Cambridge International A Level Biology Revision Guide

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Cambridge International A Level Biology 466 is used that gives blunt ends, then sticky ends need to be attached to both the gene and the plasmid DNA. The opened plasmids and the lengths of DNA are mixed together. Some of the plasmid sticky ends pair up with the sticky ends on the new gene. The enzyme DNA ligase is used to link together the sugar–phosphate backbones of the DNA molecule and the plasmid, producing a closed circle of double-stranded DNA containing the new gene. This is now recombinant DNA. Bacterial plasmids can be modified to produce good vectors. Plasmids can also be made artificially. For example, the pUC group of plasmids have: ■■ ■■ ■■ ■■ a low molecular mass, so they are readily taken up by bacteria an origin of replication so they can be copied several single target sites for different restriction enzymes in a short length of DNA called a polylinker one or more marker genes, allowing identification of cells that have taken up the plasmid. Plasmids are not the only type of vector that can be used. Viruses can also be used as vectors. A third group of vectors are liposomes, which are tiny spheres of lipid containing the DNA. There is more about these other vectors later in this chapter. Getting the plasmids into bacteria The next step in the process is to get bacteria to take up the plasmids. The bacteria are treated by putting them into a solution with a high concentration of calcium ions, then cooled and given a heat shock to increase the chances of plasmids passing through the cell surface membrane. A small proportion of the bacteria, perhaps 1%, take up plasmids with the gene, and are said to be transformed. The rest either take up plasmids that have closed without incorporating a gene or do not take up any plasmids at all. Identifying bacteria with recombinant DNA It is important to identify which bacteria have been successfully transformed so that they can be used to make the gene product. This used to be done by spreading the bacteria on agar plates each containing an antibiotic. So if, for example, the insulin gene had been inserted into the QUESTION 19.2 Summarise the advantages of using plasmids as vectors in genetic engineering. plasmid at a point in the gene for tetracycline resistance in pBR322, then any bacteria which had taken up plasmids with the recombinant DNA would not be able to grow on agar containing tetracycline. However, this technique has fallen out of favour, and has largely been replaced by simpler methods of identifying transformed bacteria (page 468). DNA polymerase in bacteria copies the plasmids; the bacteria then divide by binary fission so that each daughter cell has several copies of the plasmid. The bacteria transcribe the new gene and may translate it to give the required gene product, such as insulin. Insulin production One form of diabetes mellitus is caused by the inability of the pancreas to produce insulin (Chapter 14). Before insulin from GM bacteria became available, people with this form of diabetes were treated with insulin extracted from the pancreases of pigs or cattle. In the 1970s, biotechnology companies began to work on the idea of inserting the gene for human insulin into a bacterium and then using this bacterium to make insulin. They tried several different approaches, finally succeeding in the early 1980s. This form of human insulin became available in 1983. The procedure involved in the production of insulin is shown in Figure 19.5. There were problems in locating and isolating the gene coding for human insulin from all of the rest of the DNA in a human cell. Instead of cutting out the gene from the DNA in the relevant chromosome, researchers extracted mRNA for insulin from pancreatic β cells, which are the only cells to express the insulin gene. These cells contain large quantities of mRNA for insulin as they are its only source in the body. The mRNA was then incubated with the enzyme reverse transcriptase which comes from the group of viruses called retroviruses (Chapter 10, page 206). As its name suggests, this enzyme reverses transcription, using mRNA as a template to make singlestranded DNA. These single-stranded DNA molecules were then converted to double-stranded DNA molecules using DNA polymerase to assemble nucleotides to make the complementary strand. The genetic engineers now had insulin genes that they could insert into plasmids to transform the bacterium Escherichia coli. The main advantage of this form of insulin is that there is now a reliable supply available to meet the increasing demand. Supplies are not dependent on factors such as availability through the meat trade.
Chapter 19: Genetic technology Isolation of human gene human pancreas Preparation of vector bacteria β cells free nucleotides reverse transcriptase enzyme mRNA from β cells, some with the code for human insulin mRNA with the code for human insulin identified and isolated DNA polymerase restriction enzyme restriction enzyme plasmids from bacteria single strand cDNA Plasmids from bacteria are cut with the same restriction enzyme to produce sticky ends. cDNA mRNA is taken from β cells and used to make DNA with the help of reverse transcriptase. It is then cut to leave sticky ends. Formation of recombinant DNA, identification and cloning The gene is cloned by growing the bacterium. A transformed bacterium is identified. The recombinant plasmid is introduced into bacteria. ligase enzyme The insulin DNA and plasmid DNA are mixed together with a ligase enzyme. The sticky end bases form hydrogen bonds. The ligase joins the DNA backbone and a recombinant plasmid is produced. 467 Manufacture The transformed bacteria, containing the insulin gene, can produce insulin. These bacteria are now grown on an industrial scale in large fermenters. Insulin is extracted from the bacteria. fermenter extraction and purification of insulin pure human insulin Figure 19.5 Producing insulin from genetically modified bacteria.
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Mary Jones, Richard Fosbery, Jennif
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Index gluconeogenesis, 317 glucose,
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Acknowledgements Meiosis” in Tuat
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