Gene cloning

By-PRIYA TAMANG

Introduction

• Gene cloning is a common practice in molecular biology labs • It is used to create copies of a particular gene for downstream applications,

such as sequencing, mutagenesis, genotyping or heterologous expression of a protein.

• The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid.

• This technique is commonly used today for isolating long or unstudied genes and protein expression.

• A more recent technique is the use of polymerase chain reaction (PCR) for amplifying a gene of interest.

• The advantage of using PCR over traditional gene cloning, is the decreased time needed for generating a pure sample of the gene of interest.

• However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can be performed for further analysis.

• A clone is an exact copy of an organism, organ, single cell, organelle or macromolecule.

• Cell lines for medical research are derived from a single cell allowed to replicate millions of times, producing masses of identical clones.

• Gene cloning is the act of making copies of a single gene.• Cloning can provide a pure sample of an individual gene,

separated from all the other genes that it normally shares the cell with.

• Once a gene is identified, clones can be used in many areas of biomedical and industrial research.

• Genetic engineering is the process of cloning genes into new organisms, or altering a genetic sequence to change the protein product.

What is gene cloning?

History • In 1922, morghan and his colleagues developed the technique for

gene mapping.• In 1903,W.sutton proposed the idea of a gene residue on

chromosome.• By 1922, they had analysis the relative positions of over 2000 genes

on the 4th chromosomes of fruit fly, drosophila melongata.• Until 1940’s, there was no real understanding of molecular nature of

gene.• In 1944, the experiments of Avery, McLeod, and McCarty and in

1952, horshey and chase, stated that DNA ( deoxyribose nucleic acid) is the genetic material: up until then it was thought that genes were made up of protein.

• Discovery in the role of DNA was tremendous stimulus to the genetic research.

• Delbruck, chargeff, crick and monad, contributed in the second great age of genetics.

• Between 1952 and 1966, in this years the structure of DNA was elucidated, the genetic code cracked, and the process of transcription and translation.

• There was a period of anticlimax, some molecular biologist was in state of frustration that the experimental techniques of the late 1960’s were not sophisticated enough to allow the gene to studied in any greater extends.

• During 1971-73, there was a revolution thrown back into gear by introducing completely new methodology, recombinant-DNA technology or genetic engineering.

• This new methodology as heir core in the process of gene cloning, it sparkled as another great age of genetics.

• It led to rapid and efficient DNA sequencing techniques that enabled the structure of individual genes to be determined, reaching culmination with the massive genome sequencing projects including the Human genome project which was completed in 2000.

• In 1985, Kary Mullis invented the PCR , an exquisitely simple technique that acts as a perfect complement to Gene Cloning.

• PCR has made easier many of the technique, that were possible but difficult to carry out when gene cloning was used on it own.

• It extended to the range of DNA analysis and enabled molecular biology to find range in the field of medicine, agriculture, and biotechnology.

• With the invention of PCR , the Archeogenetics, molecular biology, and DNA Forensics have to become possible.

• 40 years passed since the dawning of age of gene cloning, but there is no end to the excitement in sight.

Fundamental steps 

• Identification and isolation of the desired gene or DNA fragment to be cloned.

• Insertion of the isolated gene in a suitable vector.• Introduction of this vector into a suitable organism/cell called

host. • The vector multiplies within the host cell, producing numerous

identical copies not only of itself but also of the gene that it carries.

• During the division of the host cell, copies of the recombinant DNA molecules are passed to the progeny and further vector replication takes place. 

• After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule.

What is PCR?• PCR is a method of copying DNA molecules. DNA replication is common in

life; for example it takes place inside your own cells every time they divide. An enzyme known as polymerase uses one strand of DNA as a template to create a complementary strand. The result is that one double stranded DNA molecule is converted into two, both identical to the first.

• PCR, or the polymerase chain reaction, adds two components to this process. The initial reaction yields twice the number of starting molecules, but then is immediately followed by a subsequent reaction, which yields twice the molecules as the first reaction. This is why PCR is known as a chain reaction. Commonly 25-40 reactions are chained together, theoretically resulting in 225 – 240 more molecules of DNA then were initially present.

• Additionally, the goal of a PCR reaction is commonly to replicate only a portion of the genome of interest. For example, somewhere between 75-1000 bases, instead of the entire human genome of 3 billion bases. As PCR produces billions of copies of only the DNA of interest, this process is known as “amplification”.

Why is PCR important?

• The amplification provided by PCR is very powerful. For example, suppose we want to detect whether a dangerous E. Coli pathogen is present in a sample of meat. That meat sample contains a huge amount of DNA from the meat source, and many non-pathogenic bacteria. Looking for the DNA from the pathogenic E. Coli, is akin to searching for a needle in a haystack.

• However a PCR reaction can be designed to amplify only the DNA from a portion of this pathogenic E. Coli. If the pathogen is present, we can make billions of copies of its targeted DNA, which will come to outnumber the overall DNA originally present in the sample, and allow us to easily detect it. If no such signal is amplified by a properly controlled reaction, we can conclude the pathogen was not present.

How is it used?• PCR and related techniques have many applications. Here

are just a few• Human Diagnostics

▫ Detecting viral infections (HIV, etc.)▫ Detecting bacterial infections (Tuberculosis, etc.)▫ Genotyping (detecting genetic variants, which can indicate

predisposition to disease)• Environmental Monitoring

▫ Water quality monitoring▫ Food safety testing

• Scientific Research▫ Preparing DNA to sequence▫ Monitoring gene expression levels▫ Manipulating DNA in genetic engineering and synthetic

biology

How does PCR work?

• The principles behind every PCR, whatever the sample of DNA, are the same.

• Five core ‘ingredients’ are required to set up a PCR. We will explain exactly what each of these do as we go along. These are:▫ the DNA template to be copied▫ primers, short stretches of DNA that initiate the PCR reaction,

designed to bind to either side of the section of DNA you want to copy

▫ DNA nucleotide bases? (also known as dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA

▫ Taq polymerase enzyme? to add in the new DNA bases▫ buffer to ensure the right conditions for the reaction.

• PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.

There are three main stages:▫ Denaturing – when the double-stranded template DNA is heated to

separate it into two single strands.▫ Annealing – when the temperature is lowered to enable the DNA

primers to attach to the template DNA.▫ Extending – when the temperature is raised and the new strand of

DNA is made by the Taq polymerase enzyme.

• These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

• A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines.

• After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

What happens at each stage of PCR?•Denaturing stage

▫During this stage the cocktail containing the template DNA and all the other core ingredients is heated to 94-95⁰C.

▫The high temperature causes the hydrogen bonds? between the bases in two strands of template DNA to break and the two strands to separate.

▫This results in two single strands of DNA, which will act as templates for the production of the new strands of DNA.

▫It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely.

▫This usually takes between 15-30 seconds.

•Annealing stage▫ During this stage the reaction is cooled to 50-65⁰C. This enables

the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding (the exact temperature depends on the melting temperature of the primers you are using).

▫ Primers are single strands of DNA or RNA? sequence that are around 20 to 30 bases in length.

▫ The primers are designed to be complementary? in sequence to short sections of DNA on each end of the sequence to be copied.

▫ Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases.

▫ The two separated strands of DNA are complementary and run in opposite directions (from one end - the 5’ end – to the other - the 3’ end); as a result, there are two primers – a forward primer and a reverse primer.

▫ This step usually takes about 10-30 seconds.

•Extending stage▫During this final step, the heat is increased to 72⁰C to

enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.

Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus aquaticus.

This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.

The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.

DNA polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37˚C (body temperature).

• 72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.▫ The result is a brand new strand of DNA and a double-

stranded molecule of DNA.• The duration of this step depends on the length of DNA

sequence being amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb).

• These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA sequence of interest.

• The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase enzyme can attach and start making DNA.

• The result is a huge number of copies of the specific DNA segment produced in a relatively short period of time.  

Why gene cloning and PCR are so important?

•Obtaining a pure sample of a gene by cloning

The problem of selection.

PCR can also be used to purify a gene

Gene isolation by PCR

Cloning applications• Gene cloning has made a phenomenal impact on the speed of biological

research and it is increasing its presence in several areas of everyday life. One of the reasons why biotechnology has received so much attention during the last decade is because of gene cloning.

 • Production of recombinant protein 

▫ Proteins that are normally produced in very small amounts include growth hormone, insulin in diabetes, interferon in some immune disorders and blood clotting factor VIII in hemophilia, are known to be missing or defective in various disorders. Prior to the advent of gene cloning and protein production via recombinant DNA techniques, these molecules were purified from animal tissues or donated human blood. But both sources have drawbacks, including slight functional differences in the non human proteins and possible viral contamination. (e.g. HIV, CJD). Production of protein from a cloned gene in a defined, non pathogenic organism would circumvent these problems. A gene for an important animal or plant protein can be taken from its normal host, inserted into a cloning vector, and introduced into a bacterium. If the manipulations are performed correctly then the gene will be expressed and the protein is synthesized by the bacterial cell. Then it is possible to obtain large amounts of the protein.But in practice obtaining recombinant protein is not as easy as theoretically it sounds. For this special types of cloning vectors are needed.

Expression of foreign genes in E.coli• Expression of a foreign gene in E.coli is dependent on the

collection of signals surrounding the gene. These signals, which are short sequences of nucleotides, advertise the presence of the gene and provide instructions for the transcriptional and translational apparatus of the cell. The three most important signals for E.coli genes are▫ 1) the promoter, at which transcription should start,▫ 2) the terminator,at which transcription should stop, and▫ 3) the ribosome binding site, a short nucleotide

sequence recognised by the ribosome as the point at which it should attach to the mRNA molecule.

• The foreign gene is inserted into a vector in such a way that the gene is placed under control of a set of E.coli expression signals. Cloning vehicles which provide these signals, and which can therefore be used in the production of recombinant protein, are called expression vectors.

  

•   An efficient expression requires a strong promoter, an E.coli ribosome binding sequence and a terminator.

• As the foreign gene is inserted into a unique restriction site present in the middle of the expression signal cluster, so a cassette is formed by these expression signals in most of the vectors.

• Ligation of the foreign gene into the cassette therefore places it in the ideal position relative to the expression signals.

• Insertion of the foreign gene into this restriction site must be performed in such  a way as to fuse the two reading frames, producing a hybrid gene that starts with the E.coli segment and progresses without a break into the codons of the foreign gene.

• The product of gene expression is therefore a hybrid protein, consisting of the short peptide coded by the E.coli reading frame fused to the amino-terminus of the foreign protein. 

Problems with the production of recombinant protein in E.coli • The problems associated with the production of protein from foreign genes cloned

in E.coli can be grouped into two categories: those that are due to the sequence of the foreign gene, and those that are due to the limitations of E.coli as a host for recombinant protein synthesis.

• The problems associated with the sequence of the foreign gene are:• 1)  The foreign gene might contain introns, this would be a major problem as E.

coli genes don't contain introns and the bacterium therefore doesn't posses the necessary machinery for removing introns from transcripts.

• 2) The foreign gene might contain sequences that act as termination signals in E.coli.• 3) The codon usage of the gene may not be ideal for translation in E.coli. These

problems can usually be solved, though the necessary manipulations may be time consuming and costly.

• The problems associated with E.coli are:• 1) E.coli might not process the recombinant protein correctly.• 2) E.coli might not fold the recombinant protein correctly. If the protein doesn't take

up its correctly folded, tertiary structure then usually it is insoluble and forms an inclusion body within the bacterium. Its nearly  impossible to convert the protein into it's correctly folded form. The protein is inactive under these circumstances.

• 3) E.coli might degrade the recombinant protein.•

        The problems associated with obtaining high yields of active recombinant proteins from genes cloned in E.coli have led to the development of expression system for higher organisms. Yeasts and fungi can be grown just as easily as bacteria in continuous culture, and may express a cloned gene from a higher organism.

Recombinant protein from yeast

• The yeast Saccharomyces cerevisiae is the most popular microbial eukaryote for recombinant protein production. Cloned genes are often placed under the control of the GAL promoter, which is normally upstream of the gene coding for galactose epimerase, enzyme involved in the metabolism of galactose. The GAL promoter is induced by galactose, regulating expression of a cloned gene.

•        Yields of recombinant proteins are relatively high, but S.cerevisiae is unable to glycosylate animal proteins correctly and lacks an efficient system for secreting proteins into the growth medium. In the absence of secretion, recombinant proteins are retained in the cell and are consequently less easy to purify. Besides these drawbacks, S.cerevisiae is the most frequently used microbial eukaryote for recombinant protein synthesis.

Recombinant protein from filamentous fungi

• Advantages of fungi in recombinant protein production lie in their good glycosylation properties and their ability to secrete proteins into the growth medium. The wood rot fungus Trichoderma reesei,in its natural habitat secretes cellulolytic enzymes that degrade the wood that it lives on. These fungi produce recombinant proteins in a form that aids purification. The fungus Aspergillus nidulans is popular for producing recombinant proteins. Expression vectors for A.nidulans usually carry the glucoamylase promoter, induced by starch and repressed by xylose.

Animal cells in recombinant protein production

• For proteins with complex and essential glycosylation structures an animal cell might be the only type of host within which the active protein can be synthesized. A problem with some animal cell lines is that they require a solid surface on which to grow, adding complications to the design of culture vessels. Rates of growth and maximum cell densities are much for animal cells compared with microorganisms, limiting the yield of recombinant protein. Two promoters that have been used in mammalian cells are the heat shock promoter of the human hsp-70 gene, which is induced at temperatures above 40°C, and the mouse metallothionein gene promoter, which is switched on by addition of zinc salts to the culture medium.

Gene cloning in medicine• Medicine will continue to be a major beneficiary of the gene

cloning revolution.

Production of recombinant insulin• Insulin, synthesized by the β-cells of the Islets of Langerhans in the pancreas, controls the

level of glucose in the blood. Insulin deficiency will result in the disease diabetes mellitus, which is a life threatening disease if left untreated. Insulin is a relatively small protein molecule, comprising two polypeptides. In humans these are synthesized as a precursor called preproinsulin which contains the A and b segments linked by a third chain C and preceded by a leader sequence. The leader sequence is removed after translation and the C chain is excised, leaving the A and B polypeptides linked to each other by two disulphide bonds.

• For synthesis and expression of artificial insulin genes, two recombinant plasmids were constructed, one carrying the artificial gene for the A chain and one the gene for the B chain. In each case, the artificial gene was ligated to a lacZ' reading frame present in a pBR322-type vector. The insulin genes were therefore under the control of the strong lac promoter and were expressed as fusion proteins, consisting of the first few amino acids of β-galactosidase followed by the A or B polypeptides. Each gene was designed so that its β-galactoatsidase and insulin segments were separated by a methionine residue, so that the insulin polypeptides could be cleaved from the β-galactosidase segments by treatment with cyanogen bromide. The purified A and B chains were then attached to each other by disulphide bond formation in the test tube. A subsequent improvement has been to synthesize not the individual A and B genes, but the entire proinsulin reading frame, specifying B chain-C chain- A chain. The pro-hormone can fold spontaneously into the correct, disulphide- bonded structure. The C chain segment can then be excised relatively easily by proteolytic cleavage.

Synthesis of other recombinant human proteins and recombinant vaccines

• Many growth factors are have been synthesized from gene cloned in bacteria and eukaryotic cells. These growth factors include somatostatin, somatotropin, factor VIII, Interferon-α, Interferon-β, Interferon-γ, interleukins etc. which are used in tratment of various diseases and also some of them having potential uses in cancer therapy. These proteins are synhesized in very limited amounts in the body, so recombinant technology is the only viable means of obtaining them in the quantities needed for clinical purposes.

•Production of  vaccines as recombinant proteins

• The use of gene cloning in this field centres on the discovery that virus specific antibodies are sometimes synthesized in response to not only to the whole virus particle, but also to isolated components of the virus. This is particularly true of purified preparations of the proteins present in the virus coat. If the genes coding for the antigenic proteins of a particular virus could be identified and inserted into an expression vector, then  the method for the synthesis of animal protein could be employed in the production of recombinant proteins that might be used as vaccines. These vaccines would have the advantages that they would be free of intact virus particles and they could be obtained in large quantities. But this approach hasn't entirely been successful, because recombinant coat proteins often lack the full antigenic properties of the intact virus. But one success has been achieved with hepatitis B virus, whose coat protein has been synthesized in Saccharomyces cerevisiae, using a vector based on 2μm plasmid. The protein was obtained in reasonably high quantities  and when injected into monkeys provided protection against hepatitis B virus. This recombinant vaccine has been approved for use in humans.

•        Live recombinant vaccine: Recombinant vaccinia viruses could be used as live vaccines against other diseases. If a gene coding for a virus coat protein, is ligated into the vaccinia genome, under control of a vaccinia promoter, then the gene will be expressed. After injection into the blood stream, replication of the recombinant virus will result not only in production of new vaccinia particles, but also in significant quantities of the major surface antigen.

• Live recombinant vaccine▫ Recombinant vaccinia viruses could be used as live vaccines

against other diseases. If a gene coding for a virus coat protein, is ligated into the vaccinia genome, under control of a vaccinia promoter, then the gene will be expressed. After injection into the blood stream, replication of the recombinant virus will result not only in production of new vaccinia particles, but also in significant quantities of the major surface antigen.

• Role of gene cloning in identification of genes responsible for human diseases▫ A genetic disease is caused by a defect in a specific gene. 

Individuals carrying the defective gene are being predisposed towards developing the disease at some stage of their lives. Gene identification may provide an indication of the biochemical basis to the disease, enabling therapies to be designed. Identification of the mutation present in a defective gene can be used to devise a screening programme so that the mutant gene can be identified in individuals who are carriers or who have not yet developed the disease. Identification of the gene is a prerequisite for gene therapy.

• Mapping the breast cancer gene BRCA1▫ Mapping of a gene accurately is required for gene isolation. Linkage analysis

involves comparing the inheritance pattern for the target gene with the inheritance patterns for genetic loci whose map positions are already known. If two loci are inherited together then they must be very close on the same chromosome. With breast cancer, the first breakthrough occurred in 1990 as a result of RFLP linkage analysis carried out by a group at the University of California at Berkeley. This study showed that in families with a high incident of breast cancer a significant number of women who suffered from the disease all possessed the same version of an RFLP called D17S74.This RFLP had previously been mapped  to the long arm of chromosome 17. BRAC1 must therefore also be located on the long arm of chromosome 17. For pinpointing the BRAC1 more accurately, the chromosomal region containing BRAC1 was examined for tendem repeat sequences, similar to those used in genetic fingerprinting, but with  very short repeat units. These repeat sequences are useful in linkage analysis as the number of repeats at a locus is highly variable, probably because occasional errors in DNA replication leads to additional units being inserted. Linkage is assessed by comparing the inheritance patterns of a locus of a particular length and the gene under investigation. This approach, using the repeat loci identified on chromosome 17, reduced the size of BRAC1containing region from 20 Mb down to just 600kb.

• Gene therapy▫ Gene therapy is the final application of cloning in medicine. Gene therapy is the

insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development.

• Somatic cell therapy• It involves manipulation of ordinary cells, usually ones which can be removed from the

organism, transfected and then placed back in the body. The technique has most promise for inherited blood diseases, with genes being introduced into stem cells from the bone marrow, which give rise to all the specialized cell types in the blood. Somatic cell therapy also has potential in the treatment of lung diseases such as cystic fibrosis, as DNA introduced into the respiratory tracts of rats via an inhaler is taken up by the epithelial cells in the lungs, although gene expression occurs for only a few weeks.

•Germline therapy

• In germline therapy, a fertilized egg is provided with a copy of the correct version of the relavant gene and reimplanted into the mother. If successful, the gene is present and expressed in all cells of the resulting individual. Germline therapy is usually carried out by microinjection of DNA into the isolated egg cell and theoretically could be used to treat any inherited disease.

•  • Before gene therapy can become a permanent cure for any condition, the therapeutic DNA

introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

•  • Gene cloning in agriculture• Gene cloning provides a new dimension to crop breeding by enabling direct changes to be

made to the genotype of a plant, circumventing the random processes inherent in conventional breeding. In gene addition, cloning is used to alter the characteristics of a plant by providing it with one or more new genes. In gene subtraction, genetic engineering techniques are used to inactivate one or more of the plant's existing genes.

Gene addition• Plants are subject to predation by virtually all other types of organisms-

viruses, bacteria, fungi and animals but in agriculture, the greatest problems are caused by insects. To reduce losses by insects, crops are regularly sprayed with insecticides. Several of these insecticides have potentially harmful side effects on the environment. An ideal insecticide should be toxic only to the insect targeted and not to any other insects, animals and humans. The insecticide should also be biodegradable, so that any residues remain after the crop is harvested, or which are carried out of the field by rain water, don't persist long enough to damage the environment.

•        Insects along with plants occasionally use bacteria as their diet. Several types of bacteria have evolved defence mechanisms against insect predation, for example Bacillus thuringiensis during sporulation forms intracellular crystalline bodies that contain an insecticidal protein called the δ-endotoxin which is highly poisonous to insects. It is an inactive precursor and after ingestion by the insect this pro-toxin is cleaved by proteinases, resulting in shorter versions of the protein that display the toxic activity, binding to the inside of insect's gut and damaging the surface epithellium so that the insect is unable to feed and consequently starves to death. Biodegradability of B.thuringiensis acts as a disadvantage so they must be reapplied at regular intervals during the growing season which is not cost effective. The δ-endotoxins that do not require regular application  are developed by modifying the structure of toxin via protein engineering to be more stable.

•     

•    Maize is a crop plant that is not served well by conventional insecticides. A major pest of the plant is the European corn borer, which tunnels into the plant from eggs laid on the under-surfaces of leaves, thereby evading the effects of insecticides applied by spraying. The first attempt at countering this pest by engineering maize plants to synthesize δ-endotoxin was made by plant biotechnologists at a Ciba-Geigy laboratory in North Carolina. They worked with the Cry1A(b) version of the toxin which had previously been shown to be a 1155-amino-acid protein, with the toxic activity residing in the segment from amino acids 29 to 607. Instead of isolating the natural gene they made a shortened version of the first 648 codons, by artificial gene synthesis. The artificial gene was ligated into a cassette vector between a promoter and polyadenylation signal from cauliflower mosaic virus, and introduced into maize embryos by bombardment with DNA coated microprojectiles. The embryos were grown into mature plants, and transformants identified by PCR analysis of DNA extracts, using primers specific for a segment of the artificial gene.

•        In the immunological tests the results showed that the artificial gene was indeed active, but the amount of δ-endotoxin being produced varied from plant to plant. This was probably due to positional effects, the level of expression of a gene cloned in a plant often being influenced by the exact location of the gene in the host chromosomes.

•Gene subtraction▫ Gene subtraction is a modification which involves the

inactivation of a gene rather than its removal. The most successful so far in practical terms is the use of antisense technology.

•Antisense technology▫ In antisense technology the gene to be cloned is ligated into the

vector in reverse orientation.When this cloned gene is transcribed, the RNA synthsized is the reverse complement of the mRNA from the normal sequence. This antisense RNA is able to prevent synthesis of the product of the gene it is directed against, this is due to the hybridization between antisense and sense copies. This block the expression by degrading double-stranded RNA by cellular ribonucleases. It prevents ribosomal attachment to sense strand.

Application• The role of polygalacturonase gene in tomato

fruit ripening▫ In tomato within six weeks after flowering, a number of genes involved in

the later stages of ripening are switched on, including one coding for the polygalacturonic enzyme. This enzyme slowly breaks down the polygalacturonic acid component of the cell walls in the fruit pericarp, resulting in a gradual softening. Partial inactivation of the polygalacturonase gene increases the time between flavour development and spoilage of the fruit.

•  • Cloning the antisense polygalacturonase gene

▫ A 730-bp restriction fragment was obtained from the 5' region of the normal polygalactujronase gene. A plant polyadenylation signal was attached to the beginning of this fragment, a cauliflower mosaic virus promoter was ligated to the end, and the construction was inserted into the Ti plasmid vector pBIN19. Inside the plant, it synthesizes antisense RNA complementary to polygalacturonase mRNA.

▫  The recombinant pBIN19 molecules were introduced into Agrobacterium tumefaciens bacteria and then the bacteria were allowed to infect tomato stem segments. Small amount of callus material collected from surface of these segments were tested for their ability to grow on an agar medium containing kanamycin. Resistant transformants were identified and allowed to develop into mature plants.

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