RECOMBINANT DNA TECHNOLOGY

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1.1 Biotechnology

1.1.1 Recombinant DNA Technology

Recombinant DNA technology is the procedure in which a segment of DNA from one organism is removed and is made to combine with DNA from some other organism of the same or different species. This is also called genetic engineering. The recombinant DNA is called heterologous DNA and such a gene as chimaeric gene.

The main feature of recombinant DNA technology is in vitro construction of recombinant DNA molecules. A recombinant DNA molecule is basically a vector DNA into which the desired DNA fragment is inserted to enable its cloning in an appropriate host.

Discoveries Responsible for Recombinant DNA Technology

Recombinant DNA technology evolved because of the following discoveries:

1. Denaturation and Renaturation of DNA: The two strands of DNA easily separate on heating by the breakdown of hydrogen bonds between their nitrogenous bases. This is called denaturation. On cooling, the two complementary strands reunite to form double stranded DNA (renaturation). Because of this property single stranded DNA segments from different sources can join, provided these have complementary base pairs. This is called annealing.

2. Artificial Synthesis of Gene: Dr. Har Govind Khorana (1970) along with his colleagues first reported total synthesis of an artificial gene, tyrosine-t-RNA, in vitro with a potential for functioning within a living cell.

3. Restriction Endonucleases or Restriction Enzymes: The restriction endonucleases, are enzymes that recognise specific nucleotide sequences in DNA and cleave the DNA double helix at or near these specific restriction sites, called the target sites. These enzymes are produced by bacteria as a protection against the entry of foreign DNA, i.e. bacteriophage DNA. These were first reported by W. II. Arber in 1962, who noticed that when DNA of a bacteriophage entered the host bacterium, it was cut into smaller pieces.

Isolation of restriction enzyme from E. coli by Meselson and Yuan and Hind II restriction enzyme from bacterium haemophilus influenzae. Rd. by Kelly and Smith (1970) were responsible for real breakthrough in recombinant DNA technology.

4. Gene Splicing or Isolation of DNA: Several techniques have been developed to isolate or synthesise the desired gene or the DNA fragment. These, are:

(a) Fragmentation of DNA by cleaving with restriction enzymes.

(b) Artificial synthesis of gene.

5. Gene Cloning: This is possible with the discovery of following steps:

(a) Joining of Foreign DNA Fragment to a Cloning Vector: The isolated foreign DNA-fragment is joined to its cloning vector to create a recombinant DNA. The vector is cut open with the help of a restriction endonuclease, the foreign DNA is inserted in the vector and cut ends are then sealed by DNA-ligase.

(b) Introduction of Recombinant DNA into Host Cell: Recombinant DNA thus formed, is introduced into the host cell where it can replicate and form clone. This is achieved either by transformation or by transduction.

(c) Formation of Clones of Recombinant DNA: The trans-formed bacterial cells, containing recombinant plasmid, multiply on culture forming clones of transformed cells.

1. Enzymes

Following types of specific enzymes are employed in recombinant DNA technology:

1. Lysing Enzymes: These are used to open the cells or dissolve the cell wall. Lysozymes are used to dissolve bacterial cell wall or render the cell membrane permeable to large molecules (recombinant DNA) or cloned DNA. The process is called electroporation.

2. Cleaving Enzymes: These are used to cleave a DNA molecule into segments or to open the circular plasmid DNA. These are:

(a) Exonucleases cut off nucleotides from 5' of 3' end of a single strand of DNA molecule.

(b) Endonucleases cleave DNA duplex at any point but not at one end. They cut only one of the two strands of DNA duplex.

(c) Restriction endonucleases cleave DNA at specific or target points recognised by palindromic sequences of nucleotides in both the strands of DNA. Restriction enzymes type-II are used in recombinant DNA technology.

3. Synthesising Enzymes or Synthetases: These help either in vitro synthesis of DNA or in the synthesis of complementary DNA.

These enzymes are:

(i) DNA polymerase helps in the synthesis of complementary DNA strands on DNA templates.

(ii) Reverse transcriptase helps in the synthesis of complementary DNA from mRNA template.

4. Ligases: These seal or join together the ends of the foreign DNA and vector DNA. Ligases are present in both prokaryotes and eukaryotes.

II. Linkers

The linkers are short double-stranded DNA segments which are formed of oligonucleotides. These contain target sites for the action of one or more restriction enzymes. The linkers can be synthesised chemically and can be ligated to the blunt ends of foreign DNA or vector DNA. These are then treated with restriction endonuclease enzyme to produce cohesive ends of DNA fragments. The commonly used linkers are EcoRI-linkers and sal-I linkers.

III. Foreign DNA

It involves isolation or synthesis of DNA which is to be transferred for cloning or to be used for the synthesis of recombinant DNA.

IV. Cloning Vectors

For transferring and cloning a foreign DNA (gene) in a suitable host or in bacterium, some vector or carrier DNA is required. The foreign DNA is inserted in the vector DNA forming recombinant vector. Plasmids, bacteriophages, cosmids and phasmids are used as cloning vectors.

1.1.2 Recombinant DNA Technology

Mechanism of recombinant DNA technology includes following steps:

1. Generation of DNA Fragments

Several techniques are adopted to isolate or synthesise the desired gene or the DNA fragment. These are:

1. Fragmentation of DNA by cleaving with restriction enzymes.

2. Artificial synthesis of gene.

3. Synthesis of complementary DNA (cDNA).

1. DNA Cleaving by Restriction Enzymes and Isolation of DNA Fragments: The desired segments of DNA from eukaryotic genome are cut by using restriction endonucleases or by mechanical shearing. In many cases, the desired DNA fragment is isolated from the genomic libraries or gene banks.

(a) DNA Fragments: The genomic DNA is extracted from any tissue of a plant or animal species. It is digested with a restriction enzyme and is cleaved into segments of different sizes. These segment are separated by gel electrophoresis. The gel used is of two types:

(i) Agarose gel used for separation of large segments of DNA.

(ii) Polyacrylamide gel used for separation of small DNA fragments differing few base pairs. It is more commonly used for DNA sequencing experiments. It is also used for the separation of RNA and proteins.

(b) Identification of DNA Fragments: The DNA fragments separated as bands can be identified by using molecular probes. These are single-stranded radioactively labelled DNAs of known nucleotide sequence and are homologous to specific DNA segments of interest. These radioactively labelled single stranded DNA probes hybridise with complementary DNA. These regions of hybridisation are detected by autoradiography and are isolated.

2. Artificial Synthesis of Gene:

It involves following two steps:

(a) Synthesis of Oligonucleotides: It is chemical assembly of nucleotides into short stranded DNA-fragments having 10-1,000 nucleotides. These small fragments are called oligonucleotides. The oligonucleotides are synthesised either by phosphodiester or phosphite triester method. In phosphite triester method, compounds with trivalent phosphorous are used in place of pentavalent phosphorous.

(b) Enzymatic Assembly of Oligonucleotides: The free hydroxyl groups present on either end of chemically synthesised oligonucleotides are 5 phosphorylated with ATP by polynucleotide-kinase enzyme. The mixture of oligonucleotides is then heated to high temperature (90-100°C). This results in annealing of oligonucleotides into double stranded DNA. These double-stranded DNA segments are joined by enzyme DNA ligase and a complete DNA molecule is formed.

3. Complementary DNA Synthesis: In this method, mRNA of the desired protein is used as a template to synthesise its complementary DNA (cDNA) by using enzyme reverse transcriptase or RNA-directed DNA polymerase. This was discovered by H. Temin and D. Baltimore in 1970.

For the isolation of desired mRNA, total RNA is extracted from a suitable tissue. Poly-A sequence is added as a primer to the 3 end of desired mRNA. The cDNA, thus formed, has a hairpin loop at one end. This hairpin loop is trimmed away by treatment with enzyme S₁ nuclease

II. Insertion of Foreign DNA Fragment to a Cloning Vector

The vector is cut open with the help of a restriction endonuclease, the foreign DNA is inserted in the vector and cut ends are then sealed by DNA-ligase. Because the plasmid DNA is circular, a single cut made in the circle opens it up. DNA to be cloned is inserted, the ends are sealed or ligased and a circular recombinant DNA molecule is formed.

There are three methods being used for this insertion:

1. Sticky or cohesive end ligation by staggered cleavage.

2. Blunt end ligation.

3. Homopolymer tailing.

1. Sticky or Cohesive End Ligation Method by Using Restriction Enzyme: In this method, foreign DNA and a plasmid vector are selected, both carrying the same recognition sites so that they can be cleaved by the same restriction endonuclease. The cuts produced by restriction endonuclease are staggered so that each strand of foreign DNA and vector DNA has a complementary single-stranded protruding 5' end with complementary palindromic base sequence. These complementary sticky or cohesive ends of foreign DNA and vector DNA join by complementary base pairing by renaturation or annealing. The nicks left are sealed by enzyme DNA-ligase, resulting in a recombinant DNA in the form of a double stranded DNA circle.

2. Blunt End Ligation Method by Using Restriction Enzyme Linkers: In some cases, blunt ends of foreign DNA fragment and its cloning vector, generated by cleaving with the same restriction enzyme are ligated with some linker DNA by using enzyme T4-DNA ligase.

The linker molecules have specific recognition sites for a specific restriction enzyme. By using specific restriction endonuclease, identical sticky ends are produced in the linkers of both the DNAs. Thus, the vector DNA and foreign DNA join together through their sticky linkers.

3. Homopolymer Tailing Method: This method of DNA splicing is based on the ability of enzyme terminal transferase to add sequences of the same nucleotides, (i.e. homopolymers) to 3’ end of DNA. Complementary homopolymer sequences are added to the ends of DNA molecules of foreign and vector DNA. For example, homopolymer-G is added to the 3 end of foreign DNA molecule. Complementary homopolymer-C is added to the cloning vector. Thus, the complementary homopolymer 3' tails... GGGG3/3' CCCC... are generated which help in their joining and thus forming recombinant DNA.

III. Introduction of Recombinant DNA into Bacterial Cell

Recombinant DNA thus formed is then introduced into suitable host cell or suitable bacterial cell where it can replicate and form clone. This is achieved by transformation. Transformation is direct uptake of recombinant plasmid by bacterial cells.

IV. Selection and Screening of Clones of Transformed Cells

The bacterial cells with recombinant DNA are selected and isolated from bacterial population either by immunochemical method or by nucleic acid hybridisation method.

The transformed cells are plated on a nutrient medium supplemented with ampicillin. The antibiotic markers present on plasmid provide resistance to an antibiotic. A single transformed cell grows to produce a colony. Each colony is a clone of cells derived from a single parent cell.

1.2 Enzymology of Genetic Engineering

1.2.1 Restriction Endonucleases

Restriction endonucleases or restriction enzymes are endonuclease enzymes that recognise specific nucleotide sequences in DNA and cleave both the strands of DNA double helix at or near these specific nucleotide sequences. These sequences are called recognition sequences or restriction sites or target sites.

Restriction enzymes are found in most bacterial cells. These enzymes are able to identify self and non-self DNA and cleave foreign DNA molecules, i.e. DNA of bacteriophages that invade the cells and destroy them. Thus, they restrict the ability of phage DNA to take over transcription and translation machinery of host bacterial cells. For this reason, these enzymes are called restriction enzymes. These enzymes were first reported by W. Arber in 1962, who noticed that when DNA of a bacteriophage entered a host bacterium, it was cut into smaller pieces. However, first restriction enzyme was isolated by Meselson and Yuan from E.coli. Real breakthrough came when restriction enzyme Hind II was isolated from bacterium, haemophilus influenzae Rd. by Kelly and Smith (1970) and Nathans.

1.2.2 Types of Restriction Endonucleases

The restriction enzymes have been classified into three categories:

1. Type 1. Restriction Endonucleases: These interact with an unmodified recognition sequence in double-stranded DNA These cleave only one strand of DNA and at an apparently random site. They recognise sequences of about 15 base pairs (15 bp) and cleave DNA about 1000 bp away from 5’ end sequence "TCA' located within the recognition site. These enzymes create a gap of about 75 nucleotides in length by releasing acid soluble oligonucleotides. These enzymes require Mg ions, ATP and s-adenosyl methionine cofactors for restriction.

2. Type II. Restriction Endonucleases: These enzymes recognise specific DNA nucleotide sequences. These cleave both polynucleotide chains within or immediately. outside the palindromic sequences. Palindromes are base pair sequences that read the same in 5 3' direction in both strands of DNA For example, these enzymes produce DNA NA fragments of defined length and sequence. These require only Mg2+ ions for restriction. More than 350 different type-II endonucleases with over 100 different recognition sites are known. ATP is not needed. Only restriction enzymes type-II are used in gene manipulation for two reasons:

(a) No ATP is needed for the cleaving action.

(b) It makes cleavage or cut in both the strands of DNA molecule.

3. Type III. Restriction Endonucleases: These cleave double-stranded DNA at well-defined sites and require Mg2 and ATP and partially S-adenosyl methionine. These enzymes are intermediate between type-1 and type-II.

Functioning or Mode of Action of Restriction Endonucleases

Restriction endonuclease enzymes cut DNA molecule recognising specific base sequences. These recognition base sequences have the same order of nucleotides on both strands in 5→3' direction but read in opposite directions on the two strands because of their antiparallel orientation. The restriction enzymes cut DNA molecule in two different styles:

1. Sticky End Style: In this style of cleavage, the restriction endonuclease enzymes recognise hexanucleotide sequence and make staggered cut in the two strands of DNA. These cuts produce complementary single-stranded protruding ends. These unpaired protruding ends are called cohesive ends or sticky ends. The single. stranded tail on the end of each such fragment can base-pair with the tail at either end of any other fragment generated by the same enzyme Restriction enzymes EcoRI, Hind II and Bgl II produce sticky ends. For example, EcoRI recognises base sequence GAATTC and cuts it leaving TTAAC tail

2. Blunt End Style: In this style, restriction enzymes cut both strands of DNA at the same point, generating restriction fragments of DNA with blunt ends. Hae III enzyme generates blunt end. It recognises tetranucleotide sequence GGCC and produce DNA segment with 5’... GG and CC...3’ ends.

1.2.3 Polymerase Enzymes

DNA Polymerases

Enzymes that add successive nucleotides to a growing DNA strand are called DNA polymerases. These play important role during DNA replication and also during in vitro synthesis of DNA. These have three sites for attachment (1) Template site for the attachment to the template DNA, (2) 5' triphosphate site for attachment of deoxyribonucleotide 5' triphosphate and (3) Primer terminus site for the attachment to the 3'0H end of the DNA primer. DNA polymerases are associated with the addition of deoxyribonucleoside triphosphate nucleotides to primer DNA in the direction from 5’-PO₄, end to the 3'-OH end.

There are three DNA polymerase enzymes that participate in the process of DNA replication:

1. DNA polymerase-I (Pol. I),

2. DNA polymerase-II (Pol. II),

3. DNA polymerase-III (Pol. III).

1. DNA Polymerase-I: This enzyme has been studied in E.coli in detail. It is roughly spherical with a diameter of about 6.5 nm. It has a molecular weight of 1,90,000 daltons and is formed of a single polypeptide chain of about 1,000 amino acid residues. It possesses a sulphydroxyl group, a single interchain disulphide and one zinc molecule at the active site.

A DNA polymerase-I molecule in reality is a complex structure. It is formed of:

(a) DNA polymerase 3'-5' exonuclease site.

(b) 5'-3' exonuclease site.

There are five specific binding sites on the spherical molecule of DNA polymerase-I.

(1) Template site for binding the template DNA.

(ii) Primer site for binding primer strand of DNA.

(iii) Primer terminus site for 3'-hydroxyl terminus of primer,

(iv) 5-triphosphate site, a locus for incoming deoxyribonucleotide 5-triphosphate group.

(v) 5'-3' exonuclease site, a locus for 5'-3' exonuclease activity situated in the path of growing chain.

Functions: DNA Polymerase-I was considered to carry out DNA replication. It also carries out a number of clean up functions during replication. Hence, it is a multi-functional enzyme. It is associated with following activities:

(a) 5' 3' Polymerase Activity: It catalyses the addition of mononucleotide units (deoxyribonucleotide residues) to the free 3' hydroxyl end of polynucleotide chain of DNA for the growth of polynucleotide chain in 5' 3' direction. A pure DNA polymerase-1 can add about 1,000 nucleotide residues per minute per molecule of enzyme at 37°C.

(b) 5' 3' Exonuclease Activity: It catalyses 5'→ 3' exonuclease activity in DNA repair by removing and replacing damaged base pairs. It also catalyses removal of pieces of RNA primers used in DNA replication from the 5’ end of each Okazaki fragment and replaces them with DNA nucleotides.

(c) 3' 5' Proofreading Exonuclease Activity: This activity of polymerase-I has a proofreading correcting function. It removes the incorrect nucleotide and places the correct one.

2. DNA Polymerase-II: It is a comparatively small enzyme with a molecular weight of about 1,20.000 daltons Its biological function is not yet fully established. It seems to repair DNA by filling gaps in polynucleotide chain that are less than 100 nucleotides. The nucleotide residues are added in 5' 3' direction. Polymerase-II cannot replicate long strands but can fill gaps in DNA duplex.

3. DNA Polymerase-III: This enzyme was discovered by T. Kornberg and M.L. Gefter in 1972. It is the largest, most complex and most active enzyme out of the three polymerases. It has a molecular weight of about 9,00,000 and consists of 18-20 subunits. Because of its primary role in replication, polymerase-III is also called replicase. It is formed of a single catalytic subunit (a-subunit) and at least 9 to 12 different associated subunits having various functions during DNA replication. Some of these subunits are β, γ, δ, θ and τ.

B-subunit of polymerase-III recognises and binds to the primer strand of parental DNA. It is also called copolymerase-III. Four of these subunits associate in pairs to form a doughnut-shaped structure that encircles the DNA and acts like a clamp. Each dimer associates with the core enzyme and acts as a sliding clamp that allows holoenzyme polymerase-III to move from one nucleotide to the next on the template strand of DNA till it reaches the end completing the synthesis of Okazaki fragment. This increases processivity of polymerase-III to 5,00,000.

The polymerising and proofreading activities reside in subunits a and & respectively. The θ subunit associates with α and ε to form the core polymerase enzyme. γ, δ, χ, Ψ form clamp-loading complex. A single clamp-loading complex consists of six subunits of five types Y₂ δ δ’ ci Ψ. The two core polymerases are linked together by a dimer of t (tau) subunits. This dimeric polymerase then associates with a single clamp-loading complex, completing DNA polymerase-III.

1.3 Vectors or Cloning Vehicles

A vector is a small DNA molecule capable of self-replication and is used as a carrier of DNA fragment inserted into it for cloning. The vector is also called cloning vehicle or cloning DNA.

Properties of Vector: A good vector must have the following properties:

1. It should be able to replicate autonomously, so that it can generate multiple copies of itself along with DNA insert within a single host cell. . A vector is supposed to have one origin of replication for DNA replication.

3. A vector should be small in size and of low molecular weight. Ideally it should be less than 10 kb (kilo base) in size, because large DNA molecules are broken during purification and present difficulty during manipulation required for gene cloning

4. It should be easily isolated and purified.

5. It should be easily introduced into host cell.

6. Vector should afford easy transformation of host cell 7. The vector should contain a suitable marker or markers to permit its detection in the host cell and selection of transformed host cell.

8. The vector should have unique target sites for as many restriction enzymes as possible, so that DNA insert can be integrated without disrupting essential functions of the vector.

9. When objective is gene transfer, the vector should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.

10. When objective is expression of the DNA insert, the vector should contain suitable control elements such as promotor, operator and ribosome binding site, etc.

11. The transformed cells containing DNA insert or recombinant DNA should be identifiable from those transformed by the vector molecule alone.

1.3.1 Cloning Vector and Expression Vector

Vector that is used only for the propagation or cloning of DNA insert inside a suitable host cell is called cloning vector. The cloning vectors contain relaxed replication control so that they can produce multiple copies in each transformed cell. A vector is termed expression vector, when it is used to express the DNA insert producing the specified protein. Such vectors contain the regulatory sequences such as promotors, operators and ribosomal binding sites. Expression vector has prokaryotic promotor and ribosome binding site just before the eukaryotic coding sequence because, the regulatory sequences of eukaryotes are not recognised in the prokaryotic cells.

Plasmid Vectors

Plasmids are extrachromosomal, self-replicating, double-stranded, circular DNA molecules, found in the bacterial cells in addition to the bacterial chromosome. These range in size from 1 ×10⁶ daltons to 200 ×10⁶ daltons. These may exist either independently or may become integrated into bacterial chromosome. Generally, plasmids are non-essential for the bacterial cells except under specific environment. Plasmids contain genetic information for their own replication. They also contain genes for antibiotics, heavy metal resistance, nitrogen fixation, pollutant degradation, etc. Plasmids in a bacterial cell may occur singly or in multiple copies. Single copy plasmids replicate and segregate with the bacterial chromosome. This is called stringent replication. The multiple copy plasmids undergo more than one replication for each replication of the bacterial chromosome. This is called relaxed replication.

Properties of Vector

1. It must have minimum amount of DNA.

2. It must have relaxed replication control.

3. It should have atleast two suitable markers for identification.

4. It should be easily isolated from the cell.

5. It should possess a single restriction site for one or more restriction enzymes.

6. Insertion of a foreign DNA molecule at one of these sites does not alter its replication property.

7. The unique restriction site must be located within one of the two selectable markers.

Plasmids having all the above mentioned characters have been prepared by modifying naturally occurring plasmids by in vitro techniques. Some of such plasmids are:

1. PBR322 Vector

pBR322 is a reconstructed plasmid. It is most widely used plasmid having 4362 base pairs. Its entire base sequence is known. It is constructed from three plasmids. It has following regions:

1. Origin of Replication (Ori): It is derived from plasmid pMBI. This plasmid is closely related to naturally occurring plasmid Col E1.

2. Genes Conferring Resistance Against Antibiotics, Ampicillin (amp^r) and Tetracycline (tet^r): Gene amp^r is derived from plasmid R1 and gene tet^r from plasmid R6.5. Both are natural populations of antibiotic resistant plasmids. These genes are used as markers.

3. Unique Recognition Sites for 20 Restriction Endonucleases: Some of these sites lie within the markers amp^r and tet^r genes. The presence of restriction sites within markers tet and amp permits an easy selection of transformed cells having recombinant PBR322. Insertion of DNA fragment into the plasmid using restriction enzyme Pstl or Pvul, places the DNA insert (foreign DNA) with the marker gene amp^r. The DNA insert (foreign DNA) within the marker gene amp^r makes it non-functional. Bacterial cells with such a recombinant pBR322 will be unable to grow in presence of ampicillin but will grow successfully on tetracycline. Similarly, when restriction enzyme Bam HI or Sal I is used, the DNA insert is placed within the marker gene tet^r. This makes tetracycline gene non-functional. Transformed bacterial cells possessing such a recombinant pBR322 will multiply on ampicillin but not on tetracycline. This process allows an easy selection of a single bacterial cell having recombinant DNA.

4. Nic-bom Region: pBR322 has a nic-bom region (bom = basis of mobility). It is responsible for mobilisation or cell to cell transfer of the plasmid. This region interferes with the replication efficiency of extra chromosomal DNA in monkey cells.

The name pBR is derived from (i) p that denotes plasmid, (ii) B is from the name of scientist Boliver and (iii) R from Rodriguez. These two scientists developed this plasmid. The numeral '322' distinguishes this plasmid from other plasmids developed in the same laboratory (such as pBR325, pBR327, pBR328, etc.)

Useful Features of pBR322: The useful features of this plasmid are (a) small size (4.4 kb) enables easy handling, purification and manipulation. (b) Two selectable markers amp^r and tet^r permit easy selection of recombinant DNA. (c) About 15 copies of them occur p cell which can be increased to 1.000 to 3,000 when protein synthesis is blocked.

pBR327 has more advantageous features than plasmid pBR32 Its 30-40 copies occur in each normal cell of E. coli It is an expression plasmid.

II. PUC Vector

pUC8 is a derivative of pBR322. It is much smaller (2.7 kb). has following two parts derived from pBR322: (a) Ampicillin resistance gene, (b) Col El origin of replication.

It also has lac Z gene derived from E. coli. A polylinker sequence having unique restriction sites lies in the lac region. When DNA insert is cloned in this region, the lac gene is inactivated. The transformed bacterial cells with recombinant DNA molecule are ẞ- galactosidase deficient and are easily selected from normal bacterial cells in a single step. pUC8 has become one of the most popular E. coli cloning vector Other vectors in pUC series are pUC9, pUC12 and pUC13, etc.

III. PGEM3Z Vector

This plasmid vector is similar to a pUC vector. It is almost of the same size and has the same functional nodules amp gene, Col El origin and lac ZX sequence.

In addition to E.coli, some Gram-negative and Gram-positive bacteria are also used as cloning vector for medical and agricultural studies. PK2 plasmid is cloning vehicle of this category. Species of Pseudomonas also have a variety of plasmids. Some of these plasmids are an excellent vector for cloning small fragments of DNA and are used as tool for establishing cDNA library.

1.3.2 Bacteriophage Vectors

Bacteriophages are viruses that attack bacteria. Several bacteriophages are used as cloning vectors. The most commonly used E.coli phages are λ (lambda) phage and M13 phage. Phage vectors have three advantages over plasmid vectors:

1. Phage vectors are more efficient than plasmids for cloning large DNA fragments. Foreign DNA up to 25 kb in length can be inserted into phage vector.

2. DNA can be packed in vitro into phage particles and transduced into E.coli with high efficiency.

3. It is easy to screen and in storage of recombinant DNA. .

λ (Lambda) Phage Vectors

The genome of A-phage is 48502 bp long, i.e. about 49 kb and has 50 genes. About half of these genes are essential. The genome remains linear in the phage head and has single-stranded protruding cohesive ends of 12 bases (5 GGGCGGCGACCC 3' and 3' CCCAGCGGCGGG5) The two cohesive ends anneal to form circular DNA molecule. The sealed cohesive ends are called cos site. It is the site of cleavage of phage DNA. For packaging in the phage head of bacteriophage the A-DNA must be larger than 38 Kb and smaller than 52 Kb. The genes for lysogeny are located in the middle part of -phage genome. For creating lambda phage vectors, this segment is removed wholly or partially. This allows the vector to accommodate large DNA inserts and also avoids chances of lysogeny. Several vectors have been produced from wild type lambda genome by mutation and recombination, as well as by recombinant DNA techniques. These vectors have the following two basic features:

1. The vector can be propagated as phages in E. coli cells enabling preparation of vector DNA.

2. They contain restriction sites, which allow removal of lysogenic segment and also provide insertion site for the DNA fragment to be cloned.

The various λ vectors are classified into two categories-Insertion vectors and Replacement vectors.

1. Insertion Vectors: In case of insertion vector, a portion of non-essential region is deleted and the two arms of λ genome are ligated. The foreign DNA is inserted at the unique restriction site. The DNA insert does not affect the functioning of phage. The insertion vectors is of the size of about 35 kb or more. It can have a DNA insert of upto 18 kb. Examples of insertion vectors are λ gt 10, λ gt 11 and λ ZAP II, etc. λ -gt 10 is 43 kb double-stranded DNA for cloning DNA fragments of about 7 kb long. The insertion of foreign DNA inactivates cl (repressor) gene. λ gt II is 43.7 kb double-stranded DNA. It clones less than 6 kb long foreign DNA. It is an expression vector. Its inserted DNA is expressed as ẞ-galactosidase fusion protein.

2. Replacement Vectors: In replacement vector, the non-essential part of λ phage DNA is substituted by foreign DNA. The deleted region is called the stuffer fragment. Replacement vectors have two recognition sites for the restriction enzyme used for cloning. These sites flank the stuffer region. The maximum size of DNA insert depends upon how much of phage DNA is non-essential. Usually 20-25 kb of genome is non-essential and can be replaced with foreign DNA. EMBL3 and EBML4 are two replacement vectors that are designed for replacing the central non-essential part of 44 kb by foreign DNA about 20-23 kb length. These replacement vectors are used mainly for preparing genomic libraries of eukaryotic genome with cloned fragments.

II. Phage M13 Vectors

M-13 phage is a filamentous bacteriophage of E. colt containing 7.2 kb long single stranded circular DNA. It has been variously modified to give rise to MP13 series of cloning vector. Phage M13 vectors are used for obtaining single-strand copies of cloned DNA. These are used for DNA sequencing. They are derived from 6.4 kb genome of filamentous bacteriophage M13 found in E.coli. Phage M13 has a single stranded linear DNA. Inside the host cells, it changes into a double stranded circular replicative intermediate DNA. Each infected E. coli cell has more than 100 copies of M13 genome and about 1,000 new phage particles are formed during each generation of infected cell.

The double-stranded form of M13 genome is used for obtaining recombinant molecules. This form is obtained from host cells. A complete series of M13 mp vectors (M13 mp 8, M13 mp 9, etc.) has been reconstructed from M13 genome.

Features of M13 Vectors:

1. Very large DNA segments can be cloned.

2. Pure single stranded copies of double-stranded DNA inserts can be obtained.

3. The single-stranded copies of both the strands are obtained because DNA inserts are accepted in either of the two orientations. It means some recombinant clones produce single-stranded copies of one strand and others form copies of its complementary strand of DNA insert.

4. The bacterial cells infected with M13 vectors remain viable because, they do not lyse the cells.

5. The recombinant DNA is obtained within stable bacteriophage particles.

6. M13 infected E. coli cells form plaques, so they could be easily selected.

1.3.3 Cosmid Vectors

Cosmids are hybrid vectors derived from plasmids having a fragment of λ -phage DNA with its cos site (the sequence yielding cohesive ends) and sequences needed for binding of and cleavage by terminase. These have minimum 250 bp of λ -DNA.

Structure and Characteristics of Cosmid

(a) Origin of replication.

(b) Unique restriction site for cleavage and insertion of foreign DNA.

(d) Cos site/sites from lambda phage has 12 bases. It enables recombinant DNA to be packed in lambda particle in vitro due to circularisation and ligation.

(e) Cosmids have a length of about 5 kb. They can be used to clone DNA inserts of upto 40-45 kb.

(f) These can be packaged into λ-particles which infect host cells.

(g) Packaged cosmids infect host cells like 2-phage particles but multiply and propagate like plasmids.

Modern cosmid vectors belonging to pWE and sCos have:

(1) Multiple Cloning Sites (MCS) for simple cloning using unselected DNA fragments, (ii) phage promoters flanking MCS to generate RNA copies of DNA inserts and (iii) unique restriction sites (Not I, Sac II or Sfi I) for cutting restriction enzymes and flank MCS and (iv) vector may contain mammalian expression modules which may code dominant selectable markers for gene transfer to mammalian cells.

Use of Cosmids: Cosmids are used for the construction of genomic libraries of eukaryotes.

1.3.4 Phagemid Vectors

Phagemids are also reconstructed plasmid vectors. Each phagemid contains origin of replication from a phage in addition to that of its own. For example, pBluescript SK(+/-) is a phagemid vector. It has 2958 bp (base pairs) derived from plasmid pUC19.

Structure: A typical phagemid has following structures:

(a) Origin of replication from phage fl or M13.

(b) A multiple cloning site (MCS) within lac Z gene.

(d) Phage T-7 and T-3 promoter sequences that flank MCS sequence.

(e) Col El origin of replication (II).

(f) amp gene for antibiotic resistance. R

DNA insert is integrated in vitro into the double stranded vector. The vector is then introduced into E.coli cells like any other plasmid vector It can be used for cloning DNA insert of upto 10 kb only. The recombinant DNA propagates inside E.coli as a plasmid using col EI origin of replication of plasmid and several copies of recombinant DNA are obtained. The phagemid vector can also be used as an expression vector because the DNA insert is transcribed along with lac Z gene. A fusion protein is formed which has lac Z gene product along with insert DNA product.

p Bluescript vector also provides in vitro production of RNA copies of either strand of DNA insert. In phagemid vector phage, T7 promoter is located on one side of MCS and promotes transcription of antisense strand of lac Z gene and DNA insert. The phage T3 promoter is located on the other side of MCS. It promotes transcription of RNA from the sense strand of lac Z gene. Where these RNA transactions are labelled with radioactivity or with non-radioactive labels, they are used as RNA probes. Because of this, these phagemid vectors are also called riboprobe vectors.

Phagemid vectors also generate single-stranded copies of DNA insert. These single strand copies are used for DNA sequencing. This is facilitated by the presence of six primer sites within and around MCS. These are T7, T3, reverser primer, SK primer and M13-20 primer.

1.3.5 Artificial Chromosome Vectors

1. Bacterial Artificial Chromosomes (BAC) Vector

BAC vectors are shuttle plasmid vectors, created for cloning large sized foreign DNA. They have origin of replication (oriS) from bacterium E. coli F-factor. This maintains a strict control on copy number of vector to one or two per cell. The low copy number of BAC avoids any possibility of recombination between different DNA inserts of multiple vectors.

Structure: The first BAC vector was pBAC108L. Other BAC vectors are pBeloBAC11, pBACe3.6, etc. The vector pBeloBAC11 has 7.4 kb and allows selection of recombinant clones by lac Za complementation.

It has following modules:

(a) OriS origin of replication from E.coli F1 plasmid.

(b) repE encodes a Rep. protein that binds to oris to initiate

replication. (c) CM³ chloramphenical resistance as a marker.

(d) cos lambda phage cos site.

(e) lacZ, ẞ-galactosidase gene as a restriction site.

(f) T7 from bacteriophage for T7 RNA polymerase driven promoter.

(g) SP6, bacteriophage SP6 RNA polymerase driven promoter.

(h) parA, parB and parC for the division of F plasmid during cell division.

(i) IoxP, site on phage P1 genome for recombination.

Significance of BAC Vectors:

(a) BAC vectors can clone DNA inserts of upto 300 kb.

(b) They are stable and more user friendly.

(c) They do not suffer from chimerism caused by variation in cloned DNA by recombination (d) The low copy number of BAC vectors per host cell maintains DNA inserts in their original form. It also avoids counter selection of cloned genes.

(e) BAC vectors are extensively used in the analysis of genomes.

The host for BAC vectors is a mutant strain of E.coli in which normal restriction and modification sites are missing.

2. Yeast Artificial Chromosome (YAC) Vectors

YAC are linear plasmid vectors that behave like a yeast chromosome. A typical YAC (e.g. pYAC3) contains following functional modules from yeast chromosome:

(a) ARS sequence for replication.

(b) CEN4 sequence for centromeric function.

(c) Telomeric sequences from yeast chromosome at the two ends against exonuclease action, consisting of 20-70 tendem repeat of 6 bases, 5'CCCCAA3"

(d) TRP1 and URA3 as the two selectable markers.

(e) Sequences from E.coli plasmid for selection and propogation of E.coli.

(f) SUP4, a selectable marker where DNA insert is integrated.

The first YAC vector developed was pYAC3. It is essentially a pBR322 plasmid vector into which above mentioned yeast sequences have been integrated. It propagates in E.coli in circular form. The DNA insert is integrated. It propagates in E. coli within SUP4 to generate linear YAC vector. The recombinant YAC is introduced into TRPI URA3 yeast cells by protoplast transformation. The recombinant clones are identified by the insertional inactivation of SUP4 detected by a simple colour test.

YACs are used for cloning very large (100-1400 kb) DNA segments They help in mapping complex eukaryotic chromosomes. But they have two disadvantages.

(a) Cloning efficiency is very low about 1000 clones per µg DNA. Thus they cannot be used for complete generation of genomic libraries.

(b) It is not possible to recover large amount of pure insert DN from individual clones.

1.3.6 Yeast Plasmid Vectors

Yeast plasmid vectors are used in the yeast cells Saccharomyces cerevisiae.

The plasmid vectors developed for the yeast cells are of two types replicative plasmid vectors and integrative plasmid vectors. The yeast replicative plasmid vectors are further of following types:

1. Yeast Episomal Plasmids (YEps): These are shuttle vectors derived from yeast plasmid or 2 um plasmid and pBR322 plasmid of E.coli. These are able to multiply in yeast cells as well as in bacterial cells. Some of these vectors contain the entire 2 um plasmid while others have only the origin of replication from this plasmid.

The 2 um plasmid is found in some strains of yeast. It is 6,318 bp long. 2 um plasmid is a multicopy plasmid. Its 70-200 copies may be present in each yeast cell. About 50% genome of this vector is associated with the maintenance of such a high copy number. YEp13 is a common yeast integrative vector. Its pBR322 segment contains:

(a) Origin of replication for E.coli host.

(b) ter^Rand amp^R as the selectable marker genes.

The portion of yeast 2µm plasmid is represented by LEU2 gene which encodes for leucine biosynthesis. The vector YEp13 contains several unique restriction sites for the insertion of foreign DNA segments. LEU2^- functions as a selectable marker in yeast cells. The cells lacking function of this gene have leu2 gene. The yeast cells can grow in minimal medium, (i.e. without leucine) only when they contain YEp13 vector with LEU2⁺ gene.

2. Yeast Replicative Plasmids (YRps): These are also shuttle plasmids derived from pBR322 and a part of yeast chromosome. They have ARS (autonomously replicating sequences) and TRPI gene from yeast plasmid. The ARS is present in origin of replication of yeast plasmid and is formed of about 100 bp sequences with a 11 bp consensus sequence. TRPI is used as a marker gene. YRp vectors are less efficient in transformation than YEp vectors. They yield 1 to 10 thousand transformed cells per µg of plasmid DNA. They are less stable and only 3-100 copies per cell are present.

3. Yeast Centromeric Plasmids (YCps) or Minichromosome Vectors: These replicative shuttle vectors behave like very small chromosomes because they replicate only once during each cell division and are distributed to daughter cells like true chromosomes.

4. Yeast Integrative Plasmids (YIps): YIps are bacterial plasmids that have one yeast gene. The yeast gene functions as a selectable marker in a suitable strain. These vectors lack an origin of replication for yeast genome to be maintained in a yeast cell. YIp5 is an example of such a vector. It is essentially pBR322 with URA3 gene from the yeast.

1.4 Genetic Engineering and its Applications

1.4.1 Recombinant DNA Technology

Recombinant DNA technology is the method of deliberate modification in an organism's genetic composition using in vitro processes.

Recombinant DNA technology is also called genetic engineering. It involves in vitro construction of recombinant DNA molecules (rDNA). A recombinant DNA molecule is produced by joining of two or more DNA segments originating from different organisms.

Objectives of Formation of Recombinant DNA Molecules

Recombinant DNA molecules are produced with following three objectives:

1. To obtain a large number of copies of specific DNA fragments.

2. To recover large quantities of protein produced by the concerned gene.

3. To integrate the gene into the chromosome of a target organism where it can express itself.

Applications of Recombinant DNA Technology

The recombinant DNA technology has opened up wide opportunities for the scientists in the field of genetics, medicines, agriculture and industry.

1. Diagnosis and Cure of Genetic Diseases (Gene Therapy): Human beings suffer from more than 5,000 different hereditary diseases, caused by single gene mutations. Such diseases are Sickle-cell anemia, haemophilia, Diabetes, Tay Sach's disease, Huntington's chorea, Leasch-Nyhan syndrome, Adenosine deaminase deficiency, Purine nucleoside phosphorylase deficiency, Hypertension and Mental illness, etc.

Scientists are anticipating that in near future, some of the hereditary diseases will be cured by the introduction of normal gene. This is called gene therapy. The normal gene for most of these diseases has been successfully cloned. Greever et.al. (1981) used genetic engineering technique to identify defective gene for sickle cell anaemia.

2. Production of Fine Chemicals and Rare Drugs (Pharmaceuticals): Genetically engineered or genetically manipulated bacteria are being used at the industrial level to produce fine chemicals and pharmaceuticals. Human genes for insulin, somatostatin (antigrowth hormone) thymosin, human growth hormone or somatotropin, β -endotrophin and blood clotting factor III and VIII, interferons, thymopoietin, etc. have been cloned to produce pure forms of these biologically important fine chemicals or hormones. In 1984, a functional gonadotropic hormone was also obtained from transformed human cells.

3. Production of Vaccines: Genetic engineering or recombinant DNA technology has revolutionised the process of obtaining cheap and effective vaccines against large number of serious infectious diseases caused by viruses, bacteria and protozoans. These include vaccines for Polio, Yellow fever, German measles, Hepatitis and liver cancer, Rabies, Foot and Mouth Disease of Cattle (FMDC), Feline leukemia causing Cancer, Cholera, Malaria, etc. Gene coding for an antigen protein is isolated from the pathogen and is cloned in a bacterial host or mouse cells, where it produces the concerned protein or the antigen. This is isolated and purified to be used as a vaccine. Hepatitis B vaccine is produced from recombinant yeast (Saccharomyces cerevisiae), bacterial cells or genetically engineered mouse cells. Edible vaccines produced from genetically engineered plants are put for clinical trials. The gene encoding an antigen may be isolated from the pathogen. These may be transferred and expressed in banana or potato. Such bananas or potatoes when eaten raw lead to oral immunisation. Vaccines in these fruits are called edible vaccines. Some edible vaccines are in clinical trial.

(a) Anti-hepatitis Vaccine: A French team of Pasteur Institute in 1980 succeeded in inducing genetically engineered bacteria and mouse cells to produce the antigen of hepatitis B virus which conferred immunity against hepatitis-B infection.

(b) Anti-rabies Vaccine: In 1981-82, the scientists from a French company "Transgene' produced genetically engineered E.coli cells which could synthesise a surface protein of rabies virus.

(c) Anti-foot and Mouth Disease Vaccine for Cattle: A Dutch firm 'Akzo' in 1982, marketed a genetically engineered vaccine against the viral disease, anti-foot and mouth disease of cattle.

(d) Cholera Vaccine (bacterial vaccine): Vaccine against cholera disease caused by bacteria, Vibrio cholerae.

(e) Malaria Vaccine: Efforts are in progress to produce vaccine against Malaria at Medical Centre of New York University. The Water Reed Institute of Colombo, Sri Lanka, Queensland Medical Research Centre, Australia.

(f) Vaccine for Smallpox Virus: The vaccinia virus (cowpox virus) can be used as the basis of smallpox vaccine, using recombinant DNA technology.

4. Biosynthesis of Interferons: Interferons are body's first line of defence against viral infection. These are proteins, released in very minute quantities by the host cells when a virus enters. These are produced by leucocytes of blood, the fibroblasts of connective tissue and the T lymphocytes of immune system. Interferons have antiviral and anticancer properties. In 1980, α-interferons were produced from genetically engineered E.coli and in 1981 these were obtained from genetically engineered yeast cells (Saccharomyces cerevisiae). γ-interferons were obtained from genetically engineered monkey cells in 1981.

5. Hybridoma and Monoclonal Antibodies (Mab): Monoclonal antibodies are antibodies of only one type of homogeneous immunoglobulins or immunological reagents of defined specificity. These are used for the diagnosis and screening. Monoclonal antibodies are being synthesised by popular hybridoma technology.

For monoclonal antibodies, hybridoma are formed by the fusion of:

(a) Antibody producing lymphocytes from the spleen of mouse immunised with red blood cells or specific antigen from sheep, and

(b) Myeloma cells (bone marrow tumour cells) from rat or rabbit which are capable of indefinite multiplication.

Monoclonal antibodies are used in the identification of blood groups, diagnosis of disease and cancer, in vaccine production and in immunotherapy.

6. Production of Novel Proteins: Genes for completely novel proteins can be constructed and then introduced into the bacterial cell for expression. For example, a synthetic gene which produces proline rich protein has been expressed in E.coli.

7. Production of Transgenic Plants and Crop Improvements:

A number of transgenic plant varieties have been developed by recombinant DNA technology. These transgenic plant varieties have novel traits like insect pest resistance, herbicide resistance, frost resistance and resistance to various biotic stresses, viruses and insects. Transgenic plants are developed to produce novel biochemicals like interferon, insulin, immunoglobulins, etc., to produce quality proteins and lipids and to produce recombinant vaccines or edible vaccines, etc.

8. Nitrogen Fixation: Nitrogen Fixation Gene (NIF) can be transferred from the genome of bacterium Rhizobium into the chromosomes of non-leguminous plants. The work is also on to alter the genome of bacteria or of non-leguminous plants so that new relationships can be developed and non-leguminous plants are able to support these nitrogen-fixing bacteria or non-leguminous plants are able to fix nitrogen directly from the atmosphere.

9. Enzyme Technology: At present, more than 2,000 enzymes have been isolated from bacteria. Out of these about 1,000 enzymes are recommended for various uses and out of them about 50 microbial enzymes have industrial use. These enzymes have use in dairy industry, in detergent and starch industry, in brewing and wire industry and in pharmaceutical industry. They also have therapeutic use and presently the enzymes are used as biosensors. In case of particular microorganism that produces a desired enzyme is pathogenic, the required genes for such enzymes can be transferred to a harmless and more suitable organism or bacterium.

10. Food Processing: Recombinant DNA technology or genetic engineering can be used in food industry in following ways:

(a) Recombinant microorganisms can be generated to be used to modify foods and to obtain desired processed food products.

(b) The recombinant microorganisms may be developed that have the capability of transforming inedible biomass into food for human or animal consumption.

(c) Such recombinant strains of microorganisms be created that produce greater amount of protein degrading enzyme like rennin or trypsin. Rennin is obtained from calf-stomach and is used in cheese industry for curdling the milk.

(d) In cheese industry bacterial starter culture of Streptococcus lactis is used which is attacked by phages. Often cheese formation does not occur because of failure of starter. By recombinant DNA technology, phage resistant bacterial strains can be created.

11. Fermentation Technology: Using recombinant DNA technology, the microorganisms can be engineered which have higher productive capacity and can produce substances beyond their natural capability or can produce substances of desired quality.

12. Oil Pollution Control: Oil spills on the surface of sea water during transportation had been a big problem and a big threat to sea Life Prof. Ananda Chakrabarty in 1979 constructed a 'superbug' a strain of bacterium Pseudomonas. It can consume different types of hydrocarbons present in the oil spills. This has a plasmid with genes incorporated from four different plasmids.

13. Pesticide-Degradating Microorganisms: Pesticide degrading plasmid mediated genetically engineered bacteria have been developed by Chakrabarty, Gunsalus, Nagana and others. They can degrade camphor, naphthalene, xylene, toluene, octanes and hexanes. Opd gene isolated from Flavobacterium species and Pseudomonas deminuta are associated with the degradation of parathion and methyl parathion. The reconstruction of genetically engineered microorganisms to degrade synthetic pesticide promises freedom from such harmful chemicals.

14. Bioremediation: Use of living organism or the genetically engineered microorganisms to degrade environmental pollutants is called bioremediation. It is to remove hazardous chemicals accumulated in the cells or to detoxify them into non-toxic forms. The removal of hydrocarbons, dyes, industrial wastes, heavy metals, xenobiotics, etc. by microorganisms is also bioremediation.

1.5 Process of Gene Transfer

The process of insertion of desired DNA or desired genes into the animal cells or early embryo is called gene transfer. It can be achieved by following methods:

1. Transfection of Animal Cells or Embryos: The retroviruses are used as vectors to transfect the animal cells and deliver the desired gene. The other viruses used for gene transfer in animal cells are vaccinia virus adenovirus, herpex virus and bovine papiloma virus. The transfected mammalian cells are cultured and are used for diagnosis of oncogene and for gene therapy. The transgenic embryos produce transgenic animals.

2. Gene Transfer through Microinjection: By this technique desired DNA or gene is injected into the cell, egg, oocyte or embryo though a glass micropipette. The pointed tip of micropipette acts like an injection needle for injecting nucleus or DNA segment into the target cell. Pipette is used for holding the target cell in position while Injection. The injected DNA integrates randomly with the nuclear DNA and expresses itself.

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