9. Recombinant DNA Technology – Essentials of Molecular Biology

9

Recombinant DNA Technology

CONCEPT OUTLINE
  • Introduction
  • DNA Isolation and Sequencing
    • DNA isolation
    • DNA sequencing Automated
    • Automated DNA sequencing
  • Tools of Recombinant DNA Technology
  • Restriction Endonucleases
    • Types of restriction endonucleases
    • Nomenclature of restriction endonuclease
    • Type II restriction endonucleases
  • Producing rDNA by Sticky-end Ligation
  • Producing rDNA by Blunt-end Ligation
    • Homopolymer tailing

       

  • Other Enzymes Used in rDNA Technology
  • Cloning Vectors
    • Types of cloning vectors
    • Cosmid vectors
    • Shuttle vectors
    • Yeast episomal plasmids
    • YAC vectors
    • Vectors for plants
    • Genes of Ti plasmid and their function
  • Nucleic Acid Hybridization and Probes
  • In Situ Hybridization
  • Molecular Cloning
    • Preparation of vector DNA
    • Preparation of target DNA
    • Construction of rDNA
    • Transport into the host cell (transfection)
    • Selection of transformed cells
  • Construction of C-DNA and Genomic Libraries
    • Genomic library

       

  • Applications of Recombinant DNA Technology
    • In pharmaceutical industry
    • Gene therapy
    • Construction of industrially important bacteria
  • Summary
  • References
INTRODUCTION

Recombinant DNA (rDNA) technology deals with the isolation and manipulation of DNA. DNA molecules from all organisms share the same chemical structure; they differ only in the sequence of nucleotides, consequently, a DNA from a foreign source can be linked to host DNA sequences, i.e., the DNAs of two different species can be linked to form the ‘recombinant DNA’ or ‘rDNA’ or ‘chimeric DNA’. For example, a plant DNA may be joined to a bacterial DNA or a human DNA may be joined with a fungal DNA. DNA sequence may be even chemically created and introduced into any of a very wide range of living organisms. Upon translation, the rDNA results in the production of ‘recombinant protein’ or ‘fusion protein’, with the help of various rDNA technology tools.

‘Molecular cloning’ is the laboratory process used to create rDNA. Using rDNA technology, desired proteins can be made to be expressed in the organisms that have short generation time such as bacteria, and thus can be amplified within short duration. The expressed proteins can be isolated and used for various purposes.

An organism containing an artificially inserted foreign piece of DNA is said to be ‘transgenic’.

DNA ISOLATION AND SEQUENCING

The first step in the rDNA technology is the isolation of the desired gene of interest for which the DNA from the cell has been isolated.

DNA Isolation

DNA can be isolated by employing gentle methods of cell rupture. Cell walls if present are digested enzymatically (lysozyme treatment) and the cell membrane is solubilized using detergent. Autoclaving is necessary to eliminate DNase activity. Once nucleic acids are released from the cell, RNA can be removed by treatment with RNase. The proteins can be removed by treatment with water-saturated phenol or with phenol/chloroform mixture. This denatures the protein but not the nucleic acids. The emulsion formed is centrifuged and the protein precipitated. The aqueous layer is recovered and deproteinized repeatedly. This is followed by centrifugation. The supernatant is recovered and treated with ethanol that precipitates the DNA. The precipitated DNA is recovered and dissolved in a buffer containing EDTA (to eliminate DNAse action) and stored at 4°C. This procedure is best suited for cellular DNA. If DNA from specific organelle or viral particle is needed, it is best to isolate the organelle or virus before extracting the DNA (Figure 9.1).

 

Figure 9.1 DNA isolation

DNA Sequencing

The nucleotide sequence of the gene of interest can be analysed. There are two main methods of nucleic acid sequencing namely:

  1. Maxam and Gilbert’s chemical method and
  2. Sanger’s dideoxy method or enzymatic method or chain termination method.

Maxam and Gilbert’s chemical method

Allan Maxam and Walter Gilbert developed a chemical method for DNA sequencing. However, the method is laborious and time-consuming.

The basic steps employed are:

  • Double-stranded DNA is separated.
  • The separated single-stranded DNA is end labelled at its 5′-end by using polynucleotide kinase.
  • The DNA strand is exposed to mild treatment with a chemical that destroys one of the four bases (e.g., only A residues). Since the treatment is mild, usually only one of the A residues in each molecule is destroyed at random. As the result, a group of DNA fragments of different lengths, reflecting the different sites at which A residues occur in the original DNA are generated.
  • These fragments are separated on a gel and detected by autoradiography and their sizes reveal the distances from the labelled end and the A residues.
  • Similar procedures are carried out simultaneously on four separate samples of the same 5′-end-labelled DNA molecule using chemicals that cleave DNA preferentially at T, C and G respectively.
  • The resulting fragments are separated on an agarose gel, giving a pattern of radioactive DNA bands from which the DNA sequence is read. The nucleotide closest to the 5′-end of the sequence is determined by looking across the gel at level 1 (at the bottom of the gel) and see-ing in which lane a band appears (T). The same procedure is repeated for level 2, then level 3 and so on, to obtain the sequence (Figure 9.2).

 

Figure 9.2 Maxam and Gilbert’s chemical method of DNA sequencing

Sanger’s dideoxy method

This method employs the following steps:

  • Double-stranded DNA is separated.
  • Separated DNA template, DNA polymerase Klenow fragment (the larger domain of DNA polymerase-I (DNA Pol-I), lacking 5′ → 3′ exonuclease activity), labelled primer, deoxyribo-nucleotides and four different dideoxyribonucleotide, each one specially added in one tube, are taken in four reaction tubes (Figure 9.3).
  • When such a modified nucleotide is incorporated into a DNA chain, it blocks the addition of the next nucleotide due to the absence of a 3′-OH group.
  • Each newly synthesized DNA strand made in a test tube by DNA polymerase will stop at a randomly selected base in the sequence.
  • This reaction, therefore, generates fragments of DNA similar to that explained previously for the chemical method. These fragments are detected by a label (chemical or radioactive) that is either incorporated into the oligonucleotide primer or into one of the deoxyribonucleoside triphosphates used to extend the DNA chain.

 

Figure 9.3 DNA sequencing by Sanger’s dideoxy method

 

Figure 9.4 Sanger’s dideoxy method of DNA sequencing

  • To determine the full sequence, four different chain-terminating nucleoside triphosphates are used in separate DNA synthesis reactions on the same primed single-stranded DNA template.
  • When the products of these four reactions are analysed by electrophoresis in four parallel lanes of a polyacrylamide gel, the DNA sequence can be derived in the same way as explained for the chemical method (Figure 9.4).

Automated DNA Sequencing

Sanger’s method was automated in 1986 by Leroy Hood and Loyd Smith. In this method, each ddNTP is tagged with a fluorophore of four different colours. Thus, instead of having four separate sequences as described in the previous methods, the reactions can be combined into one tube. The DNA fragments are separated using a polyacrylamide gel. A laser beam excites the flurophore tagged to the fragments as they reach the detector near the end of the gel. These signals are fed to a computer that reads the results as DNA sequence. About 4,800 bases of sequence can be read per day by this method. Currently more automated sequencers are used, which can detect as many as 2 million base sequences per day.

The above picture depicts the steps involved in automated DNA sequencing. The technique uses dideoxynucleotides, just as described in other methods, but the primers used in each of the four reactions are tagged with different fluorescent molecules. The products from each tube will emit a different colour fluorescence when excited by light.

 

TOOLS OF RECOMBINANT DNA TECHNOLOGY

The basic tools of rDNA technology include various:

  • Nuclear enzymes such as DNA polymerases, restriction endonucleases, terminal deoxynucleotidyl transferase, alkaline phosphatase, reverse transcriptase and ligases.
  • Vectors.
  • Linkers.
  • Adaptors.
  • Nucleic acid probes.
  • Gene libraries.
RESTRICTION ENDONUCLEASES

The term restriction endonuclease was coined by Lederberg and Meselson in 1964 to describe the nuclease enzyme that destroys or restricts any foreign DNA entering a bacterial cell. These restriction endonucleases are widely used in rDNA technology. They specifically bind to double-stranded DNA and cleave it at specific sites known as recognition sequence or restriction sites. They recognize specific sequences that are 4–6 bp in length and show a two-fold dyad symmetry. The DNA fragments produced by the action of restriction endonuclease help in the joining of DNA fragments to form new rDNA.

Types of Restriction Endonucleases

Restriction endonucleases are of three types, namely types I, II and III (Table 9.1). Their grouping is based on the types of sequences recognized, the nature of the cut made in the DNA and the enzyme structure. Type I and type III restriction endonucleases are not useful for gene cloning, because they cleave DNA at sites other than the recognition sites and thus cause random cleavage patterns. In contrast, type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites.

 

Table 9.1 Types of restriction endonuclease

 

Type I Enzymes : These enzymes attach to DNA molecule and migrate to a distance of about 1,000–5,000 nucleotides and cleave the DNA strand at a random site, creating a gap of about 75 nucleotides in length. Type I enzymes also methylate DNA, i.e., they function both as endonuclease and as methylase. They consist of three subunits—one subunit is responsible for restriction, one subunit for methylation and the third subunit for DNA binding. Examples of type I restriction enzymes are EcoB and EcoK.

Type II Enzymes : These enzymes are used for gene manipulation studies. They recognize specific target sequence and cleave the double-stranded DNA molecule within or near the recognition sequence. Their action results in DNA fragments of defined length and sequence. They require Mg2+ ions for their activity. Example of type II enzymes are EcoRI, EcoRII, etc.

Type III Enzymes : These enzymes consist of three subunits—one specifies site recognition, one specifies methylation and the other specifies cleavage. These require ATP as the source of energy and Mg2 ions as cofactor. They cleave DNA at specific non-palindromic sequences. Example of type III enzymes are HpaI, MboII, etc.

Nomenclature of Restriction Endonuclease

The restriction enzymes are named based on the following principles:

  • Restriction endonucleases are named for the organism in which they were discovered. The name of the organism is identified by the first letter of genus name and first two letters of species name to form a three-letter abbreviation, which is italicized.

    For E. coli = Eco

    For H. influenzae = Hind

  • A strain or type of the organism in which the restriction enzyme is identified is also written along with the name.
  • For example, EcoK for E. coli strain K, Hind for H. influenzae strain Rd.
  • The restriction systems genetically specified by plasmid of the organism is also indicated. For example, EcoRI, EcoPI.
  • When a strain has several restriction and modification systems, they are identified by Roman numerals. For example, Hind I, Hind II and Hind III. These should not be confused with the Roman numerals used for specifying the types.

 

Restriction enzymes and their recognition sequences

Name Source microorganism Recognition sequence
Bam HI Bacillus amyloliquefaciens G↓GATCC
ECo RI Eschericia coli RY13 G↓AATTC
Hind III Haemophilus influenzaeRd A↓AGCTT
Not I Nocardia otitidiscaviarum GC↓GGCCGC
Pst I Providencia stuartii CTGCA↓G
Sma I Serratia marcescens CCC↓GGG

Type II Restriction Endonucleases

Restriction enzymes can cut to produce:

  • ‘Blunt ends or flush ends’, i.e., they cleave both strands of DNA at same base pairs, in the centre of the recognition sequence (Figure 9.5).

    For example, Hae III (Haemophilus aegypticus, order of the enzyme III) recognizes a four-nucleotide-long palindromic sequence and cuts symmetrically both DNA strands forming blunt ends. It recognizes the sequence

    The cut is made between the adjacent G and C.

  • ‘Sticky ends or cohesive ends’, i.e., they make staggered cuts; for example, EcoRI recognizes the sequence (Figure 9.5).

The cut is made between G and A residues of each strand and produces two single-stranded complementary cut ends that are asymmetrical having 5′ overhangs of four nucleotides.

 

Figure 9.5 Restriction endonucleases producing blunt and sticky end

Isoschizomers

In general, different restriction enzymes recognize different sequences. However, a few of them recognize the same restriction sites. Such restriction endonucleases that are isolated from two different sources but possess similar recognition and cleavage sites are called ‘isochizomers’.

 

Isoenzymes

If two different restriction endonucleases produce same cohesive ends, then the two enzymes referred as isoenzymes.

For example, Bam HI and Sau3AI create the fragments with cohesive end of ‘GATCC’ by recognizing the sequence of GGATCC and NGATCN′, respectively (Figure 9.6).

 

Figure 9.6 Isoenzymes

PRODUCING RDNA BY STICKY-END LIGATION

Both the target DNA and the vector such as plasmid DNA can be cut by the same restriction enzyme, so that they produce complementary staggered ends, which are annealed together to get the rDNA (Figure 9.7).

PRODUCING RDNA BY BLUNT-END LIGATION

The blunt ends created using the action of restriction enzymes can be ligated with a similar blunt created in vectors by using:

 

Figure 9.7 Recombinant DNA production by sticky end ligation

  • T4 DNA ligase,
  • Linkers,
  • Adapters and
  • Homopolymer tailing.

T4 DNA ligase

Blunt ends can be ligated using E. coli and phage DNA ligases, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. The enzymes act similarly; however, they differ in their cofactor requirements. The T4 enzyme requires ATP, while the E. coli enzyme requires NAD+. In each case, the cofactor splits and forms an enzyme-AMP complex. The complex binds to the nick, joining the 5′ phosphate and 3′-OH group, making a phosphodiester bond (Figure 9.8).

 

Figure 9.8 Ligation of DNA fragments by T4 DNA ligase

Linkers

These are chemically synthesized DNA molecules that are covalently joined to the ends of a DNA fragment or vector, in order to produce cohesive ends. Linkers are blunt-ended molecules but contain a restriction site. To the blunt-ended DNA of interest, these linkers can be attached by T4 DNA ligase. Cohesive ends are produced when these terminal extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction enzyme can be added to virtually any DNA molecule. It is important to realize that the use of high concentrations of linkers would inevitably cause multiple linkers to be attached to either side of the DNA molecule (Figure 9.9).

Adaptors

These are also short synthetic oligonucleotides such as linkers. However, the adaptor is synthesized such that it has one blunt end and one sticky end. In case of linkers, the restriction enzyme added may cut the target DNA (gene of interest) at an internal site. To avoid cutting up the target, adapters are used. The blunt end of the adaptor is not modified but the sticky end is modified, i.e., the 5′-PO4 group is removed. Therefore, two adaptor molecules cannot be linked as DNA ligase cannot ligate 5′-OH and 3′-OH. Therefore, the only possibility is the ligation of adaptor with the target DNA, i.e., the adaptors can be ligated to the DNA molecules but not to themselves. After the adaptors have been attached, the 5′-OH cohesive ends are then modified to 5′-phosphate by adding a phosphate group using the enzyme polynucleotide kinase. The modified DNA-adaptor molecules are now ready to be inserted into an appropriate cloning vector (Figure 9.10).

 

Figure 9.9 Linkers

 

Figure 9.10 Adaptors

Homopolymer Tailing

Terminal deoxynucleotidyl transferase catalyses the repetitive addition of mononucleotide units from deoxynucleoside triphosphates to the terminal 3′-hydroxyl group of a DNA molecule. The enzyme catalyses the addition of homopolymer tails to DNA fragments. This technique called homopolymer tailing is used for creating sticky ends on blunt-ended DNA fragments (Figure 9.11). For example, when DNA fragments are treated with the enzyme terminal deoxynucleotidyl transferase, in the presence of dATP, it results in the formation of poly-tails at the 3′-end of the DNA fragment.

 

Figure 9.11 Homopolymer tailing

OTHER ENZYMES USED IN RDNA TECHNOLOGY

S1 Nuclease

It is an endonuclease, which cleaves single-stranded DNA or single-strand protrusion of double-stranded DNA with cohesive ends. Because of S1 nuclease action, cohesive ends are converted into blunt ends. Thus, S1 nuclease is used to remove the incompatible ends.

DNA Pol-I, Klenow fragment

This fragment has the polymerase activity and 3′ → 5′ exonuclease activity of DNA Pol-I but does not have the 5′ → 3′ exonuclease activity. This 5′ → 3′ exonuclease activity is often troublesome because it degrades the 5′ terminus of primers that are bound to DNA templates and removes 5′-PO4 from the terminal DNA fragments that are to be used as substrates for ligation. The Klenow fragment can be used in the end filling and synthesis of DNA in cDNA clone.

Alkaline phosphatase

The cohesive ends of restriction enzyme treated plasmids instead of joining with the target DNA, sometimes reseal without taking the insert (target DNA) and are recircularized. To overcome this problem, the restricted vector (plasmid) is treated with the enzyme alkaline phosphatase, which removes the terminal 5′-PO4 group. The restriction fragments of the target DNA to be cloned are not treated with alkaline phosphatase. Therefore, the 5′end of the target DNA can covalently join with the 3′-end of the plasmid. Ligase action completes the formation of the rDNA.

Reverse transcriptase

Reverse transcriptase is used in the synthesis of cDNA using RNA template and also for the construction of cDNA clone bank.

Deoxyribonuclease I (DNAse I)

It is an endonuclease, which digests single- and double-stranded DNAs. This enzyme is useful for a variety of applications including nick translation, DNA foot printing, bisulphite-mediated mutagenesis and RNA purification. These enzymes have a role in genetic engineering and thus produce required specifications.

CLONING VECTORS

A DNA molecule that carries foreign DNA into a host cell, replicates inside the host cell and produces many copies of itself is called a cloning vector. Cloning is frequently employed to amplify DNA fragments containing genes.

Some essential characteristics of a cloning vector are:

  • It contains a sequence that allows for the propagation of itself in the host.
  • It contains an insertion site for the foreign DNA—also called an MCS (multiple cloning site) that can be cut by several restriction enzymes.
  • It contains specific control systems such as promoters, terminators and ribosome-binding site, so that the cloned DNA is expressed properly.
  • It contains marker genes that allow for the selection of the host cells that contain the insert (DNA of interest).

Types of Cloning Vectors

The various types of cloning vectors used in rDNA technology include:

  • Plasmids vectors,
  • Bacteriophage vectors,
  • Cosmid vectors,
  • Shuttle vectors,
  • Yeast episomal plasmid (YEP vectors),
  • Yeast artificial chromosomes (YAC vectors) and
  • Ti plasmids.

Plasmid vectors

Plasmids are found in bacteria and are autonomously replicating extrachromosomal circular DNA molecules. They are small between 2 and 8 Kb and often have a high copy number. They also encode antibiotic-resistant gene for propagation in a suitable host and contain unique restriction enzyme sites for cloning purposes. Plasmids are frequently used, as they are the easiest class of vector to work with, as their isolation from bacteria, modification with enzymes, ligation and introduction into the host are quite easier.

One of the earliest plasmid vectors was pBR322 that was named after Bolivar and Rodriguez who were involved in engineering this plasmid. pBR322 is a small plasmid of 4.36 Kb in size. It replicates extrachromosomally in the bacterium E. coli. pBR322 encodes antibiotic-resistant genes for ampicillin (Amp) and tetracycline (Ter), which facilitate selection on antibiotic-containing plates. It also contains unique restriction sites for many enzymes such as Sal I, Eco R I, Bam H I and Pst I (Figure 9.12).

 

Figure 9.12 Plasmid vectors

 

The restriction digestion of pBR322 with Sal I allows the insertion and ligation of Sal I-digested foreign DNA into the middle of the tetracycline gene. This helps in the selection of the transformed bacterial cells (cells bearing the rDNA) for Amp resistance that will be intact, followed by subsequent screening for tetracycline sensitivity that arises due to the interruption of the Ter-resistant gene because of the insert. In this way, bacterial cells bearing the rDNA can be selected and subsequently cultured, thus amplifying the gene of interest. With the advanced genetic engineering techniques, the pBR322-based plasmid vectors were now modified to be more versatile and user-friendly plasmids.

The pUC family plasmids

These plasmids were developed by Messings and co-workers at the University of California. One revolutionary feature introduced in these plasmids was the presence of MCS’s. The MCS is a short stretch of DNA that contains the recognition sequence for a large number of restriction enzymes. Thus, the choice of the restriction enzyme required to digest the vector and the target DNA is increased.

The pUC plasmids also contains lacZ′ gene as the marker gene. The lacZ′ gene codes for the α-peptide portion of the enzyme β-galactosidase. When this plasmid is inserted into an E. coli, which lacks lacZ′ segment (i.e., lacZ′ mutant), the bacterial and plasmid genes complement each other to produce a functional β-galactosidase. This process is called ‘α-complementation’. If a foreign gene is inserted into this lacZ′ gene of pUC plasmids, then it cannot complement and no functional β-galactosidase is formed (Figure 9.13).

 

Figure 9.13 Cloning vector-pUC19

 

When the lacZ is expressed in a suitable host, it is capable of degrading the chromogenic substrate X-gal (5-bromo-4-chloro-3-indoyl β-D-galactopyranoside) to produce a blue colour. If the DNA of interest is introduced into the lacZ gene, the β-galactosidase protein will not be produced and hence any clone with the insert is incapable of degrading X-gal substrate. Thus, recombinant bacterial cells will appear colourless while non-recombinants will be blue in colour.

Disadvantages of plasmid vectors

  • The size of the target DNA inserted into these vectors is usually limited.
  • Recombinant plasmids can be extremely unstable in certain hosts; for example, some strains of Bacillus.

Bacteriophage vectors

Bacteriophages are viruses that infect bacteria. A number of different bacteriophages have been studied and well characterized. Many gene-cloning experiments are performed with bacteriophage lambda λ(X phage), which infects E. coli. The λ phages have a protein head that contains the phage DNA (approximately 49 Kb). It also has a tail and tail fibres. The tail fibres help in the adsorption of the phages onto the surface of the bacterium. Once inside the bacterial cell, the λ phage DNA enters either a lytic or lysogenic cycle.

 

 

In the ‘lytic cycle’, the host cell’s machinery is utilized by the λ phages for carrying out it own molecular processes such as replication, transcription and translation, following which the phage particles are assembled. Once a threshold number of phages are assembled, the bacterial cell bursts releasing the mature phages that can infect other bacterial cells. In the ‘lysogenic cycle’, the phage that enters the bacterial cell remains associated with the host chromosome (Figure 9.14).

 

Figure 9.14 Lytic and lysogenic cycle of λ phage

 

λ Phage’s DNA can be modified in several ways, thus making it suitable to be used as a cloning vector. Infection can be achieved by packaging rDNA into phage outside the natural environment by a process known as ‘in vitro packaging’.

In vitro packaging is done by isolating a combination of phage mutants that can over produce the various protein components of mature phage while lacking one of the essential proteins. These can be mixed together in the correct proportion to generate ‘packaging mixes’.

When the phage DNA is added to the packaging mix, it is packaged by a specific cleavage mechanism at a sequence termed the Cos site (cohesive site). Thus, infective phages can be generated in vitro. These are made to infect the E. coli cells. The infected cells are subcultured on agar plates. Successful infection results in the production of small lytic zones (clearings) on the agar plate, which are called ‘plaques’. The rDNA containing cells will not produce such plaques.

There are two broad classes of λ phage vectors used in gene cloning, which are ‘replacement vectors’ and ‘insertion vectors’.

 

Figure 9.15 Replacement vector

Lambda replacement vectors

These have been designed to accommodate large fragments of DNA. This is made possible by the removal of the DNA of the viral genome that is unnecessary for its function by restriction digestion. The removed fragment is termed the ‘stuffer’ fragment. The remaining DNA is termed the ‘arms’. The foreign DNA of interest digested with the appropriate restriction enzyme can then be used to replace the stuffer fragment by ligation to the arms. These recombinant fragments can then be used to infect bacterial cells after in vitro packaging (Figure 9.15).

Lambda insertion vectors

In these vectors, the DNA of interest is inserted at a single site without the removal of viral sequences. Thus, only smaller fragments can be inserted by this method when compared to the replacement vectors (Figure 9.16).

 

Figure 9.16 Insertion vectors

Disadvantages of bacteriophage vectors

The difficulties in manipulation; for example, isolating arms, preparation of packaging mixes, etc. are some of the disadvantages. However, the introduction of cloning kits has greatly reduced the laborious aspects of using the bacteriophage vectors.

Cosmid Vectors

Cosmids are novel vectors that combine the features of plasmid and λ phages cosmids are plasmids with cos site. They resemble plasmids in having an antibiotic-resistant marker and can replicate in bacterial cells. However, unlike plasmids, they can accommodate larger DNA fragments of up to 45 Kb. This is possible because of their cos site. The presence of the cos site allows the vector and target DNA to be packaged as if it were λ DNA. The packaged DNA is then used to infect E. coli cells. Once injected, the cosmid can replicate like a plasmid in the bacterial cell.

The various steps for cloning DNA fragments in cosmid vectors are (Figure 9.17):

  • The cosmid vector is first cut with a restriction enzyme and then ligated to 35–45-Kb restriction fragments of foreign DNA with complementary cohesive ends.
  • If the concentration of foreign DNA is sufficiently high, the ligation reaction generates long DNA molecules that are multiple restriction fragments of the foreign DNA separated by the cosmid DNA. These ligated molecules resemble the concatemers that form during the replication of λ phage in a host cell and can be packaged in vitro by using packaging mixes.
  • In the packaging reaction, the λ Nu1 and A proteins bind to cos sites in the ligated DNA and directs the insertion of the DNA between two adjacent cos sites into empty phage heads. Packaging will occur as long as the distance between adjacent cos sites does not exceed above 50 Kb.
  • Phage tails are then attached to the filled heads, producing viral particles that contain a recombinant cosmid DNA molecule.
  • When these recombinant virions are plated on agar plates containing E. coli cells, they bind to phage receptors on the cell surface and inject the packaged rDNA into the bacterial cell.
  • The injected DNA does not encode any λ proteins and hence no viral particles are formed in the infected cells and no plaques develop on the plate.
  • The inserted DNA forms a circular plasmid or a cosmid carrying the inserted DNA fragment in each host cell.
  • Like plasmids, the cosmid vector also replicates autonomously and is segregated to the daughter cells.
  • Cosmid vectors containing transformed cells can be selected on antibiotic plates.

Cosmid vectors are the best to clone eukaryotic genes, as many of the eukaryotic genes are of the order of 30–40 Kb in length.

Shuttle Vectors

Certain vectors can replicate in different host systems; for example, in E. coli and in yeast. Such vectors are called shuttle vectors. The shuttle vectors carry different origins of replication that enable them to replicate in different host systems. Usually, the vector is cloned in prokaryotic systems and then the recombinant vectors are grown in eukaryotic cells. One of the most common types of shuttle vectors is the yeast shuttle vector.

 

Figure 9.17 Construction of a cosmid library. Cosmids are plasmid vectors that carry the cos sites from the λ phage as well as a standard plasmid origin of replication and a drug-resistance gene [here, ampicillin (ampR)]. To clone genomic DNA into a cosmid, the vector is linearized with the restriction enzyme BamHI and the genomic DNA is partially digested with Sau3AI, which leaves BamHI-compatible ends. DNA fragments ranging from 35 to 45 kilobase pairs are isolated and ligated to linearized cosmid DNA, forming tandem arrays of vector and genomic DNA fragments, A λ packaging extract recognizes and packages any ligated DNA that carries two cos sites 35 to 45 kb apart. These cosmid virions are introduced into Escherichia coli cells by infection and replicate as drug-resistant plasmids. In this way, a vector, is available. It represents the entire genome of a microrganism

Yeast Episomal Plasmids

YEPs are shuttle vectors. They can replicate in E. coli and also in yeast; for example, pJDB219 (Figure 9.18). The artificial plasmid has the following features:

 

Figure 9.18 Shuttle vector-YEP

 

  • The 2-μm circular plasmid of yeast.
  • The entire pBR322 sequence.
  • The Leu2 gene (that acts as the selectable marker gene) of the yeast chromosome. This also carries an origin of replication.

2-μm circular plasmid

It is the plasmid found in several strains of yeast. It is 6 Kb in size and has high copy number between 70 and 200. It replicates autonomously using the enzymes provided by the host cell.

Leu2 Gene

This is a yeast chromosomal gene and encodes the enzyme isopropyl malate dehydrogenase. This is one of the enzymes involved in the conversion of pyruvic acid to leucine. When this gene is used as a selectable marker, the host yeast cell must be a mutant that has a non-functional Leu2 gene. Such cells will be unable to synthesize leucine and will grow only when this amino acid is supplemented to the growth medium (Figure 9.19).

On the other hand, cells that are transformed by the YEP containing Leu2 gene can grow in the medium lacking leucine. This enables the selection of transformed yeast cells.

The pBR322 plasmid segment of the YEP has marker genes the ampr and the terr genes, a bacterial origin of replication that will enable the YEP to replicate in a bacterial system too.

 

Figure 9.19 Selection of transformed cells using Leu2 gene as a selectable marker

 

Figure 9.20 Recombinant YEP13 molecules

 

The YEP vector is episomal in nature and can co-integrate with one of the yeast chromosomes. Integration is made possible by homologous recombination between the Leu2 gene and the yeast mutant Leu2 gene. The plasmid portion of the vector may remain integrated or it may be excised later (Figure 9.20).

The initial cloning experiments are done with E. coli cells. The recombinants are selected. The recombinant plasmids are then purified and introduced into yeast where the new gene will express.

YAC Vectors

Large DNA segments can be cloned in YAC. They are linear segments that contain all molecular components required for replication in yeast namely (Figure 9.21(a)):

  • A replication origin known as autonomously replicating sequence (ARE).
  • A centromere.
  • The telomeres.

They are called mini chromosomes. The DNAs of several hundred Kb (200–400 Kb) can be introduced into YACs and successfully cloned (Figure 9.21(b)).

Expression vectors

For a eukaryotic gene to be expressed in E. coli (in bacterial system), the gene must have a promoter, terminator, ribosome-binding site and other regulatory signals recognized by the bacteria. Bacterial RNA polymerase cannot recognize eukaryotic promoter, etc. Therefore, vectors are designed in such way that the inserted foreign gene is placed under the control of E. coli expression signals. Such vectors are called expression vectors since they allow the expression of a foreign gene.

 

Figure 9.21(a) YAC (b) The cloning strategy with pYAC3

 

An expression vector should carry a strong promoter, so that the cloned gene is transcribed at the highest rate. If the recombinant protein produced harmful effects on the host, then its synthesis must be regulated to prevent toxic levels. Some of the promoters used in expression vectors include E. coli Lac promoter, E. coli trp promoter, E. coli tac promoter (a hybrid of lac and trp promoters; stronger than lac or trp and is induced by IPTG), λPL promoter, etc. These promoters can be recognized by E. coli RNA polymerase.

The efficiency of translation of an mRNA in bacteria is dependent on the presence of a ribosome-binding site (i.e., Shine-Dalgarno sequence) and also on the distance between this site and the first AUG codon. Therefore, to express a eukaryotic gene, the Shine-Dalgarno sequence is usually included in the expression vector. The initiation codon of the eukaryotic gene should be placed downstream at the correct distance from the Shine-Dalgarno sequence (Figure 9.22). The expression vectors so designed when expressed will give a ‘hybrid protein’ containing a few amino acids from the prokaryotic protein and the remaining from the eukaryotic protein (i.e., inserted gene). This is called a ‘fusion protein’. Such fusion proteins are more stable in bacteria and are not degraded by bacterial proteases.

 

Figure 9.22 Expression vector

Vectors for Plants

Ti plasmid

The soil-borne bacteria Agrobacterium tumefaciens and A. rhizogenes causes crown gall disease and hairy root disease on the stems of numerous plants. A tumour-inducing plasmid from these bacteria is used by scientists to insert desirable genes into plant chromosomes. This plasmid is called Ti plasmid (as it induces tumour).

 

 

The size of the Ti plasmid ranges between 180 and 250 Kb. It contains a T-DNA region of about 23–25 Kb. Ti plasmid also contains regions for opine synthesis and catabolism.

Genes of Ti Plasmid and their Function

 

vir DNA transfer into plants
shi Shoot induction
roi Root induction
nos Nopaline synthase
noc Nopaline catabolism
ocs Octopine synthase
occ Octopine catabolism
tra Bcterial transfer genes
inc Incompatibility genes
oriV Origin of replication

 

The Ti plasmids can be grouped into three types based on the opine genes they possess; for example, octapine, nopaline and agropine (Figure 9.23).

 

Figure 9.23 Ti plasmid

 

A. tumifaciens attacks many dicotyledonous plants and results in the formation of a tumour. The lipopolysaccharide secreted by the bacterial cell wall helps in its attachment with the polygalacturonic fraction of the plant cell wall. From the wounded cell wall of plant, a phenolic compound called acetosyringone is secreted, which induces the vir genes of Ti plasmid. Vir genes encode an enzyme that nicks the T-DNA and mediates its transfer into the plant cell and gets integrated with the plant DNA. Ops genes encodes enzymes for the synthesis of opines in the transformed cell, which are required for bacterial proliferation. The T-DNA also encodes enzymes that are involved in the biosynthesis of phytohormones, auxin and cytokinin. This results in the disorganized proliferation of plant cells that are commonly known as callus or galls or tumour. The galls are colonized by A. tumefaciens.

The Ti plasmid can act as a very good plant vector. The DNA of interest could be spliced into the T-DNA. The Ti plasmid cannot easily be manipulated. An intermediate vector initially receives the DNA of interest and various other genes necessary for recombination, replication and antibiotic resistance. The intermediate vector can then be inserted into the Ti plasmid forming a, co-integrated plasmid.

The Ti plaarmed, i.e., its entire right-hand region of its T-DNA, including tumour genes and nopaline synthase genes are deleted, making it in capable of tumour formation. It retains the left-hand border of its T-DNA, which is used as the cross-over site for the incorporation of the intermediate vector. The intermediate vector contains multiple unique restriction sites. The gene of interest can be introduced in these sites. The intermediate vector also carries suitable antibiotic marker genes such as kanamycin-resistant gene (kanR) for the selection of the recombinants.

NUCLEIC ACID HYBRIDIZATION AND PROBES

If DNA is denatured and later allowed to renature, the two separated single strands of DNA will zipper back to reform the double-stranded DNA molecule. This ability of the complementary sequences to anneal or to hybridize one another is called ‘nucleic acid hybridization’. This technique helps in determining the gene structure and in identifying molecules that contain the same nucleotide sequence. Thus, from a complex mixture of nucleic acid molecules, hybridization techniques help in the separation of complementary sequence.

Hybridization is normally performed using one labelled sequence called the ‘probe’. Probe is a short oligonucleotide that is complimentary to the target DNA sequence.

For the identification of hybridized nucleic acid duplexes, the labelling of probe is necessary. There are various ways of labelling nucleic acids, which are:

  • Nick translation,
  • End labelling or end filling,
  • Non-radioactive labelling
    1. Biotin labelling
    2. Digoxigenin labelling,
  • Fluorescein labelling and
  • Enzyme labelling.

Nick translation

E. coli DNA Pol-I adds nucleotides to the 3’-OH terminus that is created when one strand of the double-stranded DNA molecule is nicked. In addition, polymerase I can remove nucleotides from the 5’-side of the nick. The simultaneous removal of nucleotides from the 5’-end of the nick and the addition of nucleotides to the 3’-end of the nick result in the movement of the nick (nick translation) along the DNA. By replacing the pre-existing nucleotides with 32P-nucleotides, the 32P-labelled DNA can be obtained (Figure 9.24).

End labelling or end filling

This is a gentler method that rarely causes a breakage of the DNA (nick translation can cause the breakage of DNA under some circumstances). This method can be used to label DNA molecules that have sticky ends. The Kelenow fragment of DNA Pol-I is used. If the reaction is carried out in the presence of labelled nucleotides, then the DNA is labelled (Figure 9.25).

Kinase end labelling

The 5’ terminal phosphate is replaced by 32P-labelled γ-phosphate of [γ-32P] ATP (Figure 9.25(a)).

Fill-in end-Labelling

The DNA of interest is cleaved with a suitable restriction endonuclease to generate sticky ends. These sticky ends act as primer for Klenow DNA polymerase and incorporate labelled nucleotides (Figure 9.25(b)).

 

Figure 9.24 Nick translation

 

Figure 9.25(a) Kinase end labelling of oligonucleotides and (b) Fill-in end-labelling

Non-radioactive labelling

Biotin labelling

dUTP molecules are modified by the reaction with biotin. By nick translation, it is possible to introduce 10–15 biotin dUTP per 100 nucleotides. The biotinylated probe is then be detected by treatment with avidin coupled to a fluorescent marker. Biotinylated adenosine and cytosine triphosphates can also be used instead of dUTP.

Fluorescein labelling

Fluorescein nucleotides such as fluorescein dUTP/UTP/ddUTP are incorporated into oligonucleotides by nick translation. Detection is done by an anti-fluorescein antibody, which is coupled to an enzyme.

Enzyme labels

Enzymes such as peroxidase and alkaline phosphatase can be coupled directly to DNA probes in the presence of glutaraldehyde. The enzyme-labelled probe is hybridized with a target DNA and visualized by adding its chromogenic substrate.

Advantages of non-radioactive labels

  • They are safe than radio-labels.
  • They have long shelf life.
  • There is no radioactive waste disposal problems.
  • They can be used in concentration that shortens the hybridization time.
IN SITU HYBRIDIZATION

This is used to visualize the position of a cloned gene on a eukaryotic chromosome.

The cells are treated with a fixative. They are attached to a glass slide and incubated with RNAse and NaOH to degrade RNA and denature the DNA. The chromosome exposes the segments of DNA. A sample of the cloned gen is labelled and applied to the chromosome preparation. Hybridization occurs between the labelled cloned gene and its chromosomal copy. This results in a dark spot on autoradiography if the cloned gene was radioactively labelled. The position of this spot will indicate the location of the cloned gene on the chromosome (Figure 9.26).

 

Figure 9.26 Fluoresence in situ hybridization

Fluorescent in situ hybridization (FISH)

A fluorescently labelled cloned gene is used as probe and is hybridized to chromosome preparation as described before. This method is also useful for characterizing chromosomal rearrangements and for analysing microdeletions.

MOLECULAR CLONING

Cells all of which contain the same DNA sequences are called ‘clones’. Cloning serves two main purposes.

  1. From a limited number of starting material, a large number of rDNA molecules can be produced.
  2. A second important function is purification. No other DNA molecules are present at the end of the procedure.

The various steps involved in molecular cloning can be outlined in the following sections.

Preparation of Vector DNA

Vector DNAs that originate from microorganisms are propagated and harvested from their appropriate microbial hosts. The bacterial cells grown in a nutrient broth are harvested by low-speed centrifugation (5,000 g for 20 minutes) at 4°C. After the supernatant is discarded, a large bacterial pellet is left. In order to release the plasmid DNA from this pellet, the cells must be ruptured. This can be achieved by a variety of techniques. Sonication and boiling can be used but the most common method is using lysozyme. Lysozyme degrades the peptidoglycan cell wall of bacteria very efficiently. After the cell wall has been disrupted and cell lysis occurs, the plasmid DNA needs to be separated from the cellular proteins and the high molecular weight chromosomal DNA. This can be achieved by treating the lysed cells with the detergent sodium deodecylsulphate (SDS) (1 per cent W/V) and 0.2-M sodium hydroxide. This SDS/NaOH treatment denatures the double-stranded DNA and solubilizes the protein. The alkali treatment is followed by the acid treatment with 3-M sodium acetate in a pH of 4.5. After incubation on ice for an hour, the mixture is centrifuged at high speed (17,000 g) for one hour. This procedure causes acid precipitation of the protein and high molecular weight chromosomal DNA, which are pelleted, leaving the plasmid DNA in the supernatant. The supernatant can then be treated with ethanol and incubated at 0°C-20°C in order to precipitate the plasmid DNA. The plasmid DNA can then be pelleted by centrifugation.

Preparation of Target DNA

The tissue containing the DNA of interest is crushed with a homogenizer. The macerated tissue is placed in a digestion buffer (100-mM NaCl; 0.1 mg/ml proteinase K enzyme). The tissue is digested in the presence of a detergent at 50°C for 12–24 hours. Following the digestion step, the mixture is treated with the organic mixture of phenol and chloroform in order to extract the chromosomal DNA. The DNA is retained in the aqueous phase and the proteins are denatured and extracted into the phenol chloroform organic phase. The DNA is then treated with ethanol that precipitates the DNA and pelleted by centrifugation. The pellet is then washed in 70 per cent ethanol, centrifuged and resuspended in suitable buffer.

Construction of rDNA

The vector and the target DNA are cut with appropriate restriction enzyme and are then ligated together by DNA ligase.

Transport into the Host Cell (Transfection)

There are different ways of transporting the rDNA into a host cell. Some of the methods include:

  • Using calcium chloride,
  • Electroporation,
  • Nucleofection,
  • Liposome-mediated gene transfer,
  • Particle bombardment and
  • Microinjection.

Using calcium chloride

A solution of 50-mM CaCl2causes the rDNA to precipitate onto the outside of bacterial cells (host). Then, a brief heat shock is given by raising the temperature to 45°C. Calcium ions promote cell membrane lysis that facilitates DNA transfer into the cell, subsequent heat shock also promotes the process.

 

Electroporation

This method employs the use of high-voltage electric pulse to introduce the rDNA into the host cell. The host cells are subjected to an electric pulse of 2,500 V for 3–5 m/sec. Some host cells are killed by this process; however, many survive and take up the rDNA.

Nucleofection

Nucleofection is the method of introducing DNA molecules efficiently into the nucleus of virtually any cell type, therefore, significantly increasing the chances of chromosomal integration of the transgene.

Liposome-mediated gene transfer

Polycationic lipid and a neutral lipid when mixed will result in the formation of unilamellar liposome vesicles that have a net positive charge due to the highly positive amine head groups on these molecules. Liposome-mediated gene delivery is technically easy, highly reproducible and very efficient.

Particle bombardment

This is a valuable tool for molecular biologists and permits direct gene transfer to a wide range of cell and tissue types. Some of the important applications of the process include the production of transgenic crop species including maize and soya bean and the introduction of DNA into plastids and mitochondria.

Microinjection

The host cell is immobilized by applying a mild suction with a blunt pipette. The foreign gene is then injected with a microinjection needle. Transgenic mice have routinely been generated by injecting a solution of DNA into a pronucleus of a fertilized egg. The DNA becomes integrated into one of the chromosomes. Cells descending from this fertilized egg, including germ cells, can contain the transgene DNA/rDNA (Figure 9.27).

 

Figure 9.27 Microinjection

Selection of Transformed Cells

The ligation of vector DNA with the DNA of interest may result in:

  • Unligated vectors,
  • Recircularized vector molecules without insert and
  • Host cells that had not taken up the rDNA.

Therefore, the selection of the recombinant host cells is important. This can be done by the following methods:

  • Insertional inactivation of selectable marker gene.
  • Insertional inactivation of antibiotic-resistant gene.

PBR322 plasmid has ampr and tetr genes. For Bam H I, there is a unique restriction site within the tetr gene. Therefore, if the DNA of interest is inserted and the plasmid vector is transformed into E. coli, the amps and tets E. coli hosts become ampr and tets. The various steps involved can be discussed as follows:

  • E. coli cells are transformed with the recombinant plasmid.
  • The E. coli cells are then plated on ampicillin-containing agar. All the colonies that grow in this medium will be transformed cells, as untransformed cells cannot grow in this medium.
  • The bacterial culture is then ‘replica plated’ onto an agar containing tetracycline. In this medium, only ampr and tetr cells will grow, i.e., recombinants will not grow in this medium.
  • The recombinants can be picked from the master plate.

Insertional inactivation of other gene

pUC8 plasmid carries ampr genes and lacZ’ genes. The lacZ’ gene codes for the α-peptide portion of the enzyme β-galactosidase. When this plasmid is inserted into an E. coli that lacks lacZ’ segment (i.e., lacZ’ mutant), the bacterial and plasmid genes complement each other to produce a functional β-galactosidase. This process is called ‘α-complementation’ (Figure 9.28). If a foreign gene is inserted into this lacZ’ gene of pUC plasmids, then it cannot complement and no functional β-galactosidase is formed, i.e., recombinants show no β-galactosidase activity.

 

Figure 9.28 Selection of recombinants by a-complementation

 

The various steps involved are:

  • Recombinants are first selected by growing in ampicillin-containing medium.
  • E. coli cells with normalpUC8 plasmid will be ampr and can synthesize β-galactosidase.
  • Recombinants will be ampr but cannot synthesize β-galactosidase.
  • When X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) is added to the medium, the recombinant colo-nies will appear white in colour, as they do not have β-galactosidase; while non-recombinants will appear blue in colour, as β-galactosidase breaks X-gal to give a blue colour (Figure 9.29).

 

Figure 9.29 Recombinant selection using X-gal

CONSTRUCTION OF C-DNA AND GENOMIC LIBRARIES

Genomic Library

It is a collection of clones containing every single gene present in an organism. For the construction of a genomic library, the entire genomic DNA is isolated from host cells/tissues, purified and broken randomly into fragments of appropriate size for cloning into suitable vector. DNA can be fragmented by physical shearing or by the action of restriction enzymes. These experiments with randomly cloned fragments are known as ‘shot gun cloning experiment’.

The various steps involved in creating a genomic library can be discussed as follows:

  • The chromosomal DNA of the organisms is isolated.
  • It is then treated with a known restriction enzyme.
  • The fragments are cloned using appropriate cloning vector.
  • For identifying recombinants, a marker gene is inserted while multiplying in vector.
  • The rDNA is then hybridized by two major techniques, namely colony hybridization and plaque hybridization.

Colony hybridization

The cloned colonies are transferred from culture plate onto a nitrocellulose filter paper. The filter with the colony replicas is treated with NaOH to lyse the host cells and to denature the DNA. The filter is then baked to fix the DNA. It is then treated with a radio-labelled probe that is complementary to the DNA of interest. The filter is then washed to remove the unbound excess probe. It is then autoradiographed. This indicates the host colonies that carry the desired gene. The colony is taken out from the master culture plate and then mass cultured.

Plaque hybridization

This technique is used when a phage particle is carrying the gene of interest. In this case, a culture of bacterium is infected with a mixture of chimeric phage particles. A large number of plaques develop overnight. These plaques are then treated like the colonies in colony hybridization, thus identifying and isolating the chimeric phage particle carrying the gene of interest.

As the genomic DNA of eukaryotes contains more introns, regulatory regions and repetitive sequences, the establishment of genomic library of eukaryotes is not meaningful. Hence, the cDNA library is generally established for eukaryotes.

cDNA library

For higher organisms such as plants and animals, a gene library will contain so many different clones, so that the identification of a desired clone is difficult. Here, a cDNA library is useful. Since only a few genes are expressed in any cell and only those genes that are expressed are transcribed into an mRNA, if the mRNA is used as the starting material, the resulting clones will comprise only a selection of the total number of genes in the cell. The mRNAs cannot be directly cloned because they are unstable and hence they are converted into their complementary DNAs (cDNAs). The library made from comple-mentary or copy DNA is called ‘cDNA library’. The cDNA library can be made from mRNA because they are highly processed, intron-free and have only coding sequences.

Steps involved in establishing cDNA library are (Figure 9.30):

  • Isolation of mRNA : The majority of mRNA sequence in eukaryotes contains a long polyadenylated tract at their 3’-end. Therefore, mRNA binds to an oligo-dT cellulose affinity column or poly-U sepharose column from which it can be eluted.
  • Reverse transcription : Reverse transcriptase is required for the synthesis of DNA copy of an mRNA. The mRNAs are treated with oligo-dT primer, reverse transcriptase enzyme and dNTPs. The oligo-dT primer binds to polyadenylated tail and provides free 3’-OH for reverse transcription. Reverse transcriptase adds complementary dNTPS one by one to the free 3’-OH group of the primer and thereby results in the formation of RNA-DNA hybrid. Cellular DNA and total RNA inhibit reverse transcriptase; hence, it is necessary that the mRNA must be in pure form before cloning.
  • Oligo-dC tailing : The RNA–DNA hybrid is treated with the enzyme terminal transferase and dCTP. This enzyme adds dCTP one by one to the 3’-OH group of RNA and DNA strands. As the result, a short oligo-dC tail is produced at the 3’-end of both the strands. The cDNA becomes now curved, forming a hairpin loop.
  • Alkali hydrolysis : On treatment with alkaline sucrose solution, the mRNA–cDNA strands are separated into single strands.
  • Addition of oligo-dG primer : Oligo-dG primer is then added to the reaction mixture and the temperature is maintained at 55°C. This favours the binding of dG primer to oligo-dC tails formed on cDNA. Now, cDNA acts as a template for the synthesis of double-stranded cDNA in the presence of DNA Pol-I.
  • Cloning of cDNA : The blunt-ended cDNA is treated with linkers/adaptors and subjected to restriction enzyme action creating sticky ends that are ligated to appropriate vector and transferred into a bacterium. Each bacterial cell possesses a single-stranded cDNA clone and hence the collection of all recombinant bacteria is called cDNA library.
  • Screening of cDNA clones : The cDNA clones can then be selected by colony hybridization.

 

Figure 9.30 cDNA library

 

APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

rDNA technology has gained importance in each and every aspect of modern biological researches. The rDNAs are used in basic research or in the commercial production of useful products. rDNA also find application in agriculture and industry.

In Pharmaceutical Industry

Important compounds such as recombinant insulin, which are used in the treatment of diabetes, human growth hormone (hGH)/somatotropine and interferons can be produced by rDNA technology.

Synthesis of recombinant insulin

Mature insulin has A-chain of 21 amino acids and B-chain of 30 amino acids held together disulphide bridges. It is secreted by the pancreatic β-cells. The DNA with the correct nucleotide sequences to specify the A- and B-polypeptide chains are chemically synthesized. About 63 nucleotides encode the A-chain (21 × 3 = 63) and 90 nucleotides encode the B-chain (30 × 3 = 90). A stop codon and an initiating AUG codon, along with the respective genes for A- and B-chains, were ligated into the lacZ’ gene of E. coli, which was carried in a plasmid expression vector.

The codons must be placed in correct reading frame with that of the lacZ’ gene. The recombinant plasmids were then introduced into E. coli. The recombinant plasmid vector carrying the gene of interest autonomously replicates and is transcribed and translated in the bacterial host. The protein so produced is called a ‘fusion protein’, as it contains a part of β-galactosidase fused by methionine residue to either A-chain or B-chain.

Methionine does not occur in either A-chain or B-chain. Therefore, by treatment with cyno-gen bromide, a chemical that destroys methionine, the A- and B-chains can be released from the β-galactosidase fragment. The A- and B-proteins are then purified, mixed and disulfide bridges were formed to give pure human insulin (Figure 9.31).

Synthesis of somatotropin

Somatotropin, the hGH, is secreted by the anterior lobe of pituitary and consists of 191 amino acid units. The deficiency of somatotropin has been estimated to about 1 in 5,000 children. Hence, there is a need to synthesize this hormone commercially for pharmaceutical purpose. Double-stranded cDNA, produced from mRNA precursor of hGH, was ligated to suitable vector and incorporated into bacterial cell. The synthesis of hGH is induced by an inducer of lac operon (IPTG). The hGH produced is subsequently purified. About 100,000 molecules of the hormone per cell of E. coli can be produced.

Synthesis of interferon

Interferons are proteins that exert non-specific antiviral activity. Interferon is used to cure many viral diseases such as common cold and hepatitis. There are three main classes of interferons, namely IFN-α or leucocyte interferon, IFN-β or fibroblast interferon and IFN-γ or immune interferon. The cDNA prepared from leucocytes, fibroblast and immune cells can be ligated to suitable expression vector and can be cloned in E. coli.

 

Figure 9.31 Production of recombinant insulin

Synthesis of vaccines

Recombinant vaccines for hepatitis B virus (HBV) are produced by cloning HBsAg gene of the virus in yeast cells. The HBsAg gene is introduced near the yeast alcohol dehydrogenase I promoter. The HBsAg gene contains 6-bp-long sequence preceding the AUG that synthesizes the N -terminal methionine. This is joined to ADH promoter and cloned in the yeast vector PMA-56. The recombinant plasmid is inserted into yeast cells. The transformed yeast cells are multiplied in tryptophan-free medium. The transformed cells are selected and cultured. The expressed HBsAg protein has similar structure and immunogenicity that of the HBV and elicits an immune response thus acting as a vaccines.

Gene Therapy

This is the process of treatment by which defective genes are replaced with normal ones. It is of two types, namely gene replacement therapy and gene augmentation therapy. Gene therapy requires:

  • Isolation of particular gene together with its regulatory sequence.
  • Sufficient number of cells from the patient into which the gene is to be inserted.
  • An effective way of returning these cells to the patient.

There are two main strategies of gene therapy namely:

  • Somatic gene therapy : Correcting a genetic defect in the somatic cell of the body.
  • Germ line gene therapy : Introduction of genes into the germ cells for correction of the genetic defect in the offspring.

Gene delivery strategies:

  • Viral : Replication defective retroviral safe vector is tranfected into a packaging cell line. This packages the recombinant safe vector carrying the foreign DNA, with the help of gag, pol and env proteins. This is then allowed to infect the target cell. Within the target cell, it is integrated randomly into host DNA and the foreign gene is expressed.
  • Non-viral : This includes various methods such as aerosol delivery of genes to lungs, direct DNA injection into skeletal muscle and gene-coated gold particles bombardment into liver cells.

Construction of Industrially Important Bacteria

Bacteria with novel phenotypes can be produced by rDNA technology. For example, several genes from different bacteria have been inserted into single plasmid that has been introduced in marine bacterium, making it capable of metabolizing petroleum. This organism has been used to clean up oil spills in oceans. Furthermore, many bacteria are designed to synthesize industrially important chemicals. Some bacteria are also designed to compost waste more efficiently and to fix nitrogen. An interesting example is a strain of Peudomonas fluoresecens that lives in association with the roots of maize and soya bean. A lethal gene from Bacillus thuringiensis, a pathogenic bacterium to the black cutworm has been engineered into this bacterium. Inoculation of soil with engineered P. fluorescens resulted in the death of black cutworm.

Genetic engineering of plants

Altering the genotypes of plants is an important application of rDNA technology. With the help of Ti plasmids, it is possible to introduce genes from one plant into another, thus introducing desired char-acters in required plants. In this way, crop improvements can be made.

Transgenic farm animals

rDNA technology is helpful for the production of transgenic animals. For example, caseins are the major milk proteins. By transferring genetically manipulated casein genes, the texture of cheese and heat-stable dairy products can be improved.

For clearing environmental pollution

Genes responsible for degradation of environmental pollutants such as toluene, chlorobenzene, halo-genated pesticides and other toxic compounds have been identified.

For example, OCT plasmid degrades octane, hexane and decane and XYL plasmid degrades xylene and toluenes.

  • DNA of two different species can be linked to form the ‘recombinant DNA’, ‘rDNA’ or ‘chimeric DNA’.
  • ‘Molecular cloning’ is the laboratory process used to create rDNA.
  • Upon translation, the rDNA results in the production of ‘recombinant protein’ or ‘fusion protein’.
  • An organism containing an artificially inserted foreign piece of DNA is said to be ‘transgenic’.
  • Restriction endonucleases are molecular scissors that destroy or restricts any foreign DNA entering a bacterial cell.
  • Restriction endonucleases are isolated from two different sources but possess similar recognition and cleavage sites are called ‘isochizomers’.
  • A DNA molecule that carries foreign DNA into a host cell, replicates inside the host cell and produces many copies of itself is called a cloning vector.
  • Vectors that can replicate in different host systems, for example, in E. coli and in yeast, are called shuttle vectors.
  • The ability of the complementary sequences to anneal or hybridize to one another is called ‘nucleic acid hybridization’.
  • Hybridization is normally performed using one labelled sequence called the ‘probe’. Probe is a short oligonucleotide that is complimentary to the target DNA sequence.
  • The library made from complementary or copy DNA is called ‘cDNA library’. The cDNA library can be made from mRNA because they are highly processed, intron-free and has only coding sequences.
  1. Enumerate the steps involved in DNA isolation in laboratory.

  2. What is the importance of DNA sequencing? Mention the types of DNA sequencing.

  3. Describe Maxam and Gilbert’s chemical method.

  4. Define restriction endonuclease. Explain in detail about the types of restriction endonuclease.

  5. What are isoenzymes?

  6. Describe in detail about plasmid vectors.

  7. Explain the importance of Ti plasmid as vectors for plants.

  8. Explain the concept of nucleic acid hybridization.

  9. How is labeling of nucleic acids performed by nick translation?

  10. Write short notes on biotin labeling and fluorescence labeling.

  11. Explain the various steps involved in molecular cloning.

  12. What do you understand by the term c-DNA library? List its significance.

  13. How do you establish a c-DNA library?

  14. Write in brief about the various commercial applications of rDNA technology.

MULTIPLE-CHOICE QUESTIONS
  1. In Maxam and Gilbert’s method, the separated single stranded DNA labeled at its 5’ end byusing ———.

    1. polynucleotide lyase
    2. polynucleotide hydrolase
    3. polynucleotide kinase
    4. polynucleotide ligase
  2. Reverse transcriptase is used in the synthesis of ———.

    1. mRNA
    2. cDNA
    3. tRNA
    4. rRNA
  3. Which of the following plasmids were developed by Messings et al?

    1. Ti Plasmids
    2. YEP vectors
    3. YAC vectors
    4. pUC family
  4. What type of vectors can replicate in different host systems for example in E.coli and in Yeast?

    1. YEP vectors
    2. YAC vectors
    3. Bacteriophages
    4. Shuttle vectors
  5. What is the size of Ti plasmid?

    1. 180–250 Kb
    2. 100–170 Kb
    3. 300–370 Kb
    4. 50–120 Kb
  6. Which method can be used to label DNA molecules that have sticky ends?

    1. Nick translation
    2. End labelling or end filling
    3. Non radioactive labeling
    4. Biotin labeling
  7. Cells, all of which contain the same DNA sequences is called a ———.

    1. Plasmid
    2. Bacteriophage
    3. Clone
    4. None of the above
  8. ———is the method of introducing DNA molecules efficiently into the nucleus of virtually any cell type.

    1. Cloning
    2. Transfection
    3. Nucleofection
    4. Transduction
  9. In colony hybridization, the filter with the colony replicas are treated with ———to lyse the host cells and to denature the DNA.

    1. Calcium Chloride
    2. Sodium Chloride
    3. Calcium Hydroxide
    4. Sodium Hydroxide
  10. What type of vectors are usually used for cloning larger DNA segments.

    1. YAC vectors
    2. YEP vectors
    3. Ti plasmids
    4. Bacteriophages

Alberts, B., Bray, D., Lewis, J., et al. 1994. Molecular Biology of the Cell, 3rd edition. New York, NY: Garland Science.

Berg, J. M., Tymoczko, J. L. and Stryer, L. 2002. Biochemistry, 5th edition. New York, NY: W. H. Freeman. Cooper, G. M. 2000. The Cell: A Molecular Approach, 2nd edition. Sunderland, MA: Sinauer Associates. Dubey, R. C. 2007. A Textbook of Biotechnology. New Delhi: S. Chand and Company.

Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., et al. 2000. An Introduction to Genetic Analysis, 7th edition. New York, NY: W. H. Freeman.

Hill, John E., Myers, Alan M., Koernery, T. J. and Tzagoloffs, Alexander. 1986. ‘Yeast/E. coli Shuttle Vectors with Multiple Unique Restriction Sites’, YEAST, 2: 163–167.

Purohit, S. S. 2005. Biotechnology Fundamentals and Applications, 3rd edition. Published by Student Edition, India.

Strachan, Tom and Read, Andrew P. 1999. Human Molecular Genetics, 2nd edition. New York, NY: Wiley-Liss.