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10.15. Recombinant DNA (Genetic Engineering) prof. aza 10.15. Recombinant DNA (Genetic Engineering) • The body requires a constant supply of certain peptides and proteins if it is to remain health and function normally. Many of these peptides and proteins are only produced in a small quantities. • They will be produced only if the correct genes are present in the cell. prof. aza • Consequently, if a gene is missing or defective an essential protein will not be produced, which can lead to a diseased state. prof. aza • For example, cystic fibrosis is caused by a defective gene. This faulty gene produces a defective membrane protein, cystic fibrosis transmembrane regulator (CFTR), which will not allow the free passage of chloride ions through the membrane prof. aza • The passage of chloride ions through a normal membrane into the lungs is usually accompanied by a flow of water molecules in the same direction. • In membranes that contain CFTR the transport of water through the membrane into the lungs is reduced. prof. aza • This viscous mucus clogs the lungs and makes breathing difficult, a classic symptom of cystic fibrosis. It also provides a breeding ground for bacteria that cause pneumonia and other illnesses. prof. aza • Several thousand hereditary diseases found in humans are known to be caused by faulty genes. Recombinant DNA (rDNA) technology (genetic engineering) offers a new way of combating these hereditary diseases by either replacing the faulty genes or producing the missing peptides and proteins so that they can be given as a medicine (see section 10.15.2). prof. aza • The first step in any use of recombinant DNA technology is to isolate or copy the required gene. There are three sources of the genes required for cloning. • The two most important are genomic and copy or complementary DNA (cDNA) libraries. prof. aza • In the first case the library consists of DNA fragments obtained from a cell’s genome, whilst in the second case the library consists of DNA fragments synthesised by using the mRNA for the protein of interest. prof. aza • The third is by the automated synthesis of DNA, which is only feasible if the required base sequence is known. • This may be deduced from the amino acid sequence of the required protein if it is known. prof. aza • Once the gene has been obtained it is inserted into a carrier (vector) that can enter a host cell and be replicated, propagated and transcripted into mRNA by the cellular biochemistry of that cell. This process is often referred to as gene cloning. prof. aza • The mRNA produced by the cloned DNA is used by the cell ribosomes to produce the protein encoded by the cloned DNA. In theory, gene cloning makes it possible to produce any protein provided that it is possible to obtain a copy of the corresponding gene. Products produced using recombinant DNA usually have recombinant, r or rDNA in their names. prof. aza 15.1. Gene cloning • Bacteria are frequently used as host cells for gene cloning. This is because they normally use the same genetic code as humans to make peptides and proteins. • However, in bacteria the mechanism for peptide and protein formation is somewhat different. prof. aza • It is not restricted to the chromosomes but can also occur in extranuclear particles called plasmids. • Plasmids are large circular supercoiled DNA molecules whose structure contains at least one gene and a start site for replication. prof. aza • However, the number of genes found in a plasmid is fairly limited, although bacteria will contain a number of identical copies of the same plasmid. • It is possible to isolate the plasmids of bacterial cells. prof. aza • The isolated DNA molecules can be broken open by cleaving the phosphate bonds between specific pairs of bases by the action enzymes known as restriction enzymes or endonucleases prof. aza • Each of these enzymes, of which over 500 are known, will only cleave the bonds between specific nucleosides. • For example, EcoRI cleaves the phosphate link between guanosine and adenosine whilst Pvu II cuts the chain between cytidine and thymine nucleosides. prof. aza • Cutting the strand can result in either blunt ends, where the endonuclease cuts across both chains of the DNA at the same points, or cohesive ends (sticky ends), where the cut is staggered from one chain to the other (Fig. 10.47). prof. aza • The new non-cyclic structure of the plasmid is known as linearised DNA in order to distinguish it from the new insert or foreign DNA. prof. aza • This foreign DNA must contain the required gene, a second gene system that confers resistance to a specific antibiotic and any other necessary information. It should be remembered that a eukaryotic gene is made up of exons separated by introns, which are sequences that have no apparent use. prof. aza • Figure 10.47 (a) Blunt and (b) cohesive cuts with compatible adhesive cuts prof. aza • Mixing the foreign DNA and the linearised DNA in a suitable medium results in the formation of extended plasmid loops when their ends come into contact (Fig. 10.48). • This contact is converted into a permanent bond by the catalytic action of an enzyme called DNA ligase. prof. aza • Figure 10.48. A representa tion of the main steps in the insertion of a gene into a plasmid prof. aza • When the chains are cohesive the exposed single chains of new DNA must contain a complementary base sequence to the exposed ends of the linearised DNA. prof. aza • The hydrogen bonding between these complementary base pairs tends to bind the chains together prior to the action of the DNA ligase, hence the name ‘‘sticky ends’’. • The new DNA of the modified plasmid is known as recombinant DNA (rDNA). prof. aza • However, the random nature of the techniques used to form the modified plasmids means that some of the linearised DNA reforms the plasmid without incorporating the foreign DNA, that is, a mixture of both types of plasmid is formed. prof. aza • The modified plasmids are separated from the unmodified plasmids when they are reinserted into a bacterial cell. prof. aza • The new plasmids are reinserted into the bacteria by a process known as transformation. • Bacteria are mixed with the new plasmids in a medium containing calcium chloride. This medium makes the bacterial membrane permeable to the plasmid. prof. aza • However, not all bacteria will take up the modified plasmids. • Such bacteria can easily be destroyed by specific antibiotic action since they do not contain plasmids with the appropriate protecting gene. prof. aza • This makes isolation of the bacteria with the modified plasmids relatively simple. • These modified bacteria are allowed to replicate and, in doing so, produce many copies of the modified plasmid. prof. aza • Under favourable conditions one modified bacterial cell can produce over 200 copies of the new plasmid. The gene in these modified plasmids will use the bacteria’s internal machinery to automatically produce the appropriate peptide or protein. prof. aza • Since many bacteria replicate at a very rapid rate this technique offers a relatively quick way of producing large quantities of essential naturally occurring compounds that cannot be produced by other means. prof. aza • Plasmids are not the only vectors that can be used to transport DNA into a bacterial host cell. • Foreign DNA can also be inserted into bacteriophages and cosmids by similar techniques. prof. aza • Bacteriophages (phage) are viruses that specifically infect bacteria whilst a cosmid is a hybrid between a phage and a plasmid that has been especially synthesised for use in gene cloning. Plasmids can be used to insert fragments containing up to 10 kilobasepairs (kbp), phages up to 20 kbp and cosmids 50 or more kbp. prof. aza • It is not always necessary to use a vector to place the recombinant DNA in a cell. If the cell is large enough, the recombinant DNA may be placed in the cell by using a micropipette whose overall tip diameter is less than 1 mm. prof. aza • Only a small amount of the recombinant DNA inserted in this fashion is taken up by the cell’s chromosomes. • However, this small fraction will increase to a significant level as the cell replicates (Fig. 10.48). prof. aza • Host cells for all methods of cloning are usually either bacterial or mammalian in origin. For example, bacterial cells often used are E. coli and eukaryotic yeast while mammalian cell lines include Chinese hamster ovary (CHO), baby hamster kidney (BHK) and African green monkey kidney (VERO). prof. aza • In all cases small-scale cultures of the host cell plus vector are grown to find the culture containing the host with the required gene that gives the best yield of the desired protein. prof. aza • Once this culture has been determined the process is scaled up via a suitable pilot plant to production level (see section 16.6). The mammalian cell line cultures normally give poorer yields of the desired protein. prof. aza 15.2.2 Manufacture of Pharmaceuticals • The body produces peptides and proteins, often in extremely small quantities, which are essential for its well being. • The absence of the necessary’ genes means that the body does not produce these essential compounds, resulting in a deficiency disease that is usually’ fatal. prof. aza • Treatment by supplying the patient with sufficient amounts of the missing compounds is normally successful. • However, extraction from other natural sources is usually’ difficult and yields are often low. prof. aza • For example, it takes half a million sheep brains to produce 5mg of somatostatin a growth hormone that inhibits secretion of the pituitary growth hormone. prof. aza • Furthermore, unless the source of the required product is donated blood there is a limit to the number of cadavers available for the extraction of compounds suitable for use in humans. prof. aza • Moreover, there is also the danger that compounds obtained from human sources may be contaminated by’ viruses such as HIV, hepatitis, Creutzfeld– Jakob disease (mad cow disease) and others that are difficult to detect. Animal sources have been used but only a few human protein deficiency disorders can be treated with animal proteins. prof. aza • Gene cloning is used to obtain human recombinant proteins. However, some proteins will also need post— translational modification such as glycosylation and/or the modification of amino acid sequences. • These modifications may require forming different section, of the peptide chain in the culture medium and chemically’ combining these sections in vitro. prof. aza • The genes required for these processes are synthesised using the required peptide as a blueprint. For example, human recombinant insuline may he produced in this manner (Figure 10.12). The genes for the A and B chains of insulin were synthesised separately. prof. aza • They were cloned separately, using suitable plasmids. into two different bacterial strains. One of these strains is used to produce the A chain whilst the others is used to produce the B strain. The chains are isolated and attached to each other by in vitro disulphide bond formation. prof. aza • This last step is inefficient and human recombinant insulin is now made by forming recombinant proinsulin by gene cloning. • The proinsulin is converted to recombinant insulin by proteolytic cleavage prof. aza Figure 10.42. An outline of the synthesis of recombinant human insulin. prof. aza