulated. Changes in the base sequence produce mutations, which have a profound effect on the protein synthesis.
2.1 DNA modifying enzymes
Restriction enzymes and DNA ligases represent the cutting and joining functions in DNA manipulation. All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes. These enzymes are involved in the degradation, synthesis and alteration of the nucleic acids. They are broadly categorized as nucleases, polymerases and enzymes modifying ends of DNA molecules.
Nucleases are enzymes that degrade the DNA molecules by breaking phosphodiester bonds that hold nucleotides together. There are two types of nucleases: endonucleases act on internal phosphodiester bonds within a DNA molecule, while exonucleases degrade DNA and remove nucleotides from the end of the DNA molecule (FIGURE 1). Restriction enzymes are good examples of endonucleases which forms the cutting tool of genetic engineering.
Figure 1. Types of nucleases and its action. Endonucleases cleave the double-stranded DNA at specific sequences internally while exonuclease chew up nucleotides from either end of the double-stranded DNA.
3.1 Discovery of restriction endonucleases
The major problem in dealing with gene manipulation is the relative uniformity of the DNA molecule. Due to its large size and lack of complexity, studying single gene became a daunting task; Untill the 1970s, there were no molecular techniques to cleave the DNA molecules at specific sequences. The methods available for DNA fragmentation were non-specific. The sole method (with a degree of control) was the use of mechanical shearing. Restriction endonucleases were discovered when carrying out experiments with bacteriophages and bacteria. The ability of a bacteriophage to infect two different strains of E.coli (namely strain B and strain K) were analysed during the experiment. Firstly, bacteriophages were incubated with these two strains of bacteria and their ability to kill the bacterial cells was observed. Generally, infection of bacteria by bacteriophages, results in the release of millions of bacteriophage. In the first experiment, which involved the strain B, the spilling out phages were isolated and allowed to re-infect the bacteria, which showed a successful re-infection of strain B cells. But when the same experiment was repeated with the E.coli strain K, very few phages were found to undergo replication. As the number of incubations with the E.coli strain K cells were increased, the ability to infect and kill the E.coli strain K cells increased. In addition, the bacterial cells which showed a high degree of infectivity over E.coli strain K cells reduced its ability to infect the E.coli strain B cells (FIGURE 2).
Figure 2. Experimental proof for the discovery of restriction enzymes. Bacteriophages were incubated with Strain B and Strain K cells. Incubation with Strain B cells resulted in the release of millions of bacteriophage which were isolated and allowed to reinfect the Strain B cells. While incubation with Strain K cells resulted in a less number of phages at the beginning but increased after several incubations. At the same time, Strain K cells reduced its ability to infect Strain B cells.
From these experiments, researchers came to two conclusions. Firstly, both the E.coli strains developed a sort of restrictive mechanism, which modulated the ability of the bacteriophage to infect these strains. The restrictive mechanism involves the modification of host DNA by numerous ways. One way of modification was through the addition of methyl group to cytosine nucleotide; a modification known as methylation. These modifications aid in recognizing and degrading the foreign DNA. To achieve this function a set of enzymes known as Restriction endonucleases were discovered, which were able to 'chop' the DNA but not the methylated DNA (FIGURE 3). For this remarkable discovery three scientists, Werner Arber of the Basel University for his experiments on the existence of restriction endonucleases; Hamilton Smith of John Hopkins University for his discovery of a restriction enzyme; and Daniel Nathans of John Hopkins University for his utility of restriction enzymes were awarded with Nobel Prize in the field of Medicine in 1978.
Figure 3. Activity of restriction and methylase enzymes. Restriction enzyme EcoRI cleaves within the recognition sequence if the DNA is unmethylated. On methylation by methylases, the restriction enzyme EcoRI is inhibited from cleaving within the restriction site.
Restriction endonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the bacterial enzymes that can nick DNA at specific nucleotide sequences. They were first discovered in E.coli, in which bacteriophage replication was restricted by cutting the viral DNA. Thus the enzymes that restrict the viral replication are known as restriction enzymes or restriction endonucleases.
A number of restriction endonucleases have been isolated from bacteria and some of them are commercially available (TABLE 1). The first restriction enzyme isolated was from E.coli K laboratory by Meselson and Yuan in 1968. But this enzyme was unable to use as it cleaved at non-specific sequences. Hamilton Smith and his coworkers, in 1970, isolated a restriction enzyme from Haemophilus influenza strain Rd. They showed that the enzyme was able to recognize a six base pair double-stranded DNA and cleave at specific sequences on DNA. The enzyme was designated as HindIII.
Following the discovery of HindII, yet another restriction enzyme, EcoRI was isolated from E.coli strain RY13. Over 900 restriction enzymes have been isolated till date from more than 230 species of bacteria.
A vast majority of restriction enzymes have been isolated from bacteria, which serve as a host-defence role. The advantage of the restriction enzyme is that any foreign DNA (from an infecting virus) could be attacked by the restriction enzymes. This raises the question: How is the host DNA protected from restriction enzyme attack? To avoid digestion of host DNA, bacterium synthesize another set of enzymes known as DNA methyltransferases, which methylates target DNA sequences and hence protects its DNA. This combination of restriction endonuclease and methylase is called restriction-modification system.
These occur among Eubacteria and Archea. The major biological function of these restriction-modification system is the protection of host genome against foreign genetic material. Some bacterophages possess their own methyltransferases with similar specificity to other DNA modification enzymes like hydroxymethyltransferases or glucosylases. These make them resistant to the different types of restriction endonucleases.
Restriction endonucleases are classified based on its subunit components, cofactors and enzymatic mechanism. Restriction endonucleases are very stable and all require an ionic environment (sulfhydryl agent along with Mg2+) for its enzymatic activity. They also work at an optimum temperature of 37 °C. There are three different types of restriction-modification systems namely, type I, type II and type III (TABLE 2). All the three types of restriction -modification systems are found in E.coli, H. influenza, B. subtilis and other bacteria.
Eukaryotic endonucleases do not possess the properties of restriction endonucleases. But, few site-specific recombinases like homing endonucleases possess the features of restriction endonucleases.
The type I occupies first place in its characterization. The best and well known example studied under type I is from E.coli K12. These type I enzymes are multifunctional enzymes capable of both the restriction and modification functionalities. They have a molecular weight of 400kDa or still greater possessing three protein subunits known as HsdR, HsdM and HsdS. HsdR is required for restriction; HsdM helps in transferring methyl groups and finally HsdS has dual role in which it is required for specificity of cut site recognition in addition to methyltransferase activity. The restriction enzymatic activity requires Mg2+, ATP and S-adenosyltransferase (Ado-met) as cofactors while methylation activity requires only Ado-met and the reaction is stimulated by Mg2+ and ATP. But the major drawback of this type I system is that the methylation and cleavage is both performed by the same enzyme and hence less preferred in gene manipulation studies.
The type II are the most useful restriction-modification system. Type II restriction endonucleases include EcoRI system. These are simpler enzymes with a less molecular weight of 80kDa. The restriction enzyme activity requires Mg2+ while methylation needs Ado-met. Both these activities are functionally controlled by two distinct proteins but within the same recognition sequence. Type II are entirely ATP-independent enzymes. They are further classified into various subtypes (TABLE 3).
Subtypes & Examples
They have many advantages over type I and type III restriction modification systems. In type II, restriction and modifications are catalyzed by separate enzymes and this activity do not require the cofactors. Also, type I and III make cleavage at a distance away from the recognition site while type II cleaves at specific sites within the recognition sequence.
The Type III system also shares the same features as the type I system, but are less considered in gene manipulation. Type III restriction endonucleases have a molecular weight of 200kDa and require Mg2+, Ado-met and ATP. The cleavage takes place at a distance away from the recognition site and the methylation activity requires the Ado-met.
Restriction-modification systems carry genes that encode for both restriction endonuclease and methyltransferase. Both the enzymes recognize the same sequence. Type II restriction-modification systems are encoded by genes found on both chromosome and extra-chromosomal genetic material, e.g. a plasmid. However, the genes that encode restriction enzyme and methyltransferase lie adjacent to each other. In rare cases, the genes are separated by certain open reading frame (ORF) regions. Over 100 restriction enzymes and 150 methyltransferase genes have been sequenced till date. The sequencing do not show any homology between restriction endonuclease and methyltransferase genes. Type II restriction-modification system are not lost from their host cell. Those cells that lose a restriction-modification system are unable to modify recognition site and hence can not be protected from the restriction enzymes. In some cases, a strain might lose its restriction endonuclease activity but their methyl transferase activity remains unaffected.
The methyltransferase needs AdoMet as a substrate for transferring the methyl group either to cytosine or an adenine residue within the recognition sequence. There are three different types of methylations:
These methylations are brought about by three site-specific DNA methylases:
The methylase encoded by the Dam gene, transfers a methyl group from S-adenosylmethionine to N6 of the adenine in GATC. The methylase which is encoded by the Dcm gene modifies the cytosine residues at C6 position in the sequence CCAGG and CCTGG. Methylations at these positions allow the DNA to become resistant to the restriction enzymes. In eukaryotes, CpG methyltransferase modifies the cytosine in sequences containing the dinucleotide CpG. Methylation plays a significant role during development through tissue-specific inactivation of genes.
The discovery of restriction endonucleases came with a uniform nomenclature. This naming system was based upon the proposals of Nathans and Smith. The following proposals were later amended by others. The proposals are as follows:
1. The species name of the organism is identified by the first letter of the genus and the first two letters of the species name to form a three letter abbreviation. These letters are disgnated in italics. For example, Escherichia coli - Eco and Haemophilus influenza - Hin.
2. Strain and type of the host was identified using a subscript. e.g. EcoK. The viral or plasmid-specified restriction and modification system are identified by the species name of the host while the extrachromosomal element is represented by a subscript. e.g. EcoPI
3. Multiple restriction and modification systems in a particular host strain are identified by the Roman letters. For example, H. influenzae with the strain Rd has three restriction-modification system represented as HindI, HindII, HindIII etc.
EcoRI is from Escherichia (E) coli (co), strain Ry13 ® and first endonuclease (I) to be discovered. HindIII is from Haemophilus (H) influenzae (in), strain Rd (d) and the third endonucleases (III) to be discovered.
All restriction enzymes require specific sequences (substrates), which are more or less double-stranded DNA, for its action. These substrates are known as recognition sequences or target sites.
Some of the important characteristics of recognition sequences:
1. Length of recognition sequences varies. For example, EcoRI recognizes sequence of six base pair in length while NotI recognizes a sequence of eight base pair in length
2. Different restriction enzymes have the same recognition sequence. These are better known as isoschizomers. For example, SphI and BbuI restriction enzymes has the same recognition sequence.
3. Recognition sequences can either be ambigous or unambigous.
4. Recognition sequence of one enzyme contains recognition sequence for the other. For example, the restriction enzyme BamHI also contains the recognition sequence for another enzyme Sau3AI.
5. Recognition sequences are palindromic. Palindromic sequences are those, which read the same on both directions. There are two types of palindromic sequences: mirror-like palindromic sequence and inverted repeated palindrome. In the former case, the sequence reads the same in both directions on the same strand, as in case of single-stranded DNA. In the latter case, the sequence reads the same in both directions, but they are found in complementary DNA strands, as in case of double-stranded DNA.
The symbol ‘/’ is used for the position at which a restriction enzyme cuts within the sequence; whereas, an asterisk is used for the methylated nucleotide.
Several factors affect the activity of restriction endonucleases. Under extreme pH and low ionic strength, these enzymes are unable to recognize the defined recognition sequence. These altered activity of restriction endonucleases are known as Star activity. Some of the altered activity includes substitution of bases and truncation of nucleotides in the recognition sequence.
The restriction enzymes generate three different types of ends after cleavage within the recognition sequences.
1. Sticky ends: The restriction enzymes cut asymmetrically within the recognition sequences leaving single -stranded overhangs and are also called as cohesive ends. There are two types of sticky ends.
a) 5’ overhangs: The restriction enzymes cut asymmetrically within the recognition sequence in such a way that it leaves an overhanging short single stranded 5’ end. For example,
b) 3’ overhangs: The restriction enzymes cut asymmetrically within the recognition sequence in such a way that it leaves an overhanging single-stranded 3’ ends. For example,
But, the limitation of the sticky end is that they only stick to fragments which are compatible. For example, two EcoRI fragments can join together but EcoRI cannot join with any other fragments produced by other restriction enzyme.
2. Blunt ends: The restriction enzymes cleaves exactly on the same site on both the strands without leaving any overhangs. For example,
Majority of restriction endonucleases (particularly type II) cut DNA at specific sites within recognition sequence. A selected list of enzymes, recognition sequences and their products having either sticky or blunt ends formed are given in TABLE 4. The DNA fragments with sticky ends are particularly useful for recombinant DNA experiments as the single-stranded sticky ends can easily pair with any other DNA fragment having complementary sticky ends.
Enzymes Recognition sequence
1. Alu1 5' AGCT
2. BamH1 5' GGATTC
3. EcoRI 5' GAATTC
4. HindIII 5' AAGCTT
5. KpnI 5' GGTACC
6. NotI 5' GCGGCCGC
7. PstI 5' CTGCAG
8. Sau3A 5' GATC
5'---AG CT--- 3'
Sticky ends are useful as the restriction fragments produced could easily be ligated. As mentioned earlier, they could be joined to restriction fragments, which have compatible ends. To overcome this, researchers have tried ways to change the ends of the fragments to enable it to ligate with other sites. These are trimming and filling, linkers and adaptors and homopolymer tailing.
The sticky ends produced could be ligated using various strategies. One such strategy is either to fill in the complementary strand or by trimming back the unpaired bases.
If the fragment has a 5' overhang, the 3'OH end becomes the primer for the extension through addition of bases by DNA polymerases. This fills in the recessed end thereby converting single-stranded overhangs to double-stranded DNA.
In some instances, DNA polymerase have 5'-3' exonuclease activity that removes all the unpaired bases in the overhangs. This enzyme is not suitable for the filling in modification of restriction fragment ends.
Homopolymer tailing is the general method used for joining DNA molecules through annealing of complementary homopolymer sequences. This method uses the terminal deoxynucleotide transferase enzyme or terminal transferase. This enzyme adds the deoxynuleotide triphosphate to the 3'-OH end and extends the strand.
The homopolymer tailing is catalysed by an enzyme purified from calf-thymus, terminal deoxy nucleotide-transferase. This enzyme produces the homopolymeric extensions, when provided with a single deoxynucleotide triphosphate, resulting in the addition of new nucleotides to the growing 3'-OH termini of DNA molecules. Lambda exonuclease or restriction with an enzyme like Pst1 acts as a substrate for the terminal transferase. The sticky ends produced by EcoRI can also be extended using terminal transferase. Typically, the terminal transferase enzyme adds about 10-40 nucleotides.
Homopolymer tailing method was first applied by Jackson and co-workers in 1978 when they constructed a recombinant having a fragment of lambda DNA inserted into semian virus 40 (SV40) DNA. Later on, this method was extensively used with either dA.dT or dG.dC homopolymers for constructing recombinant DNA in E.coli.
Both trimming and filling lead to the conversion of sticky/cohesive ends to blunt ends. The resulting blunt ends can be ligated with other blunt-end fragments. In some instances, the ligation of blunt ends are difficult and requires the short synthetic pieces of DNA that contains restriction sites and these are known as linkers. For example, the sequence CCGGATCCGG contains the restriction enzyme BamHI site (GGATCC) within the linker sequence. The significance of linker is that it is self-complementary and any two linker molecules which are complementary anneal to produce a double-stranded DNA fragment. If this double-stranded DNA fragment is ligated to a blunt-end insert, then the fragment carries BamHI site near each end. When this is cleaved with BamHI, it generates a fragment of BamHI with sticky ends that can be ligated with a vector cleaved with the same BamHI. Ligation is not possible if the insert has an internal BamHI site.
In order to increase the efficiency of blunt-end ligation, high concentration of at least one of the components could be used. Furthermore, since the linker molecule is very small (about 10 bases in length) the efficiency can also be increased through high molar concentration. This adds multiple copies of linker to the ends of the gene of interest. The other alternative is to use adaptors. Adaptors are pairs of oloigonucleotides that are synthesised in such a way that it anneal together to generate short double-stranded DNA fragment with different sticky ends or one blunt and one sticky end. For example, two sequences 5'-GATCCCCGGG and 5'-AATTCCCGGG anneals and produces BamHI sticky end and EcoRI sticky end.
Most of the restriction enzymes isolated from bacteria recognise short palindromes that are between 4 and 8bp in length. In order to cleave mammalian genome we need restriction enzymes which recognises DNA sequences of 16bp and above. To address this question, research started with FokI (Flavobacterium okeanokoites) endonuclease. FokI belongs to the type IIS family of endonucleases that cleaves non-specifically at 5'-GGATG-3' sequence. It is a monomer with two distinct protein domains: a sequence-specific recognition domain and an endonuclease domain.
Next, they swapped the FokI recognition domain with either naturally occurring DNA-binding proteins or artificial/synthetic DNA-binding motifs to generate novel chimeric nucleases. Chimaeric nucleases are a novel class of recombinant nucleases which have numerous applications. in vitro, they are used to recognise both low and high affinity binding sites of transcription factors, to study the recruitment of cofactors on target gene promoter sites and also for the analysis of Z-DNA conformation-specific proteins.
The three DNA-binding proteins that elicit sequence-specific DNA binding of eukaryotes are helix-turn-helix motif, the zinc finger motif and helix-loop-helix protein containing leucine zipper motif. The modular structure of zinc finger domains and modular recognition by zinc finger proteins make them the versatile DNA recognition motifs for creating artificial DNA-binding proteins. The zinc finger motif contains 20-30 amino acids that folds to contain two Cys and two His residues linked through a zinc atom. These have proved more versatile in genetic engineering as well as protein engineering applications.
The first chimeric nuclease produced was by the fusion of Drosophila Ubx homeodomain with the cleavage domain of FokI endonuclease. Later on several FokI fusions were produced with various DNA binding proteins. The FokI fused with the zinc finger DNA-binding protein is the more commonly studied FokI fusion.
Restriction endonucleases play a significant role in several applications of biotechnology. They are used in the insertion of genes into vectors during recombinant DNA technology and experiments with proteins. Generally, the plasmids used during gene cloning experiments are modified to carry a short sequence of nucleotides called polylinker sequence. These polylinker sequences are rich in recognition sites for restriction enzymes.
Restriction enzymes are also used in the field of forensic science. These enzymes distinguish gene alleles through identification of single base changes in single nucleotide polymorphisms (SNP). SNPs are nucleotide sequences that is different from one individual to other at least by one nucleotide. The strategy involved is to cleave DNA sample with restriction enzymes and then resolved by agarose gel electrophoresis. Two visible bands indicate the alleles with proper restriction sites while a single band indicates an altered restriction site. Hence restriction enzymes are advantageous in genotyping.
REBASE, restriction enzyme database provides a detailed information with respect to restriction enzymes, methyl transferases, isoschizomers and recognition sequences.
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