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Tools of Recombinant DNA Technology

Essay Contents:

1. Essay on the Restriction Enzymes
2. Essay on the Cloning Vehicles (Vectors)
3. Essay on the Competent Host (For Transformation with Recombinant DNA)
4. Essay on DNA Ligase
5. Essay on Alkaline Phosphatase (AP)

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Essay # 1. Restriction Enzymes:

Steward Linn and Werner Arber (1963) isolated two enzymes which restricted the growth of bacteriophage in bacterium E. coli. One of these enzymes added methyl groups to DNA and second one cut DNA. The second enzyme was named as “restriction endonuclease.”

H.O. Smith, K.W. Wilcox and T.J. Kelley (1968) isolated restriction endonuclease whose working depended on a particular nucleotide sequence. They isolated this enzyme from bacteria Haemophilus influenzae and called is as Hind II. It was observed that Hind II always cut DNA molecules at specific place by identifying a particular sequence of six base pairs.

Restriction enzymes belong to a larger class of enzymes called nucleases.

They are of two types:

(i) Exonucleases:

They remove nucleotides from the ends of DNA.

(ii) Endonucleases:

They make cuts at specific positions within DNA.

Thus, a restriction enzyme (or restriction endonuclease) recognizes a specific base pair sequence in DNA called a restriction site and cleaves the DNA (hydrolyzes the phosphodiester back bones) within the sequence. Restriction enzymes are widely found in prokaryotes and provide protection to host cell by destroying foreign DNA that makes entry into it.

Here they act as a part of defence mechanism called Restriction Modification System.

It bears two components:

(a) First component is a restriction enzyme that selectively identifies a specific DNA sequence and degrades any DNA bearing that sequence.

(b) In second component is a modification enzyme. It adds a methyl group to one or two bases within the sequence identified by restriction enzyme. If a base in DNA is modified due to addition of methyl group, restriction enzyme cannot identify and cleave that DNA. By this method bacteria are able to protect their chromosomal DNA from cleavage by restriction enzymes.

Thus, bacteria bear sets of restriction endonucleases and corresponding methylases.

Endonucleases are enzymes that produce internal cuts called cleavage in DNA molecules. Endonucleases cleave DNA molecules at random sites. A class of endonucleases cleaves DNA only within or near those sites with specific base sequences called restriction endonucleases. Sites recognised by them are called recognition sites or recognition sequences. These sites differ for different restriction enzymes.

Restriction endonucleases serves as the tools for cutting DNA molecules at predetermined sites, which is the basic requirement for gene cloning or recombinant DNA technology.

Types of Restriction Endonucleases:

Three main types of restriction endonucleases i.e., Type I, Type II and Type III are known with slightly different mode of action. Type II restriction enzymes are used in rDNA technology because they can be used in vitro to identify and cleave within specific DNA sequences usually having 4-8 nucleotides.

More than 350 different type II endonucleases with 100 different recognition sequences are known. They need Mg²⁺ ions for cleavage. The first type II enzyme isolated was Hind II in 1970.

The recognition sequences for Type II restriction enzymes form pallindromes with rotational symmetry. In a pallindrome, base sequence of second half in DNA strand represents the mirror image of the base sequence of first half. Due to this in DNA double helix, complementary strand also represents the same mirror image.

Pallindromes are groups of letters that form the same words when read both forward and backward e.g., ‘MALAYALAM’. As against a pallindrome when same word is read in both the directions, pallindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

However in pallindromes with rotational symmetry, second half of complementary strand in DNA double helix is the mirror image of base sequence in the first half of another strand. In such cases, base sequences in both the strands of DNA helix represents the same when read from same and i.e., either 5′ or 3′ of both strands in DNA duplex, e.g.,

Eco RI cleaves DNA molecule of two plasmids due to similar recognition sites in their DNA. The circular form of DNA becomes linear in both the cases. Such linear DNAs can stick together to form single recombinant DNA molecule.

Nomenclature:

Nomenclature of restriction enzymes is usually done by following technique:

(i) The first letter of the genus is taken in which said enzyme was discovered. This letter is written in capital.

(ii) Then, first two letters of species of that organism are written.

(iii) All the above three letters should be written in italics.

Examples:

Eco from Escherichia coli, Hin from Haemophilus inflenzae and Hpa from Haemophilus parainfluenzae.

(iv) This followed by strain or type identification e.g., Eco K.

(v) When the enzyme is encoded by plasmid, the name of plasmid is written e.g. Eco RI i.e., Eco RI comes from Escherichia coli RY13. Here ‘R’ is derived from the name of strain. Roman numbers following the names indicate the order in which enzymes were isolated from the strain of bacteria.

(vi) If an organism forms many enzymes, they are identified by sequential Roman numerals.

Example:

Enzymes formed by H. influenzae strain RD have been named as Hin II, Hin III etc.

Discovery of Enzyme Eco RI led to award of Nobel Prizes to W. Arber, H. Smith and D. Nathans in 1978.

Types of Cleavage Produced By Restriction Enzymes:

Many restriction enzymes like Smal isolated from Serratia marcescens cleave both the strands of DNA at exactly same nucleotide position almost in centre of recognition site resulting in blunt or flush end.

Smal recognizes the 6 nucleotide palindromic sequence and cleave at both the ends.

Still some other restriction enzymes cleave the recognition sequence asymmetrically. Thus due to cleavage, they produce short, single stranded hanging structures. Such ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule again. A 6 nucleotides palindromic nucleotide sequence recognised by Eco RI cleave both strands at different points.

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Essay # 2. Cloning Vehicles (Vectors):

A vector is a DNA molecule which has the ability to replicate in an host cell and into which the DNA fragment to be cloned known as DNA insert is integrated for cloning.

The cloning of a foreign fragment of DNA in bacteria is made possible due to the ability of cloning Vectors’ or “carriers” to continue their lifestyle after additional sequences of DNA have been inserted into their genome. The insertion results in a “hybrid” or “chimeric” or “recombinant” vector which consists in part of the additional “foreign” fragment of DNA.

These chimeric vectors, when cloned in bacteria, replicate in exactly the same way as the original vector and so are obtained in large amounts. In this way, the inserted foreign DNA simultaneously replicates with the remaining part of chimeric vector and copies of the original foreign DNA then can be retrieved from the progeny.

To act as vector, DNA molecule should bear following characteristics:

(i) Origin of Replication (Ori):

It represents the sequence from where replication initiates and any fragment of DNA when integrated to sequence can be made to replicate with in host cells. This sequence also controls the copy number of linked DNA. For getting several copies of target DNA, it is desirable that cloning should be carried out in a vector where origin facilitates high copy number.

It should bear origin of replication (ori) due to which it is able to multiply within the host cell i.e., it should be able to replicate autonomously. Due to this any foreign DNA introduced into vector will also replicate during this process.

(ii) Selectable Marker:

It should incorporate a selectable marker gene. This gene permits to select those host cells which bear the vector from amongst those which donot. Selectable marker helps in eliminating non- transformants and selectively permitting the growth of the transformants. In transformation DNA is introduced into host bacterium.

Examples of few selectable markers are:

(a) Genes which code for antibiotic resistance e.g., ampicillin, chloramphenicol, tetracycline or terramycin.

(b) Genes which encode enzymes like β-galactosidase (product of lac Z gene) which can be identified by colour reaction.

(iii) It should be easy to isolate and purify. Cloning vector should be relatively smaller in size. Large molecules can breakdown during purification and difficult to manipulate.

(iv) Vector should definitely bear atleast one restriction endonuclease recognition site. It will allow foreign DNA to be inserted into vector during the generation of recombinant DNA molecule.

Plasmids and phages are the vectors that are used for cloning purposes in prokaryotes, particularly bacteria.

(A) Plasmids:

Plasmids are the most widely used cloning vectors in the technique of gene-manipulation in bacteria. They are circular, double-stranded DNA molecules occuring in extrachromosomal state and self-replicating. Some plasmids may have one or two copies per cell. Plasmids may be present in greater amounts, typically about 15- 100 per cell.

However, some multi-copy plasmids are widely distributed throughout the prokaryotes, varying in size from less than 1 × 10⁶ daltons to greater than 200 × 10⁶ daltons, and are generally dispensable. The plasmids possess a replication control system that maintains them in the bacterium at a characteristic level.

There are two general types of plasmids — single copy plasmids and multi copy plasmids. Single copy plasmids are maintained at one plasmid per host genome whereas the multi copy plasmids are under “relaxed” replication control which means that they f cumulate in very large amounts (about 1000) per cell when the bacteria stop growing. These plasmids are often used to provide cloning vectors.

Three widely studied bacterial plasmids are:

(i) F Plasmids:

They are responsible for conjugation.

(ii) R Plasmids:

They bear genes for resistance to antibiotics.

(iii) Col Plasmids:

Such plasmids code for colicms, the proteins that kill sensitive E. coli cells. They bear genes which provide immunity to colicin.

The plasmid vector is isolated from the bacterial cell and cleaved at one site by restriction endonuclease. The cleavage converts the circular plasmids into a linear molecule. Now the two ends of linear plasmid are joined to the ends of the foreign DNA (the gene) to be inserted with the help of enzyme DNA ligase. This regenerates a recombinant plasmid or circular hybrid or chimeric plasmid (Fig. 11.9). The chimeric plasmid is transferred of a bacterium wherein it replicates and perpetuates indefinitely.

One of the earliest plasmid vectors to be constructed was pBR 322. This plasmid bears two different antibiotic resistance genes and recognition sites for several restriction enzymes.

By this time, many plasmid vectors have been developed e.g:

(i) Plasmid vectors of pUC family. Such vectors bear a site of the lac Z gene which codes for enzyme B-glactosidase. This site also bears a polylinker and thus, introduction of any foreign DNA into any of restriction enzyme sites will lead to a non-functional enzyme.

Plasmid vectors discussed able can replicate only in E.coli.

(ii) In eukaryotic cells, many vectors have been constructed in such that can exist in both eukaryotic cells and E.coli. Such vectors (shuttle vectors) bear two types of origin of replication and selectable marker genes. One of them acts in eukaryotic cells and other in E.coli For example, shuttle vector of yeast episosmal plasmid YEp In plants a naturally occurring plasmid of bacterium Agrobacterium tumefaciens called Ti plasmid has been suitable formed to act as vectors.

An ideal cloning plasmid vector has three properties:

(i) Low molecular weight,

(ii) Ability to confer readily with selectable phenotypic traits on host cells, and

(iii) Several sites for large number restriction enzymes.

The advantages of a low molecular weight are several. First the plasmid much easier to handle. Second, low molecular weight plasmids are usually present as multiple copies. Finally, with a low molecular weight there is less chance that the plasmid will have restriction enzyme.

(B) Phages as Vectors:

Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. Two phages which have been extensively modified for development of cloning vectors are lambda (λ) and M13.

Lambda (λ) phages provide another type of useful vector system for cloning in bacteria. Usually the DNA of phage, λ, in the form in which it is isolated from the phage particle, is a linear double-stranded molecule of about 48.5 kb paris.

DNA of wild-type phage contains several target sites for most of the commonly used restriction enzymes and so is not itself suitable as vector Derivatives of the wild-type phage have, therefore, been produced which either have a single target site in their DNA at which foreign DNA can be inserted resulting in a chimeric DNA (these are called the Insertional’ phage vectors, (Fig. 11.10) or have a pair of sites defining a DNA fragment which can be removed and replaced by foreign DNA (these are called the replacement’ phage vectors).

Many vector derivatives, of both the insertional and replacement type, have been produced in the recent past and most of them have been constructed for use with Eco RE Ban II H, or Hind III restriction enzymes, but their application can be extended to other restriction enzymes by the use of linker molecules.

What enables one to create “replacement” phage vectors is the fact that the phage ‘head’ can accomodate only about 5% more than its normal complement of DNA and so prevents too long foreign DNA from being packaged into it. To overcome this problem, a fragment of phage DNA that does not carry essential phage genes is removed to increase the space within the phage DNA and is replaced by foreign DNA.

However, the chimeric or recombinant-DNA is packaged into phage head coat in vitro. The principle of packaging in vitro is to supply the ligated recombinant-DNA with high concentrations of phage head precursor, packaging-proteins and phage tail The packaging allows the recombinant-DNA to be introduced into the host bacterium by the normal processes of phage infection (transduction) i.e., phage absorption followed by DNA injection.

Phage M13 vectors:

Such vectors are used for obtaining single stranded copies of cloned DNA, which are specially suited for DNA sequencing. M13 vectors infect only F⁺ cells. It does not kill the cells but forms turbid plaques due to growth retardation of infected cells. M13 is a filamentous phage which infects E. coli, having F-pili. Its genome is single stranded circular DNA of 6407 bp.

Foreign DNA can be introduced into genome of M13 without disrupting any of the essential genes. When M13 phage DNA enters into E. coli host replicative form (RF) a double stranded form is constituted. It replicates until 100 copies are formed. Now the DNA replication becomes a symmetric and it starts producing single stranded copies of genome and extruded from cell as M13 particles.

Uses of developing M13 vectors are:

(i) The genome of M13 is less than 10 kb in size.

(ii) Replicative can be purified and manipulated exactly like a plasmid.

(iii) Genes cloned in M13 based vectors can be obtained in the from of single stranded DNA.

(iv) Very large insects can be cloned since packaging does not depend on genome size.

(v) They form plaques like λ phage vectors making selection of the recombinant vectors rather easy.

(C) Cosmids:

Cosmids have been constructed by combining certain features of plasmid and the ‘cos’ sites of phage lambda. They have been constructed to add some of the advantages of phage vectors to the plasmid vectors’ the cos sites endeavour in vitro packaging system to the plasmid vector. The cosmid vectors, however, provide an efficient means of cloning large fragments of foreign DNA (32-48 kb of foreign DNA)—much more than a λ phage vector can accommodate.

When injected into a bacterium, the recombinant-DNA of a cosmid circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions. Cosmid vectors are particularly attractive for constructing libraries of DNA fragments of eukaryotes because of their capacity to accommodate large fragments of DNA.

(D) Phasmids:

Phasmids are also a type of plasmid vectors containing a fragment of phage DNA including its att site. Like cosmids, they have been constructed to exploit the advantages of both-plasmid vector and λ phage vector. The phasmid may insert into a phage DNA in the same way by which phage DNA inserts into the bacterial chromosome during lysogenic phase of life cycle.

(E) YAC Vectors:

YACs or Yeast artificial chromosomes (Fig. 11.3) are being used as vectors to clone DNA fragments of more than 2500 Mb in size. They are being highly used in mapping larger genomes like Human Genome Project.

(F) BAC Vectors:

BACs or Bacterial artificial chromosomes (Fig. 11.14) are used as vectors which are based on natural extra- chromosomal plasmid of E.coli the fertility or F-plasmid IHs vector bears genes for replication and maintenance of F-factor, a selectable marker and cloning sites.

(G) Shuttle Vectors:

They are the plasmids capable of shuttling genes between two organisms. One of the organisms is prokaryote like E. coli and other is a eukaryote like yeast. Such vectors should bear unique origins of replication for every cell type. They should have separate markers for transformed host cells harbouring the vector.

(H) Transposons as Vectors:

Ac and Ds transposons in corn and were earlier known to represent activator dissociation (Ac-Ds) system. Each represents a transposon with short terminal repeats enclosing (base pair) a long DNA segment. It measures about 4500 bp in Ac and 400 bp m Ds. Each bears genes including the gens enzyme carries on transposition.

A fragment of this region can be removed and transposon can be utilised for cloning foreign DNA. P element is the transposon in Drosophila. It is made up of 31 bp terminal inverted repeats enclosing a 3 kilobase protein coding region. P element codes for transposon and repressor of transposition.

Transposable elements (TEs) are being used for isolation of genes, when gene product is not as a gene tag.

Method involved is:

(i) TE is transposed to a clone gene to get an unstable allele.

(ii) After cloning of this unstable allele TE is isolated.

(iii) Thin TE is transposed to gene of interest to produce unstable allele.

(iv) DNA is extracted from this mutant. This TE sequence is utilised as a probe to isolate and clone the mutant gene.

Cloning Sites (Fig. 11.9):

For restriction enzyme, vector should bear one or very few recognition sites. Gene cloning will become difficult more complicated due to formation of many fronts, when many recognition sites are present. Ligation at restriction site of alien DNA occurs in either of two antibiotic resistance gene.

In vector pBR 322 foreign DNA can be ligated at Bam H, site of tetracyline resistance gene. Such recombinant plasmids will loose tetracycline resistance due to insertion of foreign DNA. However, they can be picked up from non-recombinant ones by plating the transformants on ampicillin containing medium. It such transformants are shifted tetracycline containing medium.

Following observation are noticed:

(i) Recombinants will grow on medium containing ampicillin.

(ii) Non-recombinants will grow on medium having both antibiotics i.e., ampicillin and tetracycline Here one of the antibiotics facilitate the selection of transformants and other antibiotic i.e. becomes ‘non-functional due to insertion of alien DNA. This helps in selection of recombinants.

Many alternative selectable markers have been developed which are able to distinguish recombinants from non- recombinants due to their capability to produce colour. In such cases recombinant DNA is inserted within the coding sequence of enzyme β-galactosxdase. Due to this enzyme gets inactivated and step is called insertional inactivation.

Chromogenic substrate imparts blue coloured colonies, in absence of insert in plasmids. And if insert is present it leads to insertional inactivation of enzyme β-galactosidase. Hence colonies fail to produce any colour and such colonies represent recombinant colonies.

Vectors for Cloning Genes in Plants and Animals:

Genes are transferred from bacterium or virus to eukaryotic cells and these genes force the transformed cell to work at their will e.g., Agrobacterium mediated gene transfer (Fig. 11.15). Agrobacterium tumefaciens is a soil bacterium which causes crown gall tumors in dicotyledons.

These tumors are formed due to insertion of T_i-plasmids into nuclear genome of the infected plant. T_i-plasmid contains T-DNA within it. This bacterium is able to transfer T-DNA to transform normal plant cells into tumour and direct these tumour cells to produce the chemicals required by the pathogen.

The T-DNA causes hormonal disturbances in the transformed plant cell. Most notable of these are increased level of growth hormones i.e., auxins and cytokinins. The tumour inducing (T_i) plasmid of Agrobacterium has been modified as cloning vector. It is not now disease causing to plants but capable of utilising the system to insert desirable genes into many plants.

Gene transfer in higher plants through T_i– plasmid can be achieved by tumour formation on intact plants or plant parts with A. tumefaciens carrying a T_i-plasmid. This can also be achieved by co-culturing protoplasts of A. tumefaciens carrying T_i-plasmid or fusion of protoplasts with spheroplasts of A. tumefaciens.

Retroviruses in animals have the capability to transform normal cells into cancerous cells. Retroviruses have also been disarmed and are being utilised to transfer genes of interest to animal cells.

The capacity of A. tumefaciens to cause tumor formation can be removed by disarming the strain through deletion of genes in T-DNA without loss of UNA transfer and integration functions.

Animal and Plant Viral Vectors:

Plant and animal viruses like Simian virus 40 (SV40) Adenovirus and Papilloma virus have been manipulated that they can be used to introduce foreign DNA into plant and animal cells. They have been used to clone genes in mammals. In plants viruses like Cauliflower Mosak virus, TMV and Gemini Viruses have been tried but with little success.

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Essay # 3. Competent Host (For Transformation with Recombinant DNA):

For propagation of DNA molecules host cells are required. Host cells like E. coli, yeast and plant and animal cells are being used.

Most popular and extensively used bacterium is E. coli due to following reasons:

(i) E. coli a gram negative bacterium is easy to handle and grow.

(ii) It can accept a variety of vectors.

(iii) Under optimal conditions, bacteria double their numbers every 20 minutes. When bacteria reproduce recombinant DNA also reproduces. Eukaryotic cells are also being used as host cells for expression of eukaryoticin. This leads to proper folding of polypeptide Cham into exact 3-dimensional form. Simple eukaryotic organisms like yeast are being widely used being single called, easy to grow and manipulate and genetically well characterized.

A cell membrane does not permit DNA to pass through being hydrophilic in nature. Bacterial host cells are made competent to take up plasmid. For this bacteria are treated with divalent cation like calcium. This enhances the efficiency of entry of DNA into bacterium through pores on cell wall.

Recombinant DNA is forced into such cells by:

(i) Incubation of cells with recombinant DNA on ice.

(ii) It is followed by giving them a heat shock (42°C) and again putting back on ice.

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Essay # 4. DNA Ligase:

DNA ligase forms diester bonds between adjacent nucleotides. It is able to link two fragments of DNA by covalent bonds. The enzyme is utilized m cloning experiment is T₄ DNA ligase which is coded by phage T₄.

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Essay # 5. Alkaline Phosphatase (AP):

This enzyme is used to check the undesired self- ligation of vector DNA molecule during cloning process when vector DNA molecule is digested by restriction enzyme, cohesive ends of vector may not join with foreign DNA and may lead to recircularization of plasmid. When cleaved DNA fragments are given the treatment of AP, terminal 5′ phosphate group is removed.

This 5′ phosphate group is essential at the nick site for ligation. Due to its absence, now self-ligation fails to occur. As DNA insert still contains. For proper ligation presence of 5′ phosphate group is required at the DNA Site. AP is used to remove phosphate group from 5′ end of DNA. This leaves a free 5′ hydroxyl group.