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Using recombinant DNA technology to make
             Human haemoglobin A
            
             Haemoglobin A (Hb) found in adult humans, is essential for their survival, as it carries oxygen around the body in the bloodstream. Because of the extremely low percentage of blood donors (due to an apathetic view towards this need) and the high risks of infected or non-compatible blood (due to diseases and different blood types), scientist have been working on a way in which they can produce Hb in transgenic tobacco plants. 
             Scientists have discovered the genetic coding of the Hb genes in humans, which have coding for the production of the necessary polypeptides. The gene coding is obtained and inserted into the plasmids of the bacteria (Agrobacterium Tumefaciens). The bacterium causes crown gall disease in the Tobacco plant, causing the plant to synthesise haemoglobin in their chloroplasts.
             The production of Hb in the chloroplasts tobacco plants has been found to be very reliable, matching Hb produced by a human.  This application will bring the result of higher survival chances for humans. It has also been found that the haemoglobin can increase the plant growth. One major concern is the usefulness of this product. Is it really better than donated blood? The general publics' acceptance is unknown, but it is thought that most would be against the idea of a product of tobacco being injected into their bloodstream.
            
            
            
            
             Relevant Genetics in Haemoglobin A Synthesis
            
             All human cells contain chromosomes, which contain information for the structure and function of humans. On them are found Deoxyribonucleic acid (DNA) which is a type of nucleic acid on which genetic information is held. DNA consists of two parallel polynucleotide chains and is in the form of a double helix, whereas Ribonucleic acid (RNA) is single stranded. If DNA is considered to be shaped like a ladder, the sides are made of sugar-phosphate chains. Linking the chains, the rungs, are the bases. There are four of these, Adenine(A), Guanine(G), Cytosine(C) and Thymine(T). Adenine always pairs with Thymine and Cytosine with Guanine, in what are known as "base pairs" This constant pairing allows DNA to replicate, producing copies of the genetic material.
            
            
            
            
            
            
             All haemoglobin genes are formed like this. These genes are Haemoglobin Beta(HBB) on chromosome 11, Haemoglobin Alpha1&2(HBA1,HBA2) on chromosome 16 (identical genes).
                          In the same way, a molecule of messenger RNA (mRNA) can be formed alongside part of the DNA molecule, and it is this mRNA that acts as the template for protein synthesis - in this case for the production of polypeptide chains. In mRNA, Uricil(U) replaces Thymine. For protein synthesis (the synthesis of a haemoglobin peptide chain), the DNA needs to be copied. This is done through the process of transcription. The enzyme polymerase splits the double helix DNA and additional nucleotides come and lock on to the DNA forming mRNA. It is an accurate complimentary of the DNA's instruction for making polypeptide chains.
            
            
            
            
            
            
            
            
            
            
             Each codon codes for a specific Amino Acid. There are specific start/stop codons that show where to start/stop the production of Amino acids. The HBB & HBA genes are 146 & 141 codons long respectively.
             The next step is the mRNA moving out of the nucleus into the cytosol and to a ribosome. There the process of translation begins. This will begin where the start signal is on the mRNA, this codon will connect to make the first amino acid in the chain. The next codon will connect an amino acid to the first one. For this to happen, the codon on the mRNA connects to its complimentary transfer RNA (tRNA) brought together by reverse transcriptase enzyme.   The tRNA being the anti-codon (the one that connects to the mRNA's codon being complimentary )  is connected to an amino acid. As more of the Amino acids are added they are connected together by peptide bonds. When the synthesis is complete (i.e. When the amino acid has been connected for the stop signal) the tRNA separate from the mRNA.
            
            
            
            
            
            
            
            
            
            
            
            
             All the genes required for the normal functioning of the bacterium are carried on the double stranded circular chromosome. In addition to the chromosome, the bacterium may have plasmids. Plasmids are small circular double stranded, extrachromosomal DNA molecules. All DNA contain the same base nucleotides, A, T, G, and C.
            
            
            
            
            
             A Hb molecule consists of a Heme molecule, 2-alpha and 2-beta polypeptide chains.
            
            
            
            
            
            
             Genetic techniques used in getting Haemoglobin coded information for synthesis
            
             The information of the haemoglobin genes needs to be attained. This is done by looking in specific cells that produce haemoglobin, and by making a copy of the (Hb) mRNA template, which are exact copies of the genes. The haemoglobin mRNA's, produced through transcription would be found in red blood cells. Because this is a specific cell for the production of haemoglobin, the mRNA found in that cell would be that for the coding of haemoglobin amino acids.
            
            
            
            
            
            
            
              The mRNA acts as a template to help with the formation of a single stranded DNA molecule, copy DNA (cDNA). An enzyme, reverse transcriptase catalyses the joining of the nucleotide subunits to the mRNA.
            
            
            
            
            
            
            
            
            
            
            
            
            
            
             A second enzyme DNA polymerase makes the cDNA into a double helix molecule.
            
            
            
            
            
            
            
                  
            
            
             The result of this will be 2 HBA DNA and1 HBB DNA strands.
            
             The bacterium, Agrobacterium tumefaciens is obtained and it is burst open.
            
            
            
            
            
             The tumor inducing plasmid rings (Ti plasmid) are obtained and are cut open using a restriction enzyme.
            
   
            
            
            
              This same enzyme is to be used to create sticky ends on each of the ends of the three DNA pieces.
            
            
            
            
            
             The cut plasmids and the Haemoglobin DNA can be mixed together with the ligase enzyme, which causes the sticky ends to rejoin.  These recombinant plasmids are mixed with the bacterium.
            
            
            
            
            
            
            
            
            
            
             Some will take up the DNA and will multiply. When the bacterium contacts the tobacco plant cells, a segment of this plasmid T DNA is transferred from the Ti plasmid to the plant cell nucleus. It causes crown gall disease, characterised by tumors. Protein synthesis will occur in the plants' chloroplasts. Tobacco plants produce their own heme molecules, which bind the chains. These tumors will contain recombinant haemoglobin (rHb), which can then be extracted.
           
            
            
            
            
            
            
            
          Biological Implications
             This genetic technique produces Haemoglobin, and when suspended in a saline solution, will allow people with low haemoglobin levels (due to accidents, etc.) to raise them to more normal, safer levels.  That is the intended benefit; also scientists have found  human haemoglobin could benefit the tobacco and other plants.
             A human, who has lost blood, can have their blood increased by an injection of rHb. Thus the levels of haemoglobin is increased to safer, healthier levels. The human's survival chances are increased; therefore then chance for reproduction is improved. By surviving, his possible usefullness in the community is increased; hence being an asset to the community. The haemoglobin won't affect the genetic make-up of the human's gametes in any way. Because all the effects of this "plant produced" element in humans are unknown, and the side effects are yet to be discovered, it isn't known if this would become a frequent, safe treatment for bloodloss.
             Scientists of the University of Luna, Sweden, have produced fast growing tobacco plants. This is done by there being haemoglobin molecules in the plants. The Haemoglobin, synthesised by the plant, increases the plant's oxygen supply, allowing it to make extra chlorophyll. It is believed that haemoglobin can be used in other plants to produce larger plants. However, "Scientists don't known how accurate the structure and function will be of these when compared to 'normal' plants".  So it mightn't be a clear benefit for plants.
            
            

            
            
          
            
                 
             Issue
             Is it safer to use recombinant haemoglobin - which has been synthesised in transgenic tobacco plants but not yet fully tested, or to rely on blood donors?
             At present, the scientists say their tobacco-derived haemoglobin could be a safe product.7 Testing of the molecule has shown its function to be the same as that of the humans'; it is able to bind to oxygen and carbon monoxide as does natural Hb.8 It is safer and less risky than that of blood donors as there are no cells in the synthetic blood, so there needn't be any concern about blood matching.9 Being produced in plants, rHb should be free of viruses and disease, and there'd be smaller risks for contamination107. Because rHb has the same functional properties as natural Hb, it can perform well and requires neither matching nor replenishment under pressure.
             However, tobacco has negative connotations, at least in the health sector. Will the public have faith in this kind of product as an alternative source of treatment produced in this plant? They'd need to be convinced such that there would be an acceptance of the alternate treatment.11 The rHb in a saline solution wouldn't contain all the essential properties of blood such hormones, antibodies, etc. which are found in donated blood.12 "Clinical trials so far of the haemoglobin-based substitute have established toxic side effects"13. The apparent risks of donors' blood seem fewer.