Madical brain Notes 10

  Introduction In the last 30 years or so, there has been a revolution in the field of genetics, which has had a profound effect on virtually every other area of biology. This has been due to the development of new techniques that have enabled scientists to analyse and manipulate DNA in a quite unprecedented way. Genetically modified crops, DNA ‘fingerprinting’ and gene therapy are just three of the many applications made possible by these ad-vances. The beginnings of genetic engineering can be said to date from the discovery, in the late 1960s, of a class of bacterial enzymes called  restriction endonucleases  (REs). These are enzymes that cleave DNA into pieces by mak-ing breaks in the sugar-phosphate backbone; in nature, they serve to destroy any foreign DNA that may enter the cell. They do not cut the DNA in a random fash-ion, however; their unique usefulness to the molecu-lar biologist lies in the fact that they break the DNA  in a precise and reproducible manne...

  Plasmid cloning vectors Two main types of vector system are used in cloning, those that use  plasmids  and those that use  bacterio-phages . Naturally occur-ring examples of these are manipulated so that they pos-sess the above properties. A popular vector in the early days of gene cloning was the plasmid pBR322; Figure 12.4 shows how it contains the features described above. Let us consider now what happens, at a molecular level, when donor DNA is ligated into a plasmid vector (Figure 12.5). Sticky ends of the donor fragment form hy-drogen bonds with the exposed compatible ends of the opened up plasmid by complementary base pairing, and DNA ligase consolidates the join. Many plasmids con-tain engineered sequences called  multiple cloning sites  (MCS); these provide additional flexibility with respect to the restriction fragments that may be accommodated.   The ligation of insert DNA into the cloning site of the plasmid is not the only possible outco...

  Bacteriophages as cloning vectors The most commonly used bacteriophage vectors are those based on  phage Lambda  ( λ ) (Figure 12.8). The genome of  λ  is 48.5kb in length; it was the first genome to have its entire sequence determined (1982). As drawn conventionally, the genome is linear, and contains 46 genes. At each 5 end of the double-stranded structure is a 12 base single-stranded sequence known as a  cos  site; these have a complementary base sequence (i.e. they are cohesive, or ‘sticky’ ends). They can join together to form a double-stranded, circular  λ  molecule, a conformation that is essential for insertion and integration into the host genome. The naturally occurring phage is unsuitable as a vector, because being relatively large, it contains multiple copies of recognition sites for a number of REs. By using genetic manipulation techniques, however, unwanted sites can be removed, allowing the DNA to be ‘opened up’ at a single l...

  Expression vectors Sometimes, the aim of a cloning experiment is not just to obtain large amounts of a specific gene, but for the gene to be  expressed . This involves using the host cell as a sort of ‘factory’, to manufacture the specific protein encoded by the cloned gene. One of the earliest applications of genetic engineering was the production of human insulin in  E. coli  (Figure 12.10). Insulin is needed in considerable quantities for the treat-ment of diabetics; for years it was obtained from the pancreas of pigs and cattle, but this had several disadvantages including immunological complications and the risk of viral contamination. Insulin generated by recombinant means is free from these prob-lems. Many proteins can now be produced in this way by microorganisms at a rate several times that of the normal host cell. In order for a gene to be expressed, it must have specific nucleotide sequences around it that act as signals for the host cell’s tran-scriptio...

  Eucaryotic cloning vectors A number of problems are associated with the expression of complex eucaryotic genes in bacterial cloning systems, some of which have just been outlined. In order to avoid these, such genes may be expressed in eucaryotic cloning systems, the model system for which is the yeast  Saccharomyces cerevisiae . This holds many attractions for the experimenter: Several types of vector have been developed for use in yeasts. These are summarised in Table 12.1. The choice of which type to use will depend on the relative importance to a particular application of factors such as yield and long-term stability. Most yeast vectors are  shuttle vectors , that is, vectors that can be replicated in more than one type of host cell (in this case, yeast and  E. coli ). Initial cloning is more easily done in  E. coli , then the recombinant vector is transferred to yeast cells, which can carry out functions such as protein folding and glycosylation that are ...

  Polymerase chain reaction (PCR) First described in the mid-1980s by Kary Mullis, PCR is probably the most significant development in molecular biology since the advent of gene cloning. PCR allows us to amplify a specific section of a genome (for example a particular gene) millions of times from a tiny amount of starting material (theoretically a single molecule!). The impact of this powerful and highly specific process has been felt in all areas of biology and beyond. Medicine, forensic science and evolutionary studies are but three areas where PCR has opened up new possibilities over the last 15 years or so. To appre- ciate how PCR works, you will need to understand the role of the enzyme DNA polymerase in DNA replication. This is the enzyme, you’ll recall, that when provided with single-stranded DNA and a short primer, can direct the synthesis of a complementary second strand. Figure 12.14 illustrates the three steps in the PCR process: Denaturation:  by heating to 95 ◦ C,...